Fully delocalised chemiosmotic or localised proton flow pathways in energy coupling?

Biochimica et BiophysicaActa 811 (1985) 47-95

47

Elsevier

BBA86120

Fully delocalised chemiosmotic or Iocalised proton flow pathways in energy coupling? A scrutiny of experimental evidence Stuart J. Ferguson Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham, B I 5 2 TT ( U.K.) (Received June 14th, 1984) (Revised manuscript received November 9th, 1984)

Contents I.

Introduction

II.

Structural studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. A nonvesicular membrane capable of energy coupling? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Chemical modification of the thylakoid and inner mitochondrial membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bacterial mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The action of certain colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Consideration of the lateral distribution and motion of membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49 49 50 52 54 55

III.

Thermodynamic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Alkalophilic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Uncoupler-resistant mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Decreases in protonmotive force that are not always accompanied by proportional decreases in the extent of A T P synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Relationship of the magnitude of the protonmotive force to the extent of bacterial substrate accumulation . . . . . . . .

56 57 58

IV.

............................................................................

Kinetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Relationships between mitochondrial respiration rate and the size of the protonmotive force . . . . . . . . . . . . . . . . . B. The effect of electron-transport inhibitors upon the extent of respiratory control . . . . . . . . . . . . . . . . . . . . . . . . . . C. Effects of changes in rates of electron transport on protonmotive force and rate of A T P synthesis . . . . . . . . . . . . . . D. Relationships of rates of bacterial transport to protonmotive force . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Effects of partial uncoupling upon protonmotive force and rates of ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . F. Effects of changes in rates of nucleotide triphosphate hydrolysis upon rates of reversed electron transfer and transhydrogenase in submitochondrial particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Effects of inhibitors of the A T P synthase upon the protonmotive force and the rate of A T P synthesis . . . . . . . . . . . H. Effects of combinations of uncouplers and inhibitors upon rates of ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . J. The combined effects of restriction of electron transport and inhibitors of the A T P synthase; the double-inhibitor experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. The kinetics of the release of protons into the internal bulk aqueous phase of thylakoids . . . . . . . . . . . . . . . . . . . .

Abbreviations: ANS, 1-anilinonaphthalene-8-sutphonate; A C M A , 9-amino-6-chloro-2-methoxyacridine; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Ap, protonmotive force (summation of membrane potential, Aft, and p H gradient,

48

60 67 68 69 70 71 73 75 76 77 79 80 84

ApH, expressed in mV units); DCCD, N,N'-dicyclohexylcarbodiimide; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; PMS, phenazine methosulphate.

0304-4173/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

48 L. Deductions from the use of 21_120in place of H20 in measurementsof photophosphorylation . . . . . . . . . . . . . . . . M. Instances of failure to detect translocated protons following pulsed turnovers of mitochondrial and bacterial electron-transfer chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. Observationsmade with fluorescent probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

86

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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V.

I. Introduction There is widespread satisfaction that the problem of energy transduction in oxidative phosphorylation, as well as in related processes including many bacterial active transport systems, has been finally solved in general terms by the chemiosmotic theory [1]. This solution tells us, taking oxidativ e phosphorylation as an example, that passage of electrons along an electron-transfer chain embedded in a membrane is linked to the translocation of protons from the aqueous phase at one side of the membrane to the aqueous phase at the opposite side. Since the membrane is continuous and presents a significant kinetic barrier to the passive diffusion of protons, an electrochemical gradient of protons, usually called a protonmotive force (Ap), is generated across the membrane. The protonmotive force provides the driving force for protons to flow back across the membrane through the proton-translocating ATP synthase with concomitant A T P synthesis. Important features of this mechanism are that there need be no physical interactions between the components of the electron transfer chain and the ATP synthase, and that the membrane itself should be regarded as acting as an insulator between the two aqueous phases. As stressed by Mitchell [1], it is a mistake in terms of the chemiosmotic theory to speak of an 'energised' membrane. Although in its original formulation definite stoichiometries of proton translocation were assigned to the A T P synthase and the electron-transfer chain, the exact values of these stoichiometries are not crucial to the general concept of chemiosmotic coupling. Nevertheless, it is important to determine these stoichiometries. Despite the increasing evidence for, and therefore acceptance of, the chemiosmotic mechanism

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during the last twenty years there has been a steady stream of reports in which doubts have been raised as to whether this mechanism does provide a completely correct description of oxidative phosphorylation and related processes. Many of the reported observations have led to suggestions that pathways of proton flow are localised within or along a membrane rather than delocalised via the bulk aqueous phases at either side of a membrane. It is the present writer's impression that at least some of these reports have been widely ignored, yet apparently very important problems have been raised. Consequently, it is considered timely to review these findings in a critical fashion in the hope of distinguishing those reports that: (i) are based on inappropriate experimentation; (ii) place constraints on mechanisms of enzymes and other protein devices that catalyse chemiosmotic energy-transduction processes but do not contradict the principles of chemiosmotic coupling; (iii) raise genuine concerns as to the validity of the chemiosmotic theory. The principal objects of this review are therefore to bring together a variety of experimental findings, and to analyse whether these findings really do disturb the widespread acceptance of the chemiosmotic theory. In general, the experimental work to be discussed falls into one of three categories, structural, thermodynamic and kinetic. Accordingly, the article deals with each of these three topics in turn, although there are instances in which the nature of the work has necessitated a somewhat arbitrary assignment to one of these topics. Whatever the outcome of the considerations in this article, and the predilection of the reader, the writer hopes that at least some of the points raised will draw attention to important but perhaps neglected features of energy-transduction processes

49 in bacterial plasma membranes, mitochondria and the thylakoids of green plants. II. Structural studies

The decisive way to falsify the chemiosmotic theory would be to prepare a well-characterised membrane preparation that is competent in energy coupling and yet does not form an ion-tight seal around an aqueous lumen or internal space. By definition a protonmotive force could not be set up across such a membrane. Over the years a number of claims of such preparations have appeared, but to this reviewer's knowledge they have never been substantiated or reproduced in other laboratories. In this review only the most recent of these reports will be considered. Other structural studies are less direct, but we must consider here both the outcome of work on the chemical modification of the thylakoid membrane under a variety of conditions and the information available about certain mutants of Escherichia coli and other bacteria that raises some interesting questions. Many of these studies that have led to the conclusion that a chemiosmotic mechanism might not operate. Thus an alternative mechanism for energy coupling is required. It is usual to invoke localised pathways for protons which would allow protons to flow from, say, electron-transfer chains to ATP synthases without contact with the bulk aqueous phases, but rarely is any positive experimental evidence for such a mechanism provided.

11A. A nonvesicular membrane capable of energy coupling? A preparation of inner mitochondrial membrane fragments from rabbit skeletal muscle has been proposed to have a nonvesicular structure but to be capable of energy-transduction [2-4]. The evidence for the latter is based on four types of experiment: (i) the uncoupler 1799 (bis[hexafluoroacetonyl]acetone) stimulates the rate of respiration with either NADH, glycerol 3-phosphate or succinate as substrate [2]; (ii) addition of 1799 results in an increase in the steady state level of oxidation of cytochrome b in the presence of any of these substrates [2]; (iii) uncoupler-sensitive release of protons from the membranes is linked to

electron-transfer reactions [3]; (iv) an uncouplersensitive response of an acridine dye fluorescence is established by electron-transfer reactions and is enhanced by lipophilic anions including thiocyanate [4]. All these observations would be taken today by most investigators to mean that at least some of the membrane fragments were sealed vesicles capable of generating a protonmotive force. The argument that these fragments might not be sealed vesicles but rather open membrane fragments strips, and therefore unable to generate such a protonmotive force, is based on the following observations, together with the accepted view that in intact mitochondria the active sites of the dehydrogenases for NADH and glycerol 3-phosphate are on the matrix and cytosolic sides, respectively, of the inner membrane which is impermeable to both substrates. With the fragments from rabbit skeletal muscle mitochondria it was found that either NADH or glycerol 3-phosphate could fully reduce both b- and c-type cytochromes, thus indicating that the respiratory chains of all the fragments were equally accessible to both substrates [2]. This observation is inconsistent with the fragments being solely inverted vesicles (with respect to the original mitochondria) because this type of vesicle should not be able to oxidise glycerol 3phosphate. Similarly, the vesicles could not have retained the orientation of the mitochondria because in this case NADH should not be oxidised. Nor could the fragments be mixtures of these two types of vesicle because in this situation an additive effect of NADH and glycerol 3-phosphate upon the reductions of cytochromes could be expected. Consequently, it has been argued that the fragments must be sufficiently permeable to allow both NADH and glycerol 3-phosphate to have ready access to their dehydrogenases. Such high permeability of the membrane to hydrophilic and charged molecules would be incompatible with the generation of a protonmotive force, and these preparations from skeletal muscle mitochondria might reasonably be regarded as open fragments. However, there is perhaps an alternative explanation for the behaviour of these membrane fragments based on a precedent reported for membrane vesicles from Escherichia coli. It has been argued that certain dehydrogenases can sometimes relocate from one side of the membrane to the

50 other during the preparation procedure [5]. Thus the vesicles are considered to be scrambled [5], but the relocated dehydrogenases are still able to feed electrons into the respiratory chain. Whilst this type of process has only been documented for bacterial membranes, it is a possibility that nevertheless must be recognised also in the case of mitochondrial membrane preparations. Hence, the skeletal muscle mitochondrial membrane fragments might be essentially sealed vesicles that are uniformly inverted with respect to the mitochondrion, but with glycerol 3-phosphate on the external surface of the vesicles owing to its migration during preparation of the fragments. This point needs to be checked. The suggestion of a relocation of the glycerol 3-phosphate dehydrogenase enzyme could account for all the experimentally observed features of energy coupling described above, but if this explanation is put aside for the moment the question is whether the data so far obtained with the mitochondrial fragments from skeletal muscle preparations are really sufficient to throw doubt on the validity of the chemiosmotic theory. According to the present writer's assessment, these data would only be given considerable weight if further work were done, including the following (see also comments of WikstriSm in Ref. 6): (i) additional evidence is required to show that the preparations are nonvesicular. This might include electron microscopy, demonstration of the absence of a sucrose (or other suitable marker) impermeable space and relative accessibility of antibodies to specific membrane proteins; (ii) additional indicators of energy conservation including ATP synthesis and attempts to detect the uptake of permeant ion indicators of membrane potential, e.g., SCN or Rb ÷ in the presence of valinomycin (and related methods for the pH gradient), would be helpful, especially in assessing whether the membranes are essentially inverted with a relocated glycerol 3-phosphate dehydrogenase. These extra methods of characterisation are essential because the weight of evidence from a great range of other experiments indicates that nonvesicular membrane preparations are not capable of energy transduction. Certainly, these observations with skeletal muscle mitochondrial membrane fragments ought not to be left in the literature neither substantiated nor

refuted. The present reviewer can offer only a hypothetical rationale for the properties of these membranes, and it would be of value if the work were to be repeated and extended in another laboratory. HB. Chemical modification of the thylakoid and inner mitochondrial membranes Acetic anhydride acetylates amino and other potentially nucleophilic groups of proteins more rapidly when the pH of the surrounding aqueous medium is sufficiently high for such groups to be deprotonated. This acetylation reaction has been used to probe the pH experienced by proteins of the thylakoid membrane under a variety of conditions. The extent of labelling of thylakoid proteins and a parallel inhibition of water-splitting activity (Photosystem II) by modification with acetic anhydride in the dark has been shown to be enhanced by the presence of protonophores [7-9]. This enhancement has been suggested to be related to proton efflux from the thylakoids and consequent elevation of the pH around the polypeptides that react with acetic anhydride. Illumination of thylakoids in the presence of low concentrations of protonophores results in protection of Photosystem II from inhibition and a decrease in the general extent of acetylation. This result can be rationalised in terms of a reduction of the pH in the lumen of the thylakoids and protonation of the polypeptides as the protonophore is present at sufficiently low concentration to permit some net light-driven proton uptake. A result that at least at first sight is inconsistent with full delocalisation of protons within a bulk aqueous phase of the thylakoid lumen was noted when conditions were arranged in which either photosystem I or Photosystem II was operating alone. Proton uptake driven solely by Photosystem I was ineffective at both protecting Photosystem II from inactivation by acetylation and inhibiting incorporation of acetyl groups into proteins. In contrast, when only Photosystem II was working, and under conditions in which the net uptake of protons was the same as in the experiments with just photosystem I functioning, protection of Photosystem II against inhibition by acetic anhydride was observed. These experiments were taken to mean that the protons

51 taken up during operation of Photosystem I were not deposited into the same internal space as those taken up by Photosystem II. Extents of chemical modification are, however, sensitive to conformational changes, and, as McCarty [10] has pointed out, conformational changes during the operation of Photosystem II could be responsible for the protection against inhibition by acetic anhydride. On the other hand further work has now shown that ATP-dependent proton uptake in the dark (after activation of the proton translocating ATP synthase) resembles the operation of Photosystem II by protecting Photosystem II from the inhibitory effects of acetic anhydride [11]. All these observations have been taken as evidence that there exist in the thylakoid membrane domains into which protons released by operation of Photosystem II are delivered, and from which they can travel directly to the ATP synthase enzyme. Protons released by Photosystem I are suggested not to have access to this domain and thus protection of Photosystem II from the effects of acetic anhydride is not observed when proton uptake by thylakoids is driven solely by Photosystem I [7-9,11]. Accepting for the moment the above interpretation of the effects of acetic anhydride, the question next arises as to the possible nature of this proton domain for protons. This matter has been explored by analysing which protein is most heavily labelled by acetic anhydride. The outcome is that the highest degree of labelling is detected in a polypeptide of molecular weight 8000 in the F0 sector of the ATP synthase enzyme [8-9]. Furthermore, the labelling of this polypeptide is decreased by Photosystem II activity, but not by Photosystem I activity. On the basis of these results it has been suggested that this protein of F0 shares a restricted domain with the site of proton release by the water-splitting reaction [9]. This restricted domain could be an intramembrane channel for protons. It is nevertheless difficult to imagine the nature of this channel, especially as it would probably have to stretch over quite large distances. Dilley has also considered whether the results of chemically labelling the thylakoid membrane might also be explained in terms of the electrodic view of energy transduction put forward by Kell [12], in which a kinetic barrier to proton movement from the

surface of the membrane to the bulk aqueous phase is envisaged. The latter mechanism does not appear to provide a satisfactory explanation, because treatments with acetic anhydride take tens of seconds which might reasonably be expected to be longer than the time for equilibration of protons between the membrane surface and the bulk phase [9,11,13]. It has been suggested that buried amine-buffer groups bind the protons within the postulated intra-membrane domains [14]. The conclusion from chemical modification studies that protons from the water-splitting reaction were not rapidly deposited into a homogeneous bulk interior aqueous phase of thylakoids seemed originally to be at variance with data obtained from experiments with the pH probe neutral red. Changes in the absorbance of the latter had indicated very rapid release of protons into the interior of the thylakoid, fully consistent with the tenet of a delocalised protonmotive force as the intermediate between electron flow and ATP synthesis [15]. The notion that protons taken into the thylakoid by different parts of the electron transfer chain are not fully equivalent is inconsistent with the findings of Bowes and Crofts [16] that operation of either photosystem had equivalent effects on delayed fluorescence. The approach of testing for localised patterns of proton flow by chemical modification reactions is novel, but the difficulty is that it is an indirect approach and inevitably relies upon the pH in the immediate vicinity of a protein being the sole determinant of the reaction with acetic anhydride. Nevertheless, these experiments have prompted further experiments using neutral red as a probe for the kinetics of proton release into the interior of the thylakoids, with the somewhat unexpected outcome that is described in subsection IVK. Before leaving the topic of chemical modification, we should take note of a set of experiments. which rendered improbable the specific association of the two types of photosystem with discrete groups of ATP synthase molecules. This study monitored the light-dependent and irreversible inhibition of the thylakoid ATP synthase by either N-ethyl maleimide or sulphate [17]. It was shown that either photosystem acting alone had an equal effect on the extent of inhibition of the rate of ATP synthesis measured subsequently with the

52 driving force provided by the sole operation of the other photosystem. Clearly if operation of, say, Photosystem II were to permit irreversible inhibition of a group of ATP synthases that were associated with Photosystem II, the rate of ATP synthesis driven in subsequent experiments by Photosystem I acting alone would have shown no inhibition. Grebanier and Jagendorf [17] thus concluded from these experiments that localised proton flow from electron-transfer reactions to ATP synthases was unlikely. Tu et al. [18] have reported that chemical modification of mitochondria by molecules related to fluorescamine results in an inhibition of both ATPand electron-transport-dependent protein translocations. The modification does not cause a general enhancement in the proton permeability of the membrane [18]. Such experimental observations could reflect inhibition of a proton-pumping activity without concomitant inhibition of the exergonic reaction that drives the proton pumping, as reported for the action of N,N'-dicyclohexylcarbodiimide (DCCD) upon cytochrome oxidase [19]. However, fluorescamine-type compounds also abolish the secondary effects of electron-transport chain inhibitors upon the ATPase activity of the F0F 1 ATP synthase [18]. The mechanism whereby electron transport inhibitors influence the ATPase activity of intact mitochondria has not been resolved with certainty but at least in some instances it could be related to inhibition of the generation by respiration of a residual Ap that activates the ATP synthase. Tu et al. [18] prefer an explanation based upon a putative direct conformational interaction of respiratory proton pumps and ATPase proton pumps in the mitochondrial inner membrane. It is this interaction that is proposed to be abolished by chemical modification with fluorescamine derivatives. The existence of such local and direct interactions would need further supportive evidence before it could be accepted as a key feature of the structural organisation of the inner mitochondrial membrane. HC. Bacterial mutants

The topic of this section is consideration of the properties of mutants whose energy transduction reactions have, at least in some aspects, been sug-

gested to be not fully consistent with a delocalised protonmotive force as intermediate between electron transfer and ATP synthesis. Excluded from this section are uncoupler-resistant mutants which are more appropriately discussed under the category of thermodynamic studies (Section 3b). Kay and Bragg [20] reported that membrane vesicles from strain HfrA of Salmonella typhimurium LT2 differed from similar preparations from the wild type strain. An ATP-dependent transhydrogenase activity was absent, whereas other features including respiration-dependent transhydrogenase, ATP-dependent quenching of atebrin fluorescence and specific activity of ATPase were essentially identical in vesicles from the wild type and the HfrA strain. Both wild type and HfrA cells were able to accumulate amino acids at the expense of ATP hydrolysis. The conclusion from this work was that the singular failure of ATP hydrolysis to drive the transhydrogenase reaction in the HfrA vesicles, despite the evidence that ATP hydrolysis generated Ap and that the transhydrogenase reaction could be energy-linked to succinate respiration, was not really compatible with a fully delocalised intermediate in energy coupling as envisaged in the chemiosmotic theory. In the nine years since the publication of this work there appear to be no reports of either confirmation or rebuttal from other laboratories. Assuming that the results are reproducible, can they be rationalised in terms of the chemiosmotic theory? A first, and rather unsatisfactory, suggestion is that an allosteric effect of ATP inhibits the transhydrogenase in HfrA but not in the wild type. A second possible explanation is based upon a theme that will recur frequently throughout this article. In chemiosmotic terms the rate of the transhydrogenase reaction must be a function of the magnitude of Ap. The possible nature of this relationship has not been investigated although there is indirect evidence (Section IV) that the rate of the transhydrogenase reaction depends less sharply on Ap than does, for example, the rate of ATP synthesis. In the case of the vesicles from HfrA it can be suggested that ATP hydrolysis generates a steady state Ap that is lower than that generated in wild-type vesicles, and that a non-linear relationship between the rate of transhydrogenase and Ap results in complete loss of the transhydro-

53 genase reaction in the vesicles from HfrA. Admittedly, the extents of the ATP-dependent atebrin fluorescence quenching in the wild type and HfrA appeared to be similar, but it is not certain how closely the extent of this quenching can be taken as a guide to the relative magnitudes of Ap. If protons flow from electron transfer reactions to the ATP synthase, or to other proteins such as those involved in active transport, without equilibrating with the bulk aqueous phases at either side of the membrane it is necessary to propose a mechanism that would prevent such equilibration. An intramembrane domain as suggested by Dilley and coworkers as a result of their work on chemical modification of the thylakoid membrane is one possibility. Another line of proposed support for such structures in the membrane has come from an interpretation by Kell et al. [21] of the properties of two types (classes) of mutant of E. coll. A brief description of the properties of these mutants follows. Hong and co-workers have described a series of temperature-sensitive mutants in energy coupling that they have designated as ecf mutants [22,23]. At the non-permissive temperature one of these mutants (JSH 212) could establish only very low accumulation ratios of a variety of nutrients that are normally actively transported with Ap as the source of energy. The membrane potential generated under similar conditions was reported to be slightly (86 mV against 94 mV) lower than that generated in the wild type, in which active transport of the nutrients was readily observed at the same temperature. As the ecf mutation maps at a defined position (minute 64) on the E. coli linkage map, it was proposed that the ecf gene product had an essential role in coupling-active transport to the membrane potential [22,23]. Hong considered that the properties of this mutant could be accommodated within the requirement of a delocalised Ap as the driving force for active transport if the ecf gene product acted as the proton carrier in conjunction with a variety of other proteins that would bind the nutrient to be transported and hence confer specificity on transport processes [23]. Thus all active transport linked to Ap would be abolished by the mutation in the ecf gene. This view of the role of the ecf gene product now appears untena-

ble because in recent work it has been shown that a single polypeptide can reconstitute proton/ lactose symport in phospholipid vesicles [24,25]. An alternative role for the ecf gene product was suggested to be a common transducer of Ap into conformational energy which would act as the immediate driving force for a number of active transport system [23]. The new observations on the purified lactose carrier system also rule out this mechanism. Proponents of localised energy coupling could nevertheless argue that the relatively low rates of symport catalysed by such reconstituted systems are a manifestation of the lack of one or more polypeptides (products of the ecf gene) that are responsible for the higher rates in the native membrane [78]. Plate and coworkers [26,27] have isolated a very similar mutant of E. coli termed cup, which must be different from the ecf mutant because it maps at around minute 86 on the E. coil linkage map. Essentially the eup phenotype is very similar to that of the ecf phenotype with pleiotropic deficiencies in active transport systems. Proposals by Plate and Suit [27] that the cup gene product is involved in transporting protons whilst another polypeptide carried the nutrient can be discounted, at least for lactose transport, for the reasons given above. Kell et al. [21] have argued that the existence of ecf and cup mutations provides evidence for specific proteins, coded for by the ecf and cup genes, that are involved in transporting protons from the electron transfer chain to the ATP synthase and active transport systems. As part of this argument is based on the properties of colicins, further consideration of this matter is postponed until the next subsection (IID). Can the properties of the ecf and eup mutants be reconciled with the chemiosmotic mechanism? In the first instance we can enquire whether the mutations in the cup and ecf genes render the plasma membrane unusually permeable to protons. Tests for such enhanced permeability in the mutants have proved negative [23,26]. A related but distinct explanation is that these mutants are able, for unidentified reasons, to maintain a maximum Ap that is smaller than the Ap generated by the wild type bacteria. This is observed for the ecf mutant JSH 212 (see above). As is discussed in

54 Section IV, small decreases in the magnitude of Ap can possibly cause large decreases in the rate of active transport. A phenomena of this kind, coupled with a mechanism for solute efflux from the cells, could account for the failure of the mutants to accumulate solutes at the nonpermissive temperature despite the apparently sizeable Ap. Before leaving this section attention is drawn to a strange feature of the transport of metabolites by the ecf mutant. At the nonpermissive temperature there was a brief period of solute uptake after initiation of electron transfer. This was followed by solute efflux. The reasons for this were not understood but were suggested to be related to the inhibitory effects of metabolites of glucose [23]. It might be significant that the membrane potential showed a similar tendency to decay that was not evident in the wild type which took up solutes normally. Therefore, it cannot be ruled out that the collapse of the membrane potential, for whatever reason, was the cause of the solute efflux from the cells. In this case there is no inconsistency with the chemiosmotic theory although the interesting question remains as to the possible molecular nature of the mutation. Other possible explanations for the properties of the eup mutants have been discussed by Booth et al. [28]. In general, it must be admitted that the molecular basis of the eup and ecf mutations cannot be satisfactorily rationalised by the present writer. Proton translocation, as conventionally measured, is not impaired in the eup mutant [29]. Only by detailed characterisation of the ecf and eup gene products is a thorough understanding of the properties of these mutants, and the possible general implications, likely to emerge. When the F~ part of the ATP synthase enzyme is removed from inside-out membrane vesicles from, for example, Paracoccus denitrificans [30] or many strains of E. coli [31], it is generally considered that electron transport by the vesicles is able to sustain only a small Ap owing to proton leakage from the lumen of the vesicles via the F0 part of the enzyme which is 'opened' following removal of F 1. Cox et al. [31] have recently reported some observations on strain AN 1928 of E. coli, in which leucine -31 in the c subunit of F0 has been replaced by phenylalanine. Vesicles from AN 1928 had similar ATPase activities, rates of ATP

synthesis and P / O ratios as a control strain. Thus the F0 F 1 ATP synthase was functional. The unusual feature of AN 1928 vesicles was that after removal of the F 1 part of the ATP synthase enzyme the Ap generated by electron transport, as judged by the quenching of quinacrine fluorescence, was not diminished in contrast to that with the control strain. An interpretation is that in AN 1928 F0 depleted of its F 1 does not enhance the proton permeability of the membranes of the vesicles. From this it can be suggested that the normal function of F0 is not to provide a route for passage of protons from the lumen of the vesicles to F1, but rather to transfer (localized) protons that remain within the membrane to the F1 sector for ATP synthesis. It is interesting to note that removal of F~ from vesicles prepared from Rhodopseudomonas capsulata has been known since 1970 not to enhance appreciably their proton permeability [32], although little attention has been paid to this apparently atypical finding. This might imply that F0 of this organism does not form a proton conductor that spans the membrane, and should be considered in the context of the observations made with Rps. capsulata (subsections IVD, E and J) which have suggested a localised pathway for protons in this organism. An alternative explanation, with less profound implications, can be offered to explain the failure of F0' when stripped of F~, to conduct protons over the whole width of the plasma membranes from either E. coli AN 1928 or Rps. capsulata. It is known that removal of F~ from thylakoid membranes results in a gradual closing of the F0 proton channels [33]. Possibly the F0 disaggregates following removal of F 1 or becomes sealed by lipid molecules [33]. It is an entirely speculative suggestion, but possibly the F0 sectors of E. coli AN 1928 and Rps. capsulata behave similarly, although Cox et al., [31] consider it unlikely that substitution of leucine-31 by phenylalanine would cause the postulated structural instability of F0 from AN 1928 compared with its counterpart in other strains of E. coli.

liD. The action of certain colicins Observations on the mode of action of certain colicins have been suggested to add to the evi-

55 dence for specific proteins in the bacterial plasma membrane that have the role of providing localised pathways for proton flow from exergonic to endergonic reactions [21]. Here we first briefly summarise what is known about these colicins and then consider to what extent their mode of action might be inconsistent with the idea of a fully delocalised Ap. It is widely agreed that colicins of the E1 type kill E. coli cells by interfering with energy transduction processes at the levels of the plasma membrane. There is reasonably good evidence that the effects of the colicins include the loss of respiration-dependent membrane potential, induction of large amounts of K ÷ efflux from the cell, and depletion of cellular ATP levels. The loss of membrane potential and cellular ATP could be explained if the colicin rendered the plasma membrane permeable to small ions, which would result in collapse of the membrane potential owing to influx of cations and/or efflux of anions. In these circumstances the proton-translocating ATPase could hydrolyse ATP without any thermodynamic constraint imposed by Ap, thus accounting in part for the depletion of this metabolite. Matters are not necessarily as simple as this because the large K ÷ efflux from the cell would tend to generate a membrane potential, positive outside, which would be collapsed only by compensating ion movements to maintain electroneutrality. As yet the nature of these ion movements, if they occur, has not been established. Kell et al. [21] have argued that the following features of the action of colicins mean that their effects cannot be simply to enhance the ionic permeability of the cell membrane, but can be better understood in terms of proton-conducting channels within the membrane, known as protoneural networks: (i) the observed K ÷ efflux; (ii) responses of fluorescence probes of membrane structure to the presence of colicins; (iii) the evidence that the specific mutation in either the ecf gene or the eup gene referred to in subsection IIC renders E. coli insensitive to colicin K but not to colicin El; (iv) colicin E1 mimicked either valinomycin plus K ÷ or SCN- by enhancing the extent of proton extrusion form E. coli following introduction of a small amount of oxygen into an anaerobic suspension of cells. The argument is that

the primary action of colicins E1 and K is to disrupt a protoneural network [21]. This could explain why proton release into the bulk aqueous phase is enhanced by colicins, because, if the ecf and eup genes were to code for proteins that contribute to this protoneural network, it is not unreasonable to suggest that mutations in these genes might create resistance to the action of colicins. However, the hypothesis of protoneural networks does not appear to solve all the problems of colicin action. First, the phenomena that a small number of colicin molecules can kill a cell means that the action of the colicin must probably be sought in terms of a cooperative inactivation of the protoneural networks. Second, if colicins block the postulated protoneural networks we are left with no simple mechanism to account for massive and rapid K ÷ efflux from cells. At least the concept that colicins, in combination with a receptor that might be altered by mutations in eup and ecf genes, provide an ion channel in the membrane can qualitatively account for this observation. Third, if colicins inactivated these proton channels, their action in some respects would be similar to that of oligomycin or DCCD which block the well-characterised proton channel of the ATP synthase. In this case it would be predicted that colicins would inhibit the ATP hydrolysing capacity of the enzyme because passage of protons from the enzyme into the protoneural networks would be blocked. Inhibition by colicins of ATP synthases working in the hydrolytic direction is not usually observed. Although the present writer concurs with Kell et al. [21] in considering that a complete description of colicin action has not yet been given, it is difficult to accept that the incompleteness of our present understanding necessitates the postulate of protoneural networks. A useful perspective on whether the action of colicins E1 and K can be understood in terms of a receptor-mediated increase in proton permeability of the membrane has been given recently by Cramer et al. [34]. liE. Consideration of the lateral distribution and motion of membrane proteins

If the concept of local proton flow within or along membranes is to have any ultimate physical

56 interpretation it will have to be reconciled with what is known about the disposition and relative motion of the proteins involved in energy transduction. The current view of the inner mitochondrial membrane, and probably by analogy also the bacterial plasma membrane, is that it is organised into well-defined complexes each made up of several polypeptides. These complexes are thought to diffuse relatively slowly but randomly in the plane of the membrane. Electron transfer is thought, but not definitely proven, to be mediated by small molecules such as ubiquinone and cytochrome c that diffuse faster [35]. Given this picture of the membrane what sort of pathway for protons might be envisaged? The idea of a protoneural network is not easily accommodated because it would presumably have the effect of cross-linking the membrane proteins into large aggregates, unless the networks were to be formed only transiently. In the latter case the ends of the protoneural proteins would be open and thus the flow of protons into the surrounding membrane would have to be controlled. The random diffusion of membrane proteins would not seem necessarily to preclude a preferential channelling of protons along the surface of the membrane from the site of generation to a nearby site of consumption. Indeed Mitchell has implicitly considered this possibility [36]. He has suggested that the Ap-generating molecules must cause a small local hump in the Ap within ther immediate vicinity. Molecules that consume Ap would cause a converse local hump. Although the size of any such local field deformations could not be estimated, it was suggested that significant lateral dipole-dipole attractive interaction between generators and consumers of Ap might occur [36]. The thylakoid membrane appears to differ from the mitochondrial and bacterial membranes. The current view is that the ATP synthase and Photosystem I complex is largely restricted to the unstacked stroma lamellae part of the thylakoid, whereas Photosystem II is mainly found in the stacked grana region [37]. When chloroplasts are unstacked by transfer of thylakoid lamellae to solutions containing low concentrations of salt, the ATP synthase is thought to become uniformly distributed over the whole membrane. Thus the probable picture is that the intrinsic fluidity of the

thylakoid membrane is similar to that of its mitochondrial and bacterial counterparts, but that the portion of the ATP synthase, and perhaps also that of the Photosystem I, which protrude beyond the bilayer prevents free diffusion of the proteins into the appressed membrane regions. These structural features of the thylakoid membrane have consequences for the pathway of protons from electron transfer to ATP synthesis. If the Photosystem II complexes in the stacked thylakoid membrane are some considerable distance from the ATP synthases it is difficult to envisage the nature of any localised proton channelling from Photosystem II to the ATP synthase. As far as is known the stacked form of the chloroplast is fully competent in driving ATP synthesis. On the other hand the close juxtaposition of the ATP synthase and Photosystem I might permit a localised reaction. The structure of the thylakoid presents problems because when different types of experiment are compared, it is not always clear that the degree of stacking of the thylakoids has been the same. Variations in this feature might obviously alter the outcome of some experiments. Furthermore, the tight stacking of the granal region of the thylakoid raises the question as to whether it is meaningful to describe the internal phase as truly aqueous. This point is discussed again in subsection IVK.

11I. Thermodynamic studies There is no escaping the requirement that for Ap to be the intermediate in ATP synthesis it must be thermodynamically competent to drive ATP synthesis with an acceptable, and preferably experimentally established, proton per ATP ratio [38]. In this section consideration is given to a number of studies that have suggested that measured values of Ap are too small for Ap to be considered realistically as the intermediate in oxidative phosphorylation. If the experiments and conclusions in some of these studies are valid it hardly needs stating that the chemiosmotic theory would be unsound. Such a negative conclusion about the chemiosmotic hypothesis does not provide any evidence in favour of localised pathways of proton flow but it naturally prompts their possible role to be given serious consideration.

57

IliA. Alkalophilic bacteria Measurements of Ap in intact cells of Bacillus alkalophilus have shown that when the external pH is 11, which permits good rates of growth, the internal pH is approx. 9.0. Under these conditions the membrane potential was estimated from the distribution of a phosphonium salt to be 135 mV [39]. Hence the total Ap was only 15 mV and hardly sufficient to drive ATP synthesis or active transport processes. The inadequacy of Ap to drive active transport is circumvented by the cells because a sodium electrochemical gradient has been shown to be involved in at least some transport processes [40]. The problem of how to drive oxidative phosphorylation when Ap is as low as 15 mV is not avoided in this way as the available evidence indicates that sodium gradients are not involved [41], and that ATP synthesis is inhibited by the usual range of uncouplers and inhibitors [41]. Consideration has been given to the possibility that alkalophilic bacteria might possess intracytoplasmic vesicles designed to carry out oxidative phosphorylation at the usual values of Ap. In this way oxidative phosphorylation, but not active transport, could become independent of the high external pH and the low Ap across the cytoplasmic membrane. A study with the electron microscope of thin sections of two obligately alkalophilic bacteria has shown intracellular vesicular structures to be absent [42]. It is sometimes suggested that the pH in the aqueous phase immediately adjacent to the external surface of the cytoplasmic membrane might be much lower than that of the bulk external aqueous medium, and therefore the true Ap experienced by the cytoplasmic membrane might be within the normal range. This is a false argument because if the surface of the membrane were to be sufficiently negatively charged to reduce the pH in its immediate vicinity from, say, 11 to 7, the electrical profile across the membrane would also be reduced in compensation (e.g., Ref. 43). Thus the Ap across the cytoplasmic membrane could not be increased by a mechanism of this kind unless there exists an active transport mechanism for reducing the pH in this region with respect to the bulk aqueous phase. Such mechanisms are not known and in any case could not account for the observa-

tions of oxidative phosphorylation at low Ap in membrane vesicles. At present, work with both whole cells and right-side out vesicles of B. alkalophilus poses a dilemma for the chemiosmotic mechanism. If the available data are correct the occurrence of oxidative phosphorylation in these bacteria can only be reconciled with the low steady state Ap if either the H + / A T P is very high or if the cellular [ATP]/ [ADP]/[Pi] is very low. The present writer is not aware of any estimates of phosphorylation potential (AGp) in whole cells, but despite the difficulty in making this determination (free and not total concentrations are strictly required) it would be worth checking this point. For instance, an unusually high internal Pi concentration right contribute to a low AGp. In the studies with vesicles only modest values of AGp were generated [41] but these are very difficult experiments because of the limitation of the intra-vesicular volume that can be used. (Vesicles with the same orientation as cells were employed. They were loaded with ADP and Pi during preparation.) There are perhaps two ways in which to proceed with these results from studies on alkalophiles. The first is to accept that they demonstrate that a truly chemiosmotic mechanism is not operating in these bacteria and that therefore an alternative localised pathway of protonic coupling must be given serious consideration. The second approach would be to argue that the evidence for chemiosmotic mechanisms in other systems is so overwhelming that we must seek an error in the measurements of Ap made with the alkalophiles, assuming that the idea of very high H + / A T P or very low AGp is discounted. A reason for suspecting that Ap is not drastically underestimated is that the alkalophilic bacteria use a sodium rather than a proton electrochemical gradient to power active transport, suggesting that Ap is inadequate in this respect. On the other hand, it is possible that the requirement of a sodium-exit/protonentry antiporter for cytoplasmic pH control has led to the use of the sodium gradient in active transport processes. The prime candidate for the source of error in determinations of Ap must be the membrane potential as it seems improbable that the cytoplasmic pH could be much higher than 9 even when the external pH is 11 or higher.

58 The membrane potential in alkalophiles has been determined from the distribution of a triphenylalkyl phosphonium cation. There are still grounds for concern over the accuracy of this method, and later in this section we shall have to return to this point. A recent study by Kashket [44] suggests that the accumulation of a phosphonium ion in E. coli may tend to underestimate the membrane potential at higher values of external pH, although other recent studies have suggested that phosphonium salts grossly overestimate the potential in some bacteria unless substantial corrections for the binding of the probe are made [45]. At this point it is also as well to keep in mind the observation that carotenoid band shifts often indicate higher membrane potentials than does the uptake of a phosphonium salt [38]. We are not yet in a position to discount entirely the possibility that the carotenoid band shifts are the more accurate indicator, and that the phosphonium salts are unreliable with an inherent tendency to underestimate. Further advances in the technique of using microelectrodes for measuring bacterial membrane potentials would obviously be of great value in studying the bioenergetics of alkalophiles. In a very recent study, Guffanti et al. [46] have compared respiration and potassium diffusion potentials as driving forces for ATP synthesis by starved cell suspensions of Bacillus firmis RAB. At an external pH of 7 an increase in the ATP content of the cells was observed upon initiation of respiration by addition of DL-malate or imposition of the diffusion potential. The increase in ATP was greater when respiration provided the energy source, but in each case ATP synthesis was prevented by treatment of the cells with DCCD which was assumed to be a specific inhibitor of the ATP synthase. In contrast, when the starved cells were incubated at pH 9, the potassium diffusion potential was completely ineffective at driving ATP synthesis, whereas malate respiration generated a higher intracellular concentration of ATP at external pH of 9 than was observed when the external pH was 7. Again, ATP synthesis was inhibited by DCCD. It was clearly shown that the procedure for generating a diffusion potential was successful at pH 9 because the potential detected from the uptake of either T P M P + or Rb + was of the anticipated size and comparable to the membrane

potential generated by malate respiration at this pH. Furthermore, the diffusion potential was competent to drive the uptake of ct-aminoisobutyric acid, which is transported in symport with Na + [47], at pH 9.0. The conclusion from these findings was that the membrane potential seemingly generated by respiration at external pH of 9 is not exactly equivalent to a diffusion potential [46]. These findings were taken as strong evidence that the pathway for protons between the respiratory chain and the ATP synthase is direct or localised [46]. The present writer cannot formulate a simple explanation that is consistent with the expectations of the chemiosmotic hypothesis. The conclusions so far drawn about the alkalophilic bacteria might not be applicable to all such organisms. In the marine bacterium Vibrio alginolyticus it has been suggested that electron transport is directly coupled to the electrogenic extrusion of Na + from the cell, especially when the external pH is relatively alkaline. If this is truly the case then it could be that in this organism ATP synthesis is catalysed by a Na+-dependent enzyme that is driven by the sodium electrochemical gradient [48 49]. Chernyak et al. [50] have presented evidence that in V. alginolyticus motility is dependent upon the sodium rather than the proton electrochemical gradient and that the organism possesses a vanadate-sensitive but DCCD-resistant ATPase. Thus current evidence indicates that V. alginolyticus, unlike alkalophilic Bacillus species, might not present any apparent inconsistencies with the chemiosmotic principle because ATP synthesis is not driven directly by the proton electrochemical gradient. Maintenance of a substantial sodium electrochemical gradient is probably easier for alkalophiles than maintenance of a proton electrochemical gradient of comparable size. For the moment at least, the bioenergetic data with alkalophiles have to be taken as presenting a strong reason for considering an alternative to A p as the intermediate in oxidative phosphorylation, but these observations offer no direct guide as to what might be put in its place. Studies with mutants could be informative.

IIIB. Uncoupler-resistant mutants Some years ago Decker and Lang [51] isolated a mutant of Bacillus megaterium that was able to

59 grow in the presence of high concentrations of the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Consequently this was termed an uncoupler-resistant mutant. In their original work Decker and Lang reported that uncouplers were equally effective in both the wild type and the mutant in collapsing the membrane potential and the pH gradient across the cytoplasmic membrane [51]. It was also possible to show that in the mutant, ATP synthesis could be driven by rapidly lowering the external pH [52]. This acid-jump induced ATP synthesis was sensitive to both inhibitors and uncouplers which also inhibited respiration-dependent ATP synthesis when the external pH was 5.5. Uncouplers were less effective at inhibiting ATP synthesis when the external pH was 7.5 in the mutant. A curious feature was that in the mutant, but not in the wild type, uncouplers reversed inhibition of ATP synthesis by either DCCD or valinomycin plus potassium. The principal conclusion from this work was that when the external pH was 7.4 oxidative phosphorylation was occurring in the presence of CCCP with negligible Ap [51] and that the observations were not readily consistent with a chemiosmotic mechanism. Guffanti et al. [53] have recently extended the work of Decker and Lang. They confirmed that in the mutant 5 /~M CCCP collapses Ap, as in the wild type, but that the intracellular phosphate potential is much less reduced in the mutant than in the wild type. However, before attempting to interpret these data it is instructive to consider the magnitudes of the phosphorylation potentials that were reported. In the absence of an uncoupler, malate respiration generated a phosphorylation potential (AGp, defined in Table I) of only 390 mV which is equivalent to approx. 38 kJ-mo1-1. After addition of CCCP (5 /~M), the measured phosphate potential fell to 250 mV (23.4 kJ. mol-1) in the wild type but only to 34.3 kJ- mo1-1 in the uncoupler-resistant mutant. These values for phosphorylation potential are rather lower than anticipated in general for bacteria and are similar to the values that can be expected from the equilibrium of the adenylate kinase reaction in the absence of high rates of ATP hydrolysis (see Table I and subsection IIIC). It is worth noting that in the uncoupler-resistant mutant the ATPase activity of

cytoplasmic membrane preparations is rather lower than in comparable preparations from the wild type. Consequently, it is not altogether out of the question that the apparent difference observed between the effects of CCCP on the two types of cell is an artefact. Possibly during the quenching of cells that is necessary for phosphorylation potential determination more ATP is hydrolysed in the suspension of wildtype cells and a lower value for AGp ensues. In both the wild-type and mutant cells it is difficult to ensure that adenylate kinase activity has not contributed to the production of ATP. Although the above explanations have not been explicitly considered, it has been recognised that a problem in working with intact cells is to separate oxidative phosphorylation from other possible modes of ATP synthesis, including the so-called substrate level phosphorylation and adenylate kinase reactions. Consequently, experiments have also been done with right-side out vesicles from the uncoupler-resistant mutant and wild-type strains of B. megaterium [54]. The vesicles were loaded with ADP and Pi during their preparation so that ATP synthesis could be studied. In the first series of experiments it was shown that ascorbate plus phenazine methosulphate (PMS) oxidation by the vesicles was linked to ATP synthesis. Two differences between the wild type and the mutant were noted. First, a greater amount of ATP synthesis (expressed as nmol ATP per mg vesicle protein) was seen with the mutant. Second, the ATP synthesis by the vesicles from the mutant was less sensitive to CCCP than was the same reaction in vesicles from the wild type. In themselves, these two sets of observations cannot be taken as evidence against the chemiosmotic mechanism. If the vesicles from the mutant were able to pump protons faster than those from the wild type, then a higher Ap would result. This could give both a higher extent of ATP synthesis and would probably require more uncoupler to collapse Ap and thus abolish ATP synthesis. This explanation cannot be tested because it was not possible to measure the membrane potential developed by electron transport with a phosphonium cation probe. Such failure may reflect the possibility that only a small fraction of the vesicle population was competent to generate a membrane potential from electron transport.

60 The second set of experiments conducted with the vesicles was to measure the extent of ATP synthesis with a potassium diffusion potential. In this case it was found that vesicles from the wildtype made more ATP than those from the mutant which sometimes made none at all. In this type of experiment it was possible to detect the generation of a membrane potential from the uptake of a phosphonium salt. Note that all vesicular structures, regardless of orientation and competence to carry out electron transport would in this type of experiment be able to sustain a diffusion potential and thus take up the phosphonium salt. Thus the detection of a membrane potential by phosphonium ion uptake following a diffusion potential but not during respiration is not necessarily significant. The result from experiments with vesicles thus complemented those from experiments with cells in that the mutant was apparently less able to synthesize ATP from an induced Ap, independent of electron transport, but showed greater resistance to uncouplers when electron transport drove ATP synthesis. As noted by Guffanti et al. [54] it would be valuable to be able to measure ATP synthesis in inverted vesicles, since then it would be possible to monitor not only Ap but also both extents and rates of ATP synthesis. In the above discussion an attempt has been made to suggest how the measurements of ATP synthesis in experiments with both cells and vesicles from the uncoupler-resistant mutant might be rationalised in terms of chemiosmotic theory. To maintain a balanced argument let us now consider how the observations on B. megaterium might be understood in a non-chemiosmotic context. For CCCP to dissipate Ap but not ATP synthesis in the mutant, two modes of action of this reagent might be considered. The first would be its accepted behaviour as a protonophore. The second mode might be to interfere with a specific pathway for proton flow from the electron-transfer chain to the ATP synthase. Such a mode of action might speculatively be related to the uncoupler binding site located within the ATP synthase complex for mitochondria and some bacteria [55]. Thus the mutation in B. megaterium, which is judged by frequency of reversion to be a single point mutation, might affect the protein that provides CCCP,

and other uncouplers, with this specific binding site [56]. This explanation includes the premise that the tocalised flow of protons from electron transfer to ATP synthase is not unduly influenced by decrease in the protonmotive force, and yet by definition the activity of electron-transfer chains is responsible for the generation of this force. This is difficult to accept. Furthermore, the specific uncoupler binding protein is only associated, as far as is known, with the ATP synthase and therefore the question remains as to how active transport and other energy-linked processes are rendered insensitive to CCCP in the mutant. At present no satisfactory explanation for the physiology of the CCCP-resistant strain of B. megaterium can be given. When the cells are growing in the presence of CCCP it is possible that they rely solely on glycolysis (they are usually grown on fructose plus malate) to generate ATP and storage polymers. The availability of the latter, and ATP synthesis linked to their breakdown, might account for the ATP synthesis detected in washed suspensions of the cells. Any detoxification mechanism for CCCP possessed by the cells would not account for the properties of the membrane vesicles prepared from the mutant. In short, the uncoupler-resistant mutant of B. megaterium, and perhaps the similar uncoupler-resistant mutants of E. coli [57-58], pose a problem for future work. As stated by Date et al. [57] 'conventional views of the mode of action of CCCP suggest that resistant mutants should not occur'.

HIC. Decreases in protonmotioe force that are not always accompanied by proportional decreases in the extent of A TP synthesis One type of experiment has been especially prominent in raising the possibility of an important role for localised proton flow between respiratory chains and ATP synthases. Several investigators have used a variety of procedures to attenuate the protonmotive force generated by mitochondria, and have in parallel measured the maximum value of the phosphorylation potential that was attained. In general it has been found that AGo declines less sharply than Ap, with the consequence that the thermodynamic competence of Ap comes under increasing suspicion at lower

61

values of Ap, unless the H + / A T P increases to rather high values [38]. Before discussing such experiments further, it is as well to recognise that with mitochondria, and also of course with other systems such as intact bacterial cells, reactions other than oxidative phosphorylation or photophosphorylation can contribute to ATP synthesis. Ubiquitous amongst these is adenylate kinase activity, and we discuss first how, at least in principle, the sole operation of this enzyme can generate quite high values of the phosphorylation potential (AGp). It is obviously essential to be certain that this reaction does not perturb the relationship between AGp and Ap in experiments where the latter is varied. Table I shows what values of AGp will be observed if ADP is added to adenylate kinase at different concentrations of Pi, and using two extreme values of the equilibrium constant that are taken from the literature. The values of AGp are not low. In studies with mitochondria or bacterial membrane vesicles, oxidative phosphorylation frequently, but not always, raises the AGp above the level that can be generated by the action of adenylate kinase. In such cases there is no ambiguity when the Ap is compared with AGp. However, sometimes results are obtained in which AGp is close to values given in Table I. In principle equilibration between AGp and Ap should still occur because the ATPase activity of the ATP synthase

ought to hydrolyse the ATP produced by adenylate kinase activity until equilibrium for both reactions is achieved. However, this cannot be guaranteed because a sluggish ATPase activity, especially when concentrations of ADP and ATP are approximately equal (Table I), could prevent this equilibration, or at least equilibrium might be reached rather slowly and thus escape detection even when measurements are made over a series of time points. A further problem introduced by adenylate kinase is that it can be more resistant to irreversible inactivation by acid (S.J. Ferguson, unpublished observations) than is the oxidative phosphorylation system. Thus adenylate kinase could conceivably alter the [ATP]/[ADP] ratio during the quenching of the mitochondria or bacteria before analysis of extracts for adenine nucleotides and phosphate. The problem that adenylate kinase could cause, and one method to control against this problem, can be illustrated by reference to a report from Westerhoff et al. [59]. In the experiments shown in Table II of that paper oxidative phosphorylation with succinate as substrate was studied under several conditions with an initial ADP concentration of 0.55 mol per 1.55 ml (Methods section), i.e. 335 #M. In two sets of experiments malonate was present to inhibit electron transport partially. With malonate concentrations of 15 and 33 mM, the final total concentrations of ATP plus ADP that

TABLE I ILLUSTRATION OF THE POSSIBLE EFFECT OF ADENYLATE KINASE ACTIVITY IN GENERATING A PHOSPHORYLATION POTENTIAL, AGp = AG°' + RT In [ATP] [ADP][Pi] [ADP]added (mM) Using K 0.2 0.2

[ADP] final (rnM) [AMP][ATP] [ADP] 2

[ATP] final (mM)

[Pi ] final (raM)

AGp a (kJ. tool- 1)

0.057 0.057

1.0 10.0

46.0 40.6

0.08 0.08

1.0 10.0

49.0 43.1

0.441188] 0.086 0.086

Using K = [AMP][ATP] = 4 [189] [ADP] 2 0.2 0.2

0.04 0.04

a Calculated assuming AGO'= 30.1 kJ.mo1-1 and T = 25°C; two values for K are shown because of the discrepancies in published values.

62 were used for calculation of AGp were 153 and 42 #M, respectively. Thus a significant part of the added ADP had presumably been converted into AMP. A similar feature is apparent in experiments in which FCCP was present. The final total ATP plus ADP was 134/~M at 0.27 ~M FCCP, and 181 # M (the ADP concentration was misprinted: it should have been 122/~M; Westerhoff, H.V., personal communication) at 0.67 /~M FCCP, when again the starting concentration of ADP was 355 /tM. Even in the control experiment with no added malonate or FCCP the total ATP plus ADP was 209 #M. Thus again part of the added ADP was again missing, presumably as AMP. The values of AGp calculated from these data were in the range 46.9-44.4 kJ. mol-1 and thus close to the values that might be generated by adenylate kinase (Table I) if the rate of ATP hydrolysis were slow either for the reason already mentioned or because of the presence of ATPase inhibitor protein. Thus as it stands the data shown in Fig. 2 and Table I of Westerhoff et al. [59] might be thought inappropriate for comparisons of AGp with Ap, because the value of AGp cannot automatically be taken to be generated by oxidative phosphorylation. However, Westerhoff et al. [59] reported similar results, though with ascorbate plus T M P D as substrate, when experiments were done with AMP as the sole added nucleotide. In this case traces of ATP within the mitochondrial preparation were presumably sufficient to convert some of the added AMP to ADP via the action of adenylate kinase. In this type of experiment, in which AMP is the added nucleotide, it is difficult to see how the observed values of AGp could have been generated except through the action of oxidative phosphorylation. Therefore it is probably coincidental that the values of AGp obtained in the work of Westerhoff et al. [59] are close to those that could be obtained by the operation of the adenylate kinase reaction. The point of this illustration is simply to emphasise that unless clear controls have been done AGp values as high as 43.9 kJ. mol 1 cannot automatically be taken as the output of the oxidative phosphorylation apparatus. It may also be remarked that the mere omission of Mg 2÷ from the reaction mixture might not prevent adenylate kinase activity owing to possible leakage of Mg 2÷ from mitochondria; this omission is clearly impos-

sible in other systems, e.g. submitochondrial particles, where added Mg 2+ is essential for oxidative phosphorylation. The first experiments in which decreases in Ap brought about by addition of increasing concentrations of an uncoupler, 2,4-dinitrophenol in this case, were not found to be accompanied by exactly proportional decreases in AGp were reported by Wiechmann et al. [60] in 1975. On raising the concentration of this uncoupler from 0 to 30 ~M Ap dropped from 194 mV to 139 mV whilest the external value of AGp changed from 52.3 to 48.34 kJ. mo1-1. Subsequently, these experiments were extended by studying the effect of increasing external K ÷ concentration on these two parameters; a drop in Ap from 195 to 115 mV was accompanied by a decrease in AGp from 52.3 to 47.3 kJ. m o l - I [61]. The same laboratory later reported that limited titration with FCCP reduced Ap from 175 to 153 mV and then to 85 mV as the concentration of FCCP was raised, and that AGp fell from 46.9 via 45.6 to 45.2 k J - m o l 1 [59]. Rather similar results were also obtained by Azzone et al. [62] who progressively reduced A p from 170 to 50 mV by titration with FCCP but found that AGp decreased only from 52.7 to 43.5 kJ • moi 1. At this point we should enquire whether oxidative phosphorylation occurred in some of these experiments. Section IV discusses observations that small attenuations in the protonmotive force are accompanied by relatively much larger decreases in the rate of ATP synthesis. On this basis, it would be expected that at a Ap of less 100 mV the rate of ATP synthesis could be so low that the contribution of oxidative phosphorylation to net ATP synthesis during a reaction time of several minutes would be very small. This consideration warrants attention in future work. The most recent and ostensibly most thorough study in which systematic changes in Ap generated by mitochondria were not accompanied by corresponding changes in AGp is that of Wilson and Forman [63]. In this work three methods were employed to give progressive attenuation in Ap. The first of these was to increase progressively the concentration of propionate from 0 to 3 mM, whereupon ApH, as measured from the distribution of acetate, was found to decrease from 0.92 to 0.1 units, whilst the membrane potential, esti-

63 mated on the basis of the uptake of triphenylmethyl-phosphonium cation, remained constant at approx. 130 mV. Thus, apparently the effect of adding propionate was to decrease Ap from 188 to 132 mV, yet the phosphorylation potential developed remained constant, and well outside the range that could be developed by adenylate kinase activity (Table I), at around 63 kJ-mo1-1. In chemiosmotic terms the unexpected feature of this experiment was that as ApH was dissipated by adding propionate to increase the buffering power of the matrix, Aft should have increased to compensate. But there is a curious feature of the experiments of Wilson and Forman [63] that requires consideration. Although the pK a values of acetic (4.7) and propionic (4.9) acids are similar, the effect of 0.1 mM propionate in dissipating ApH by 50% was mimicked only by 3 mM acetate, whereas if these compounds were serving only to buffer the matrix more or less equivalent effects would be expected for a given concentration of weak acid. These observations on the relative efficacy of the two acids might be taken as an indication that the weak acids were not simply buffering the matrix pH, but rather might have been competing with acetate for binding sites on the mitochondria, despite the evidence that was presented to show that acetate binding or metabolism, as opposed to accumulation in the matrix space, was insignificant [63]. Such an explanation requires that ApH under the experimental conditions used was close to zero and that A~p was the sole component of Ap. This would leave Ap rather too small to account for the magnitude of AGp. In a second series of experiments Wilson and Forman [63] progressively reduced the osmolarity of a mitochondrial suspension. The resultant swelling of the mitochondria was detected as an increase in the sucrose impermeable space, but even after allowance was made for the swelling the value of Ap fell from 148 to 88 mV over the range of osmolarity studied. AGp was approx. 63 kJ. mol-1 at all osmolarities tested, so that once again changes in Ap and AGp were not in proportion. It is worth noting that measurement of A~ and ApH as a function of osmolarity should in principle be an experiment with which to estimate any binding of the probes for ApH and A~p to the mitochondrial membrane. This is because the ex-

tent of binding of probes should not alter significantly with variation of mitochondrial volume. In contrast, the uptake of probes of A~ and ApH in response to a constant Ap should alter in direct proportion to the changes in internal volume that result from alterations of external osmolarity. Hence, appreciable binding of probes would tend to cause a greater overestimation of Ap at the higher values of osmolarity. An effect of this kind could provide an explanation for the apparent decline in Ap reported by Wilson and Forman [63]. However, Wilson and Forman [63] did find that the rate of respiration increased upon swelling which is consistent with a decrease in Ap. This decrease was due to a greater extent to the decrease in ApH than to a change in A~k. This is perhaps a little surprising as swelling, if anything, might be expected to increase the ionic permeability of the membrane, and thus tend to cause an increase in ApH at the expense of A~b. The third approach used by Wilson and Forman [63] to decrease Ap was to increase progressively the K + concentration in the presence of valinomycin. Qualitatively in line with chemiosmotic expectations, this procedure was found to decrease A~k and to increase ApH. In these experiments there was a disproportionate decrease in Ap (54% at the maximum K + concentration tested) compared with the change in AGp (8% decrease at the maximum K + concentration). An odd feature of the series of experiments with valinomycin and increasing K + concentration was that the control experiment for the series with valinomycin plus K + was done under almost the same conditions (0.2 mM EGTA instead of 0.4 mM) as the control experiment for the series in which the effects of increasing propionate concentration were investigated. Yet, in the former experiment ApH was 0.1 unit, but in the latter 0.9 units. Other parameters, AGp, internal volume and respiration rate were similar under both sets of conditions. This is a disturbing variation in the results. All the data discussed so far in this section, as well as those in the earlier related study by Hollian and Wilson [64] not discussed here, can be interpreted in several ways. These fall into two classes, explanations that are qualitatively consistent with the chemiosmotic mechanism and those that are not. We consider each class in turn.

64 The simplest explanation in this category is that the methods for determining A~p and ApH are unreliable. A particular source of difficulty in experiments where perturbations lead to faster respiration rates might be that a more rapid onset of anaerobiosis has occurred during centrifugation of the mitochondria to separate them from the suspending reaction medium. Consequently, Ap might be underestimated owing to leakage of probes from the mitochondria. To counter this argument it has been claimed [61] that at least in some experiments very similar data has been obtained using the flow dialysis technique in which the components of Ap can be determined without separation of the mitochondria. It would be valuable if further studies using this technique, or alternatively ion-selective electrodes, were to be done. It can always be argued that the lack of proportionality between changes of AGp and those in Ap reflects the inadequacies of the available methods for determining Ap. Here one must balance defeatism against realism. It is indeed a temptation to dismiss thermodynamic inconsistencies relating to the magnitude of Ap on such grounds. However, if the standpoint is taken that Ap cannot be measured with any degree of quantitative reliability then a key basis for the acceptance of the chemiosmotic hypothesis is undermined. Measurements of Ap that are not in accord with expectation must not be dismissed without justification and the quest for validation of such measurements must continue. In principle, the strategy of varying Ap and studying the resultant effects on AGp should be a good method for overcoming systematic errors in the determination of Ap. But not all errors need to be systematic, and for instance, the binding of the probes for A~b and ApH might well vary with the total Ap for reasons discussed elsewhere [45]. Such nonsystematic errors might account for the apparent effects of propionate on ApH that were discussed earlier. Strictly speaking, the chemiosmotic hypothesis only requires that Ap be greater than zero because the H + / A T P ratio determines the maximum attainable value for AGp at any given value of Ap. Hence, it can be argued that the failure of Ap and AGp to change in strict proportion is indicative of

a variable stoichiometry with high values for H +/ ATP at low values of Ap. There is no direct experimental evidence against this possibility but it is considered improbable, at least in the context of chemical reaction mechanisms. A variable stoichiometry would make the task of relating the steady-state kinetics of ATP synthesis to changes in Ap very difficult indeed. As we have seen, the maximum value of AGp reached under the conditions in which the maximum values of Ap were measured was in many instances [59-62] only in the range 52.7-46.9 kJmo1-1. These values are considerably below the maximum AGp that mitochondria have been observed to generate (68 kJ. mol-~) [65]. Thus it is possible that the failure of AGp to decrease upon attenuation of Ap is that until the uncoupler concentration has become quite high the mitochondria are not generating, for unidentified reasons, their maximum phosphorylation potential. This explanation could not apply to the work of Wilson and Forman [63] in which values of AGv in the range of 51.9 to 59.8 kJ. mo1-1 were reported. The final type of basis for the discrepancies between Ap and AGp to be considered is the possibility that reactions other than oxidative phosphorylation have contributed to the attainment of the observed value of AGp. The problems that adenylate kinase can cause in this context have already been dealt with at length; we leave the reader to scrutinise the papers to see if adequate control experiments have been done. Another reaction that could contribute is matrix substrate level ATP synthesis especially when glutamate plus malate is the added substrate, but probably not when succinate in the presence of rotenone is used. However, when glutamate plus malate was used very high values of AGp were found [63] and thus it is unlikely that these were not maintained by oxidative phosphorylation. Finally, it is very difficult to eliminate the possibility that mitochondria are heterogeneous in respect of their ability to synthesise ATP. Such behaviour can lead to artefactual distortions in the relation between AGp and Ap. For instance, if some mitochondria were able to generate a A p that was insufficient to activate the ATP synthase, then it is possible that they would neither hydro-

65 lyse nor synthesise ATP. These mitochondria would then lower the measured value of Ap but only the unknown Ap truly generated by the phosphorylating mitochondria could be compared with AGp. Such behaviour is hypothetical. If the discrepancies between values of Ap and those of AGp are accepted and the foregoing 'chemiosmotic' explanations rejected, then the idea of a bulk-phase protonmotive force acting as the intermediate between electron flow and ATP synthesis becomes untenable. The question then arises as to what can be put in its place, but these experiments in which thermodynamic discrepancies have been reported offer no positive guide. Van Dam and co-workers [61] suggested that there exists a localised pathway of proton flow from electron-transfer complexes to the ATP synthase. More explicitly it was suggested that there was a resistance to proton flow into the bulk aqueous phase, and therefore dissipation of Ap by stimulating the movement of protons from the bulk phase by addition of an uncoupler would not be accompanied by a directly proportional effect on the real driving force for ATP synthesis. This proposal has now been withdrawn because the addition of various titres of malonate to mitochondria oxidising succinate, also causes a greater reduction in Ap than in AGp, and yet there is no reason to believe that malonate should stimulate movement of protons from the bulk phase [59]. We must conclude that if thermodynamic evidence were to signal the demise of the chemiosmotic mechanism we are left with no simple rational substitute to put in its place, although proposals have been made [38,105]. Comparisons of AGp with Ap in intact bacterial cells are beset with even more problems than the corresponding experiments with mitochondria. Prominent amongst these problems are the possibilities that AGp is measured incorrectly owing to significant binding of ADP and ATP to cellular components and that the probes for measurement of Ap are even less accurate owing to their binding to the cell wall as well as to membranes and other components of the cells. Furthermore, ATP produced by oxidative phosphorylation will be turned over by the cell so that AGp is always likely to be considerably below its equilibrium value with Ap. Apart from the data with the uncoupler

resistant mutant of B. megaterium (discussed in Section IIIB), the most important reports of studies on relationships between Ap and AGp have been on Halobacterium halobium. Michel and Oesterhelt [66] found a poor correlation between values of Ap and the extent of ATP synthesis observed in cells that were illuminated in the presence of a protonophore. They concluded that this meant that Ap was not the real driving force for ATP synthesis. Although it was not proven in that work that a substantial value of AGp was generated under the conditions tested, it is difficult to see how the observed ATP synthesis could have been catalysed other than by the action of the proton pumping bacteriorhodopsin and the ATP synthase. A light-dependent adenylate kinase was not, however, definitely eliminated from consideration. Fortunately, this original work has now been confirmed in a second laboratory by Helgerson et al. [67,190]. The reported that in the presence of two ionophores, triphenyltin to exchange C1- for O H - and the protonophore CCCP, illumination of cells generated a value for the energy charge, defined as ([ATP]+ ½[ADP])/[ATP] + [ADP]+ [AMP]) of 0.96. As by definition adenylate kinase can achieve an energy charge of 0.5, these observations indicated substantial photophosphorylation in the presence of a low protonmotive force. Thus any doubts about the conclusions of Michel and Oesterhelt [66] have been seemingly dispelled, and it must be accepted that at least as indicated by the uptake of triphenylmethylphosphonium cation and benzoic acid, measured either by flow dialysis or by rapid centrifugation through silicon oil [67,190], the Ap in illuminated cells of H. halobium does not correlate well with the ATP synthesis. This is a dramatic result because it is generally accepted that the light-activated proton-pumping bacteriorhodopsin forms patches on the cell membrane that are spatially separate from the ATP synthase. How then can protons pumped by bacteriorhodopsin be transferred to the ATP synthase without generating Ap? The present writer has no answer to this question, because even if the protons are not fully transferred into the bulk aqueous phase a bulk-tobulk transmembrane protonmotive force ought to generated for the reasons discussed by Ferguson and Sorgato [38].

66 Before leaving work on bacterial cells we should consider a recent paper by Kasket [44]. She measured Ap in E. coli by conventional methods but over a range of external pH values. These data were compared with the AGp and it was found that as the external pH was increased the ratio of AGp/Ap increased from about 2.5 to 4. When another indicator of Ap was used, however, this ratio was constant at about 3. The alternative method for determining Ap was to assume that lactose is accumulated by E. coli in symport with one proton over the entire range of external pH values studied. Thus in a fl-galactoside negative mutant the accumulation ratio of lactose could be taken as equivalent to Ap. Of course, this is a circular argument in some respects because one could equally argue that the discrepancy between Ap measured from the uptake of triphenyl phosphonium and benzoate and that measured from lactose accumulation is indicative of a discrepancy in the thermodynamics of lactose uptake, or that the stoichiometry of protons symported per lactose alters at higher external pH values. Nevertheless, the contention of Kashket that there are nonsystematic errors arising from use of the probes for estimating Ap cannot be dismissed. Some of the complications that arise from the use of intact cells can in principle be overcome if inside out bacterial membrane vesicles are used for studies of the relationship between Ap and AGp. The first extensive study of this type was conducted by Baccarini-Melandri et al. [68] on chromatophores from Rhodopseudomonas capsulata. Unlike most of the work discussed in this article, the A~k component of Ap was estimated not on the basis of permeant ion uptake but rather from the extent of the carotenoid band shift, a method that is only applicable to a limited number of photosynthetic bacteria. At a higher range of values of Ap measured in this way it was found that controlled reduction of Ap from its maximum values by a variety of treatments that led to dissipation of Ap were accompanied by proportional changes in AGp. The data was consistent with H + / A T P = 2, in good agreement with the detailed expectations of the chemiosmotic theory. However, when Ap was reduced to an estimated value of 218 mV, it was found that net ATP synthesis took place to an extent that was inconsistent with

H + / A T P = 2 and a value of 2.3 was the minimum consistent with the data. In these experiments control experiments clearly showed that photophosphorylation and not adenylate kinase was responsible for the observed ATP synthesis. Bearing in mind that it is probable that the method used for estimation of Ap in that work probably gave overestimates [38], those data again raise the problem of substantial net ATP synthesis at apparently rather low values of Ap. Membrane vesicles from Paracoccus denitrificans have also been used for studies of relationships between Ap and AGp. The first anomaly noted in this instance was that in the presence of nitrate in millimolar concentrations, but in the presence of azide to block nitrate reduction, the measured aerobic Ap was found to decrease very considerably without any change in the AGp generated [69]. Subsequent work has shown, using CIO4 rather than SCN as the permeant ion for estimation of membrane potential, that nitrate is seemingly more effective at decreasing the membrane potential as detected by SCN uptake than the membrane potential detected by CIO4- [70]. This is clearly a difficulty because the basis of the use of these permeant ions requires that each should give the same estimate of membrane potential. Nevertheless, there seems to be little doubt that the magnitude of Ap does drop quite considerably without any concomitant change in AGp, and recently work with a variety of methods has shown that the presence of nitrate does not increase ApH at the expense of A+ [70]. This work has been complemented by measurements in which Ap was progressively reduced by titration with an uncoupler and AGp was measured [71]. Here again the now familiar pattern of decreases in Ap, not being fully parallelled by changes in AGp, was observed. Possible explanations of these data are similar to those discussed earlier in this section when experiments with mitochondria were discussed, and a fairly extensive discussion is given in the original paper [71]. Suffice it to say here that the deviations from pure chemiosmotic behaviour were less striking in the work with P. denitrificans than in the work with mitochondria. It had been hoped that work with the P. denitrificans vesicle system might have more decisively either followed or deviated from chemiosmotic behaviour. Instead a somewhat

67 'in between' result was obtained. In general thylakoids are believed to show almost ideal chemiosmotic behaviour in respect of their photophosphorylating behaviour. Unfor, tunately therefore, there is, to this writer's knowledge, only one report of comparisons of Ap and AGp over a range of values. In this work [72] it was found that at lower light intensities the ratio AGp/ Ap increased slightly but was at all times less than 3 so that no serious discrepancies with chemiosmotic theory were observed. It might be of value if an uncoupler titration of both Ap and AGp in thylakoids were to be studied in future work. To summarise, the relationship between Ap and AGp remains a problem, and unless one abandons any notion of being able to quantitatively estimate Ap it must be recognised that at present there are serious indications that Ap, as measured, is not always competent to account for the extent of ATP synthesis (AGp) unless extraordinary high values of H + / A T P are accepted. Unfortunately, such a conclusion offers no clue as to what to put in the place of Ap. For that reason alone, the reported thermodynamic discrepancies discussed here must be continually scrutinised and subject to further study in case they are a cruel deception generated by some unrecognised errors in the methods for estimating Ap. Tedeschi would argue that the permanent ion method for determining A~ is indeed a cruel deception because he has repeatedly reported his failure to detect r e s p i r a t i o n - d e p e n d e n t mitochondrial membrane potentials with microelectrodes. In a recent paper [73], Tedeschi and colleagues have addressed the suggestion that after impalement by an electrode mitochondria rely on a large pH gradient rather than a membrane potential to drive ATP synthesis. Pyranine, a pHsensitive fluorescence probe, was micro-injected into giant mitochondria. Succinate oxidation by such a giant mitochondrion was found to drive ATP synthesis with the development of only a very small pH gradient (0.3 units). Thus it was concluded that impaled giant mitochondria drive oxidative phosphorylation in the absence of a sizeable Ap, as judged by the microelectrode method for A~k and the pyranine fluorescence for ApH. Obviously Tedeschi's data are inconsistent

with a delocalised proton electrochemical gradient as an intermediate in oxidative phosphorylation. Thus there is a choice for a reader. He or she must either convince him/herself that Tedeschi's experimental approach is not misleading or accept the weight of evidence from other directions that a sizeable mitochondrial membrane potential exists in respiring mitochondrial. Denial of this membrane potential would raise the question of why nonphysiological permeant ions are accumulated by mitochondria. Tedeschi's explanation for this phenomenon is not watertight [38].

1111). Relationship of the magnitude of the protonmotive force to the extent of bacterial substrate accumulation There has been a number of reports that the relationship between Ap and the substrate concentration ratio between the interior of right-sideout bacterial membrane vesicles or of bacteria is not constant [74]. These have often been interpreted in terms of an increased stoichiometry of proton translocation in symport with the substrate as the magnitude of Ap drops [74]. Such results might also be taken as evidence of either the inadequacy of the methods for determining Ap or that Ap is not the direct driving force for active transport. In this section we take as an illustration the report of Ahmed and Booth [75]. They found that the accumulation of lactose by cells of E. coli at lower values of Ap exceeded the value that was allowable on the basis of the accepted stoichiometry of one proton translocated per lactose transported. As these authors tend to eschew the idea that the stoichiometry of proton translocation may change at lower values of Ap, they were forced to consider the possibility that Ap is not the sole determinant of lactose accumulation [75]. Although it might be possible to devise a mechanistic scheme to link electron transfer to ATP synthesis without involving the bulk aqueous phase, it is more difficult to do this for active transport, because the driving force obviously has to move the substrate from one bulk phase to another. This argument is particularly applicable when the substrate carries a charge. Thus one could argue that the fact that discrepancies crop up in active transport is a pointer for being suspicious of the meth-

68 ods for determining Ap, rather than taking those measurements as being correct and doubting the validity of the chemiosmotic mechanism. We might also recall in this context the results of Kashket [44] that were discussed earlier. These also clearly show a variation in the relationship between Ap and AGp on the one hand and Ap and the lactose accumulation ratio on the other. We are left with no answer at the moment to the question as to which is correct. IV. Kinetic studies

The only kinetic prediction explicit in the chemiosmotic theory is that the protonmotive force should satisfy the criterion of kinetic competence. It has been shown that when a protonmotive force is almost instantaneously imposed across the thylakoid membrane by raising the pH of the suspending medium in a rapid mixing apparatus, the synthesis of ATP begins at least as quickly as when darkened thylakoids are switched to illuminated conditions [76]. Analogous experiments have been done with submitochondrial particles in which case the pH jump was supplemented with a potassium diffusion potential. In those experiments it was found that ATP synthesis began sooner following the imposition of Ap than when electron transport was started to drive oxidative phosphorylation [77]. Taken together with the finding that the initial rates of ATP synthesis in these two systems were similar to those found during photophosphorylation or oxidative phosphorylation, the experiments with imposed protonmotive forces would seem to supply strong evidence that a bulk phase Ap, exactly as described in the original chemiosmotic theory, comes rapidly into equilibrium both with the electron-transfer chain and the ATP synthase, and therefore is a kinetically competent intermediate. In the present article we are concerned with reports which suggest that such rapid equilibration does not occur because, in some circumstances, the magnitude of Ap does not correlate with the kinetics and thermodynamics of ATP synthesis. Is there then any alternative explanation for the result of experiments in which Ap is rapidly imposed and the ensuing kinetics of ATP synthesis measured? Two possibilities have been mooted. The first is that the

values of imposed Ap required to drive ATP synthesis are significantly greater than those detected during steady-state oxidative phosphorylation or photophosphorylation [78]. This argument naturally supposes that the estimate of the initial imposed Ap can accurately be made from information about the jump in the external pH, and from estimates of the magnitude of the K + diffusion potential. Unless information is available about the initial rates of decay of the ApH and Aq~ components of Ap, it is difficult to specify the value of Ap that actually activates ATP synthesis in these experiments. The second possible alternative explanation of these experiments is to propose that the rapid change in the medium pH and imposition of the diffusion potential somehow charges the localised pathways of proton flow directly and not via Ap. These latter explanations are generally considered improbable, although Hangarter and Good [78] have recently presented data that they interpret to mean that 'the energised state of the membranes caused by illumination is definitely not the same as the energized state developed when the membranes are incubated in acid and then exposed to more alkaline media'. Evidence for the kinetic competence of Ap has also come from experiments in which photosynthetic membranes have been subjected to very short flashes of light. A change in the spectrum of the endogenous carotenoid pigments of these membranes is thought to reflect the membrane potential, and thus by spectroscopic observation the rise and decay of the membrane potential can be followed. Using this method it has been shown that a very fast rise in A+ is followed by a decay in A~k that can be specifically associated with the synthesis of ATP [80,81]. This experiment is further evidence for the kinetic competence of Ap unless it is argued that the changes in carotenoid spectrum reflect factors other than the bulk phase membrane potential. The pros and cons for the proposition that carotenoid band shifts reflect only bulk phase membrane potentials are discussed elsewhere [38]. The chemiosmotic theory made no predictions as to the possible relationships between the magnitude of Ap and the rates of reactions that either generate or dissipate Ap. It is in this area of steady-state relationships between, for example,

69 the magnitude of Ap and the rate of ATP synthesis, that many data said to be inconsistent with the original version of the chemiosmotic theory have been obtained. The major part of this section is concerned with discussing these types of experiment, starting with the question of respiratory control, going on to discuss the effects of an inhibitor or ionophore acting alone, and followed by discussion of experiments in which combinations of inhibitors and ionophores have been used. The kinetics section is concluded with discussion of experiments that have sought to follow the appearance of the proton in the bulk aqueous phase followed electron-transport events, the use of isotopic substitution of H20 by 2H20 and fluorescence probes. IVA. Relationships between mitochondrial respiration rate and the size of the protonmotive force

In the absence of ADP well-coupled mitochondria respire at a relatively slow controlled rate. When either ADP in the presence of Pi, or a protonophore, is added to a suspension of mitochondria this control is released and the rate of respiration is substantially increased. The chemiosmotic theory suggests that the control on respiration is exerted by Ap and that therefore a given reduction in Ap by any means should be associated with a particular increase in the respiration rate. This expectation has been realised with brown rat mitochondria in which either a limited titre of protonophore or addition of ADP gave both an identical acceleration in rate and decrease in Ap [82]. Increasing concentrations of protonophore gave further stimulation of the respiratory rate although respiratory control was fully released once Ap had fallen below a quite substantial pseudothreshold level [82]. A different result has been reported for rat liver mitochondria. Three groups have found [83-85] that the onset of ATP synthesis was accompanied by a smaller diminution in Ap than was seen when respiration was increased to the same extent with an uncoupler. A similar but smaller discrepancy can be seen in the work of Kiaster et al. [86]. In these studies the maximum rate of respiration that could be induced by a full titre of uncoupler was greater than that observed in the presence

of either ADP or the limited titre of uncoupler needed to induce the same rate. Therefore the explanation of this type of result cannot be that Ap had fallen below the point at which it exerts respiratory control and into the region where the respiration rate does not correlate uniquely with a particular value of Ap. These discrepancies between the effects of uncouplers and of ADP on the respiratory rate and Ap were the first query about the chemiosmotic theory that emerged from studies of the behaviour of Ap. Padan and Rottenberg [83] proposed that their original results meant that there had to be at least a parallel and Ap-independent pathway from electron transfer to ATP synthesis in addition to the route via Ap. Eleven years later we are still left with the observation and this implication, but is there any alternative rationale? Apart from suggesting an experimental artefact, the focus of attention probably ought to be directed again towards the question of what exactly controls respiration. The conclusion of Padan and Rottenberg, and of others, assumes that Ap is the sole consideration. We must come back to the point that 2 A E - n A p might be the key term [38], where AE is the span of redox potential difference over a segment of the respiratory chain that translocates n protons upon the passage of two electrons. Thus, tentatively, we might suggest that the addition of protonophore induces a slightly different AE term than does the addition of ADP. This might arise because of control by adenine nucleotides of dehydrogenases in the respiratory chain or of enzymes in the Krebs' cycle. In the most recently reported study of this topic, O'Shea and Chappell [85] have found that whereas addition of ADP to respiring mitochondria from rat liver caused a drop in Ap from 190 to 180 mV and an almost 8-fold increase in the respiration rate, addition of the concentration of FCCP that also caused Ap to drop to 180 mV resulted in only a 10% increase in the respiration rate. The faster rate of respiration, and thus of proton translocation out of the mitochondria, in the presence of added ADP compared with added FCCP, must be accompanied by a faster rate of proton return into the matrix in the former case. That is, in the experiments cited the proton conductance is higher in the presence of ADP (i.e., the

70 ATP synthase is functional) than in the presence of FCCP. Thus O'Shea and Chappell [85] suggest that not only Ap but also the proton conductance has an influence on the steady-state of respiration. But the difficulty with this suggestion is to account for the mechanism whereby, at a given value of Ap, the electron-transfer chain can respond to the difference conductances, provided on the one hand by the operation of the ATP synthase and on the other by submaximal titres of a protonophore uncoupler. A mechanistic link appears to be missing. There are also other reported failures of the value of Ap to correlate with the respiratory rate. For example Wilson and Forman [63] showed that when Ap was attenuated by four procedures, addition of propionate, decrease of medium osmolarity, increasing external K + and titration with FCCP, quite different relationships between respiratory rate and Ap were found. In this and other examples a possible explanation to consider is that the perturbations intended to affect Ap alone have also had a secondary effect on the activity of the respiratory chain itself. For example, if FCCP has a minor inhibitory effect on the electron-transport chain, then the form of a titration with FCCP of both respiratory rate and Ap might be rather complex. Another study with discrepancies between simple expectation and experimentally observed relationships between protonmotive force and energycoupling parameters has come recently from Rottenberg [87]. He studied the effects of two general anaesthetics, chloroform and halothane, upon respiration rates and protonmotive force in rat liver mitochondria. It was reported that these substances stimulated respiration and reduced the P / O ratio rather like classical protonophore uncouplers of oxidative phosphorylation. However, in the range of concentrations over which the anaesthetics had this effect they caused no change in the magnitude of the protonmotive force. These experiments used succinate as substrate and E G T A plus Mg 2+ was present. These results were taken to mean that the anaesthetics were disrupting a control on respiration that was linked somehow directly to the ATP synthase. This view was adopted because the anaesthetics stimulated the FoF1-ATPase activity (oligomycin sensitive) even

in the presence of protonophores, thus suggesting perhaps that these substances disrupted an extra control mechanism upon the ATPase. The principal unexpected feature of these results is clearly the failure of the protonmotive force to decrease at all under conditions in which respiration is increased by up to 2.5-fold. This is difficult to explain unless it is a consequence of a compensating experimental artefact. For instance increased binding of the probes used for measurement of protonmotive force as the titre of anaesthetic was increased might compensate for decreased uptake of the probe owing to decrease in protonmotive force. This is clearly hypothetical. If it is true that a pure chemiosmotic model cannot account for the control of the respiratory rate in mitochondria, then the importance of the result is such that it should be followed up by similar experiments in other systems and using other methodologies for determining Ap. Otherwise, criticism of technique, for example that the centrifugation method might underestimate A p more severely in the presence of uncoupler than in the presence of ADP because of the tendency of uncoupler to concentrate within the pellet of mitochondria, will linger on untested.

I VB. The effect of electron-transport inhibitors upon the extent of respiratory control It was reported by Lee et al. in 1969 [88], and again by Lee and Storey in 1981 [89], that the rate of electron t r a n s p o r t catalysed by submitochondrial particles is stimulated by an uncoupler to the same extent almost irrespective of the extent to which electron transport is inhibited. In other words the respiratory control ratio is constant. This observation has been taken to be difficult to interpret in terms of Ap being the determinant of respiratory control, because in that case Ap, and hence the respiratory control index, would be expected to decline as the respiratory rate decreases. Indeed on the basis of closely related experiments Hinkle et al. [90] concurred with this view because they found, in contrast to the results of Lee and colleagues, that the respiratory control index did decrease with increasing inhibition of electron transfer, just as they expected if a protonmotive force that decreased linearly with

71 reduction in electron-transfer rate controlled the electron-transfer rate. In fact the interpretation of either type of experimental result is not straightforward because it is now clear that substantial inhibition of electron-transfer rates is usually not accompanied by proportional drops in the Ap maintained by submitochondrial particles [91]. Unfortunately, A p was not measured in the experiments where the respiratory control ratio was measured as a function of the electron-transfer rate. However, in a series of experiments with inside-out membrane vesicles from Paracoccus denitrificans both respiratory control and protonmotive force were measured with N A D H as substrate [92]. In this case it was found that the respiratory control ratio did decrease with increasing titre of the inhibitor rotenone, but that there was no appreciable drop in the magnitude of Ap that was sustained by controlled respiration [92]. The controlled rate of respiration was nevertheless reduced upon the addition of the first titres of rotenone. The first question to be addressed is whether this pattern of behaviour is that expected from by a system conforming to perfect chemiosmotic behaviour. If Ap alone is the determinant of the respiratory rate than it might be predicted that the initial titres of rotenone would have no effect on the rate because the rate-limiting step would not be the respiratory chain but the dissipation of Ap. This is not the behaviour observed because the controlled rate is inhibited by the initial titres of rotenone, although to a lesser extent than the uncoupled rate when the respiratory chain presumably must be rate-limiting. Consequently, the respiratory control ratio decreases with increasing titre of rotenone. Why then is there any decrease in the controlled rate of respiration seen during the initial stage of a rotenone titration? The answer may lie within the contention that the controlled rate of respiration is determined not by Ap alone but rather by the difference 2 A E - n A p . Thus when rotenone is added, the decrease in rate and also in extent of respiratory control would have to be related to this term. As Ap remains almost constant the 2AE term would have to increase. Until suitable measurements of both AE and Ap have been made, this explanation remains conjectural, but it is worth noting that an explanation of this type might also

account for a constant stimulation of respiration by an uncoupler over the range of respiration rates studied by Lee et al. [88-89]. The idea that a constant respiratory control ratio over a range of electron-transfer rates is evidence for a localised mode of energy transduction is also difficult to reconcile with notion that ubiquinone acts a mobile pool in the mitochondrial membrane. Such behaviour means that electrons originating from succinate or N A D H dehydrogenases are not transferred to a particular electron-transfer chain. Thus, even if a localised mode of energy coupling were to operate, a decreased respiratory control ratio might be predicted at the lower electron-transport rates. This is because under these conditions a smaller fraction of the cytochromel components would be inhibited by the back pressure of a putative localised proton intermediate under steady-state conditions. At this point we raise the question as to whether it is really possible to assign one step in a multicomponent system as being rate-limiting. Kacser and Burns [93,94] have argued that such a situation is unlikely, and that all one can hope to do is assign a control strength to a particular reaction. Although in principle a control strength of unity is possible, and corresponds to a singular rate-limiting step, in practice for a series of reactions, values of less than one will be found. We return to this point later (subsection IV J).

IVC. Effects of changes in rates of electron transport on protonmotive force and rate of A TP synthesis The steady-state value of Ap is determined by relative rates of electron-transport linked proton translocation and of proton backflow down the proton electrochemical gradient either through a transducing enzyme (e.g., the ATP synthase during ATP synthesis) or via nonspecific leakage through the membrane. There is now considerable evidence to show that Ap remains almost constant over a wide range of electron-transport rates in mitochondria, submitochondrial particles, bacterial membrane vesicles and thylakoids [38]. The underlying mechanism whereby this homeostasis of Ap is achieved (see, e.g. Ref. 38, 95, and 96) is not of major concern here, but the requirement for such homeostasis is not difficult to imagine. If

72 variations in the rate of electron transport were accompanied by directly proportional changes in Ap, as would be the case if the resistance of the nonspecific proton leak pathways in membranes was ohmic in character, then the value of AGp and the extent of other endergonic processes that are believed to be driven by Ap, including active transport, would fluctuate widely with deleterious consequences for the maintenance of ion and substrate gradients, as well as AGp, over wide ranges of electron-transfer rates, likely to be encountered in vivo. On first consideration it seems entirely predictable that as the rate of mitochondrial or bacterial electron transport decreases the rate of ATP synthesis should show proportionate changes, thus keeping the P / O ratio unchanged. Indeed this result has been observed experimentally in several systems (including rat liver [97] and plant [98] mitochondria), chromatophores [99] and vesicles from P. denitrificans [100]. However, the maintenance of a constant P / O ratio places a constraint on the chemiosmotic mechanism because reductions in the rate of electron transport are accompanied by at most slight, and sometimes even no detectable, change in the magnitude of Ap [38,69,98-99, 101-102]. The kinetic question therefore is how, when Ap changes so little, the rate of ATP synthesis varies very exactly in order to match proportionate changes in the rate of electron transport. There are two possible extreme explanations to consider. The first is to argue that the mechanism of conversion of Ap into ATP synthesis is such that quite tiny changes in Ap bring about large changes in the rate of ATP synthesis [38,99]. This would mean, of course, that large changes in the rate of ATP synthesis must occur over a rather narrow range of values of Ap and that the rate of ATP synthesis would approach zero at appreciable values of Ap. Mechanistic schemes that would account for such a characteristic have been proposed [103,104]. Indeed, one might go further and argue that as homeostasis of Ap and AGp over a range of electron-transport rates is essential for survival of cells under a variety of different nutritional states, the mechanism of the ATP synthase has had to evolve to permit very small changes in Ap to be translated into large changes in the rate of ATP synthesis.

Thus the observation of proportional changes in the rate of ATP synthesis when electron transport is inhibited can be accommodated within the chemiosmotic hypothesis, provided that ultimately the relation between Ap and the rate of ATP synthesis is related to the mechanism of the ATP synthase. The situation becomes more complicated, however, when the effects of ionophores on the rate of ATP synthesis are compared with those of inhibitors. We defer discussion of this point until subsection IVD. The second explanation for the apparent virtual independence of the rate of ATP synthesis from the magnitude of Ap strictly applies only when there is no detectable change in Ap. In that case it is difficult to avoid suggesting that the rate of ATP synthesis is controlled not only by Ap but also by other factors [38,98,101,102]. The problem here is to define what those other factors might be, although Westerhoff et al. [105] have recently made detailed proposals concerning possible routes of proton flow from electron transfer chains to ATP synthases. Mitchell [106] has recognized that it might be necessary to consider whether the redox state of one or more components of the electrontransport chain might determine the fraction of the ATP synthases that are in an active state. Before concluding this section three other matters warrant attention. The first is consideration of whether the methods used for detecting Ap might have given misleading results and that Ap really drops more than suspected upon partial inhibition of electron transport. Evidence that Ap truly must change only marginally upon attenuation of the electron transport rate has come from the demonstration that with P. denitrificans membrane vesicles inhibition of the rate of NADH oxidation by up to 80% did not cause any change in the value of AGp that was generated by vesicles form P. denitrificans [100]. It is noteworthy that small changes in AGp are readily detectable because they require relatively large changes in the concentrations of ATP, ADP and Pi which contribute to AGp through the logarithmic term. Thus provided AGp approaches its equilibrium value with Ap, the absence of any decrease in AGp as the electron-transport rate is lowered argues against any possibility that the probes for A~k and ApH are insufficiently sensitive to detect putative de-

73 creases in Ap that might have accompanied inhibition of electron transport, but escaped detection. The second matter concerns decreases in K m (ADP) for ATP synthesis that are seen upon reduction of the electron-transfer rate in submitochondrial particles and thylakoids [107-109]. If oxidative phosphorylation or photophosphorylation is thought of as two enzyme systems, electron transport and ATP synthase, acting sequentially, then the K m (ADP) value measured will only be a true K m if the ATP synthase is the rate-limiting step, otherwise there will be a tendency to underestimate Km. Hence, one interpretation of the decreases in K m that are seen upon reduction of the electron-transport rate is that the ATP synthase becomes less rate limiting. We shall return to this point later and also to the comparison with the effects of uncouplers on K m (ADP). The third point concerns the use of subsaturating light intensities to modulate the rate of electron flow in photosynthetic systems. As pointed out in particular by Clark et al. [99], this procedure introduces heterogeneity into a sample owing to self-shading. This might be a particular problem in experiments where rates or extents of ATP synthesis are measured in a whole sample, but Ap is measured by a spectroscopic method only in the region of the measuring beam. This is a factor that needs to be kept in mind. Consideration of the consequences of progressive inhibition of electron transport upon the magnitude steady-state of Ap and of the rate of ATP synthesis have raised interesting questions about the control of oxidative phosphorylation and the mechanism of the ATP synthase. Only some of the data obtained call into question the chemiosmotic concept, but comparisons with the effect of uncouplers raise more doubts in this direction (see subsection IVE).

IVD. Relationships of rates of bacterial transport to protonmotive force There are some interesting points of comparison between the limited range of studies on correlations between rates of bacterial transport and size of protonmotive force and the more extensive studies on relationships of protonmotive force to

rates of ATP synthesis just described (subsection IVC). The initial rate of alanine transport into cells of Rhodopseudomonas capsulata has been studied as a function of A~b under conditions arranged such that this was the only component of the protonmotive force, and that a protonmotive force could only be generated by light-driven cyclic electron transport [110]. It was found that the rate of alanine transport depended very sharply on the magnitude of the membrane potential with an apparent threshold value below which no transport was observed. Experiments were also done under conditions such that selected values of membrane potential were attained at a range of light intensities by introduction of a suitable titre of uncoupler. It was considered strikingly unexpected that at constant membrane potential the light intensity drastically affected the initial rate of alanine uptake [110]. This result is clearly closely analogous to the observation that the rate of ATP synthesis by chromatophores from Rhodopseudomonas capsulata decreases as the rate of cyclic electron flow is lowered by addition of antimycin, yet the membrane potential remains essentially constant [68] (see subsection IVC). Certainly these data might suggest that the membrane potential or protonmotive force is not the sole factor that controls either the rate of alanine transport or ATP synthesis in this organism. However, in any study in which light intensity is varied below its saturation level there is the danger that the sample is not uniformly illuminated. Consequently when transport activity is reduced by lowering light intensity it is possible that there is a heterogeneity within the sample in respect of membrane potential a n d rate of transport. If transport is very sharply dependent upon the membrane potential, it follows that only those cells closest to the source of illumination will drive transport, whereas all cells will generate a sizeable membrane potential owing to the tendency of membrane potential to remain almost unaltered at a range of electrontransport rates. Thus it is difficult to be certain that the results of the study of alanine transport rates in Rps. capsulata do require, as suggested by Elferink et al. [110], a link of some kind between electron-transport rate and active transport to account for the apparent closer relationship of the

74 rate of transport to the electron flow rate rather than to the magnitude of the membrane potential (see also Clark et al. [99]). However, it has now been found that when respiration is used to generate Ap in cells of Rps. capsulata the rate of alanine transport decreases linearly with inhibition by cyanide of the respiration rate [111,112]. Thus a close relationship between the rates of electron transport is not only observed under conditions of limiting light intensity. Comparable results were also reported by Elferink et al. [111,112] for proline and lactose uptake by E. coli. In the previous subsection (VIC) we have discussed why homeostasis of Ap might be a priority in the energy-transduction process. If we follow that line of argument than we are led to the view that the dependence of the rate of substrate transport upon Ap must be very sharp so that changes in rates of electron transport can be matched by proportional changes in substrate uptake. Two additional recent findings concerning bacterial transport might be supportive of this view. First, it has been found that when the lac operon is transferred from E. coli to Rps. capsulata, the conferred capacity for light-driven lactose uptake shows the same dependence on light intensity at constant Ap as does the 'native' alanine uptake [113]. It seems unlikely that any specific localised pathways for protons, or other allosteric effect of electron transport on a transport system, would carry over its effect onto a 'foreign' protein. On the other hand, the dependence of lactose transport on Ap in E. coli (see below) is apparently different when the lac operon functions in its native E. coli rather than in Rps. capsulata. The second recent development has been the observation that the rate of Na÷-dependent glutamate transport in E. coli shows the same linear dependence on the rate of respiration as do the proton-linked transport systems [113]. It is difficult to interpret this finding in terms of localised proton flow from electron transport to ATP synthases or transport systems. Rather it appears that there must be a regulatory effect of electron transport upon the transport system or that very small changes in the membrane potential cause the observed changes in transport rate. The postulate of a very sharp dependence of rates of active transport upon Ap is of course

negated if Ap really remains constant as the rate of electron transport is decreased. The sole dependence of the rates of Ap-dependent processes upon the magnitude of Ap also raises the question of how the distribution of Ap between different consumers (e.g., ATP synthesis and active transport) is achieved. It is possible that the dependence of rates of Ap-dependent processes have evolved so that appropriate rates of particular reactions are achieved at a given value of Ap. In some instances the rate of solute transport might be less sharply dependent upon Ap than the rate of ATP synthesis (e.g., rate of lactose uptake in E. coli; see below). This situation might mean that if Ap tended to drop the rate of essential nutrient uptake, from which ATP could subsequently be made, would be less affected than the rate of ATP synthesis. In earlier studies of the consequences of changes in protonmotive force upon rates of transport into bacteria, Kaczorowski et al. [114] found that, although substituting succinate by perdeutero-succinate reduced the rate of electron flow to oxygen by a factor of 2.2, the steady rate of proline uptake did not alter provided transport was measured several minutes after initiation of electron transport. Under these conditions an identical protonmotive force was detected with either substrate. Thus in this case there appeared to be no relationship between rates of electron flow and rates of transport, and presumably the protonmotive force was not reduced at the lower electron-transport rate because the passive conductance of the membrane was adjusted (see earlier discussion). This interpretation naturally implies, of course, that the coupling ratio (substrate transported per 2e) improved at the lower electron-transport rate. When the size of the membrane potential was attenuated by titration with an uncoupler it was found that transport was fairly sharply dependent on the magnitude of membrane potential although less so than in some studies of relationships between rate of ATP synthesis and size of membrane potential. In a somewhat related study by Robertson et al. [115] it was reported that doubling the size of the membrane caused a 4-fold increase in the rate of lactose uptake into E. coli vesicles. Sodium efflux from envelope vesicles prepared from Halobacteria halobium has been found to

75 require a minimum threshold value of protonmotive force and above the threshold a steep linear relationship between rate of efflux and protonmotive force was found [116]. In this work the protonmotive force was adjusted by changes in the light intensity, and so the same reservation as expressed above about use of light intensity might apply to the interpretation of these results with H. halobium vesicles. Work on relationships between rates of substrate transport and magnitudes of protonmotive forces obviously needs to be extended because at present no consistent pattern has emerged, and because in a sense active transport ought by definition to behave as an archetype chemiosmotic process.

IVE. Effects of partial uncoupling upon protonmotiue force and rates of A TP synthesis In all systems tested it is found that introduction of a protonophore decreases both Ap and the steady-state rate of ATP synthesis. The problem, in chemiosmotic terms, arises when the effects of uncouplers are compared with those of inhibitors of electron transport. In both mitochondria [98,102] and chromatophores from Rps. capsulata [68] it has been reported that a given rate of ATP synthesis is associated with a smaller Ap in the presence of an uncoupler than in the presence of an inhibitor of electron transport, or alternatively subsaturating light intensities in the case of photosynthetic systems [68]. Even more dramatic is the report of Zorratti et al. [102] that low concentrations of valinomycin in the presence of millimolar concentrations of K + cause reduction of Ap in mitochondria, but with no change in the rate of ATP synthesis. As these observations stand they are very difficult to accommodate within the framework of the basic chemiosmotic hypothesis. A factor to consider in their interpretation is that Ap is an intensive variable as well as the substrate for the ATP synthase. Hence the problem of how to write kinetic schemes arises. Molecular substrates for enzyme can be described in terms of concentrations or activities. These are extensive terms. Nevertheless, it is reasonable on the basis of the chemiosmotic hypothesis to expect a unique relationship between Ap and the rate of ATP

synthesis. There is indeed no sign in the literature of any dissent from this view. Thus we are left with a serious deviation from the original chemiosmotic theory, although it is worth noting that similar deviations have not been observed in work with thylakoids [117]. Is there any possibility that the discrepancies betweeen the effects of electrontransfer inhibitors and of uncouplers can be attributed to experimental problems? In subsection IVC it was explained that irrespective of the actual measurements of Ap, there is good reason to believe that Ap decreases little, if at all, upon inhibiting electron transport. Therefore, if we are to find an experimental problem it seems more likely to be associated with either the measurement of Ap under conditions of partial uncoupling or of the rates of ATP synthesis. The present writer cannot identify any possible faults with the latter experiments, but Ap measurements might suffer from the admittedly hypothetical difficulty discussed earlier with respect to the comparative effects of uncouplers and ADP on the Ap and the extent of respiratory control (subsection IVB). At the moment the wisest counsel is probably to suggest that a comparison of the effects of uncouplers and inhibitors of electron transport be extended to other systems with as wide a variety of methods as possible. It should also be mentioned here that if the rate of ATP synthesis were to be regulated by factors other than Ap (see subsection IVC), then it is conceivable that there is not a unique relationship between Ap and the rate of ATP synthesis. If this is the case then research is clearly needed to identify these factors. A point to consider is that in many instances the ATP synthase is seemingly not only driven, but also activated, by Ap (e.g., Ref. 99 and 121). There are some puzzling reports concerning the relationship between the size of the protonmotive force and the rate of ATP synthesis by thylakoids. Giersch [118-120] has found that low concentrations of amines or of nigericin in the presence of K + stimulated the rate of photophosphorylation catalysed by unbroken chloroplasts, and yet under both sets of conditions a decrease in the protonmotive force accompanied the stimulated rate. This is surprising because other studies with thylakoids [117] have indicated that small decreases in Ap are accompanied by large decreases in the rate of ATP

76 synthesis. Giersch was led to the conclusion that localised proton fluxes connected electron transport reactions to those of ATP synthesis [120]. There are now reports that reduction by titration with an uncoupler of the rate of ATP synthesis catalysed by thylakoids [108], submitochondrial particles [107,109,121] and vesicles from P. denitrificans [121] is accompanied by increases in K m (ADP), although there are also indications for no change in K,~ (ADP) from some other work on thylakoids and thylakoid ATP synthase [121,122]. Reduction of the rate of ATP synthesis by titration with an electron transport inhibitor has, in contrast to the effects of an uncoupler, been found to be accompanied by decreases in the K m (ADP) (subsection IVC). It is not easy to rationalise this type of result because a particular change in Ap, however achieved, could be expected to have an equal effect on a kinetic parameter in accordance with the chemiosmotic theory [108]. A possible explanation for the decrease in K m (ADP) as the electron-transport rate is attenuated was discussed earlier (subsection IVC). Why though, should g m (ADP) increase in the presence of an uncoupler? In terms of rate-limiting steps such a result would suggest that the ATP synthases, by being in competition with the protonophore, become more rate-limiting under these conditions. Another possibility that is difficult to exclude rigorously is that the uncouplers bind to the ATP synthase in a process unconnected with their primary mode of action, and that this binding influences the kinetic parameters of the enzyme [121]. Yagi et al. [109] have presented an alternative explanation for the opposite effects of partial uncoupling and restriction of electron flow upon K m (ADP).

IVF. Effects of changes in rates of nucleoside triphosphate hydrolysis upon rates of reversed electron transfer and transhydrogenase in submitochondrial particles In submitochondrial particles ATP hydrolysis can drive an energy-dependent transhydrogenation of N A D P by N A D H . Substitution of ATP by ITP decreased by 50% the rate of nucleoside triphosphate hydrolysis and also resulted in a proportional decrease in the rate of transhydrogenation [123]. Similarly, if the rate of ATP hydrolysis was

decreased by addition of oligomycin or ATPase inhibitor protein, directly proportional changes in the rate of transhydrogenase were seen. A different pattern was observed when energy-dependent-reversed electron transport from succinate to NAD was studied. Substitution of ATP by ITP has usually been found to result in almost complete inhibition of reversed electron transport [123]. Yet when the rate of ATP hydrolysis was reduced by titration with the inhibitor protein of the ATP synthase, the loss of reversed electron transport followed very closely the inhibition of the ATPase [123,124]. In further experiments it was found that both the protonophore FCCP and an antibiotic inhibitor of the ATPase, oligomycin, were more effective at inhibiting reversed electron transport than transhydrogenase [123]. The difficulty in interpreting these results is that little is known about how the rates of the two reactions depend upon Ap. Nevertheless, the finding that the transhydrogenase reaction rate is considerably less sensitive to FCCP than reversed electron transport may be taken as at least token evidence that the rate of the former reaction might show a less sharp dependence upon Ap than the latter. Thus a small drop in Ap, following replacement of ATP by ITP, or partial inhibition of ATP hydrolysis by oligomycim could conceivably produce a large change in the rate of reversed electron transport, but could only cause a smaller change in the transhydrogenase rate. On this basis the finding that changes in the latter are very similar to the changes in the rate of nucleoside triphosphate hydrolysis can be suggested to be fortuitous and not a reflection of discrete assemblies containing both ATPase and transhydrogenase enzymes. Not all observations on the kinetics of reversed electron transport are compatible with the above picture. These are the effects of the ATPase inhibitor protein [123,124], the lack of correlation between the rates of hydrolysis of a variety of nucleoside triphosphates and their efficacy in driving reversed electron transport [125] and also the effects of ADP and AMP-PNP in actually inhibiting ATP hydrolysis activity more effectively than reversed electron transport [125-127]. Of these, only the close parallelism between the inhibition of both reversed electron transport and ATPase activity by the inhibitor protein can be taken as

77 indicative of localised interactions between ATPases and the reversed electron transport that is catalysed by Complex I of the respiratory chain. Nevertheless, is must be recognised that in none of these cases has Ap been estimated and that at least in principle Ap can depend on both the rate of nucleoside triphosphate rehydrolysis and on the value of AG for the hydrolysis [128,129]. The latter could be affected by triphosphate resynthesis through adenylate kinase activity which would vary according to how well a particular nucleoside diphosphate fitted into each of the two substrate sites on adenylate kinase. Any further understanding of the dependence of the rates of reversed electron transport and transhydrogenase on the turnover of the ATPase, and the confirmation or rebuttal of localised schemes for energy coupling, requires measurements of Ap to be made under the same reaction conditions. Recently, some progress has been made in this direction. First, it has been shown that ITP and ATP drive reversed electron transport at very similar rates when the membrane potential generated by hydrolysis of either nucleotide is the same [128]. This condition is satisfied when the concentration of ATP or ITP equals that of ADP or IDP. At higher concentrations of ATP a larger membrane potential is generated and a faster rate of reversed electron transport observed [128]. As discussed by Sorgato et al. [128,129] the exact membrane potential generated by nucleotide triphosphate hydrolysis is probably a complex function of the rate and of the free energy of hydrolysis. A second set of results that warrants attention has been obtained from experiments using submitochondrial particles in which the F1F0-ATPase was inhibited to defined extents by covalent modification with the photoaffinity label 8-azido-ATP [130]. It was shown that inhibition of reversed electron transport was linearly proportional to the inhibition of ATP hydrolysis, but that the protonmotive force was not altered by the inhibition of ATP hydrolysis activity [130]. Hence, it was concluded that changes in the protonmotive force were not responsible for the inhibition of reversed electron transport and that therefore the F0FlATPase and the NADH: ubiquinone oxidoreductase (Complex I) were directly coupled without a

delocalised intermediate [130]. Although the published account does not make clear whether the protonmotive force was measured under conditions in which ATP hydrolysis was driving reversed electron flow, the paper by Herweijer et al. [130] is an important contribution, and at first sight defies a chemiosmotic explanation. As usual the retort from a pure chemiosmotic standpoint has to be that measurements of protonmotive force are insufficiently precise to eliminate explanations based on the theme that very small charge in protonmotive force cause large changes in rates of protonmotive-force-dependent processes. It is worth noting that if this is the case then clues may be provided about the mechanism of operation of complex I and transhydrogenase within a strictly chemiosmotic framework. It is appropriate to conclude this section by stating that some, but not all, of the observations could in principle be readily accounted for on the basis of a mechanistic refinement of the chemiosmotic theory. However, other results are consistent with energy transduction within discrete coupling units but do not provide any direct evidence for this concept. It is also useful at this point to recall the failure of ATP hydrolysis to drive transhydrogenase in a mutant of Salmonella typhimurium under conditions where ATP hydrolysis was apparently competent to generate a pH gradient (subsection IIC). Taken together with the considerations about the rate of transhydrogenase in submitochondrial particles this could be evidence for some type of direct interaction between ATPases and transhydrogenase enzymes. But the nature of any such interaction is a mystery.

IVG. Effects of inhibitors of the A TP synthase upon the protonmotioe force, and the rate of A TP synthesis Later in this section on kinetic studies we shall be discussing the results of experiments in which inhibitors of ATP synthases have been used in combination with methods for reducing the rate of electron flow. The results of such experiments are frequently discussed as possible evidence for localised modes of energy coupling, but a prerequisite for their analysis is to understand the effects of ATP synthase inhibitors on both Ap and the rate of ATP synthesis.

78 It is known that Ap is lowered in most systems upon commencement of ATP synthesis. Therefore, when an inhibitor of the ATP synthase is introduced to a system catalysing oxidative phosphorylation, there should be a tendency for Ap to rise towards the value observed in the absence of ATP synthesis. But, as discussed earlier (subsection IVC), if the rate of ATP synthesis catalysed by the ATP synthase enzyme rises very sharply as Ap is slightly increased, then those ATP syntheses that do not have the inhibitor bound should increase their turnover rate. Thus the shape of a titration of the rate of ATP synthesis by an inhibitor of the ATP synthase might be rather complicated [131]. It is certainly possible that the extent of inhibition of the ATP synthesis rate by a given titre of such an inhibitor might be less than the extent of inhibition of ATP hydrolysis measured in the absence of Ap, and also not directly proportional to the fraction of the ATP synthase molecules that have inhibitor bound. In general terms this description could account for the shape of a titration of the rate of mitochondrial ATP synthesis with oligomycin. It has often been observed [102, 132-134] that the initial additions of oligomycin cause little inhibition so that the overall titration tends towards a sigmoidal shape. It is interesting to note that Lardy et al. [132] suggested that this result indicated that a delocalised intermediate connected electron transfer to ATP synthesis. On the other hand Zorratti et al. [102] mentioned that it might be indicative that the ATP synthase was not the rate-limiting step in oxidative phosphorylation catalysed by mitochondria. The alternative explanation for such a sigmoidal titration profile renders equivocal such a conclusion about a ratelimiting step. What is known about comparisons of the binding of inhibitors of the ATP synthase with the profile of their inhibitory action against the rate of ATP synthase? Unfortunately there are few studies directed to this point. Kahn [135] showed that the specific binding of radiolabelled tributyltin to the ATP synthase of thylakoids from Euglena gracilis paralleled the inhibition of the rate of ATP synthesis linked to non-cyclic electron flow at saturating light intensities. Unfortunately, this work was done before it was realised that trialkyltin compounds could also inhibit by catalysing on O H - / C I ex-

change across the thylakoid membrane, and so the results are not unequivocal. Possibly the least ambiguous comparison of the effect of an inhibitor on the rates of both ATP synthesis and hydrolysis was made using vesicles from P. denitrificans and the well-defined inhibitor 4-chloro-7-nitrobenzofurazan [136]. It was found that this compound, which modifies an essential tyrosine on the F 1 segment of the ATP synthase [137], caused parallel inhibition of both reactions. As covalent modification with this inhibitor completely abolishes the activity of the enzyme, the loss of ATPase activity can be taken as a measure of the binding of the inhibitor to the enzyme. Hence, the rate of ATP synthesis must have been inhibited in direct proportion to the extent of modification of the enzyme. Recently, a similar result was obtained for submitochondrial particles. The ATP synthase was inhibited by covalent modification through reaction with the photoaffinity label 8-azido ATP and ATP synthesis driven by N A D H oxidation was reported to be proportionally inhibited compared with ATP hydrolysis [130]. Furthermore, it has been shown that an ATP synthesis and hydrolysis are inhibited equally by a given amount of covalently incorporated D C C D in studies with mitochondria [138]. The general pattern of behaviour shown by inhibitors of the ATP synthase is thus that ATP synthesis and hydrolysis are equally inhibited. The insensitivity of the rate of mitochondrial ATP synthesis to the initial titres of oligomycin appears to be peculiar to that inhibitor and is not, for example, duplicated by the titration profile for venturicidin as an inhibitor of the ATP synthase in P. denitrificans [30]. Thus the majority of the results obtained with inhibitors of the ATP synthase indicate that the value of the protonmotive force normally attained during oxidative phosphorylation is sufficient to drive all of the ATP synthase enzymes at their maximum rate. Any increase in the steady-state value of the protonmotive force following inhibition of a proportion of the enzyme molecules cannot therefore be translated into a faster turnover rate by those enzyme molecules that are not inhibited. Alternatively, one could argue that an exact correspondence between the fraction of the enzyme molecules that have an

79 inhibitor bound and the inhibition of the rate of ATP synthesis argues for a localised mode of energy coupling. Further measurements of titration profiles, Ap, and, where possible the binding of inhibitors, are needed to clarify how changes in Ap during such titrations affects the rate of ATP synthesis.

IVH. Effects of combinations of uncouplers and inhibitors upon rates of A TP synthesis In a recent series of papers Hitchens and Kell [139-141] have investigated the titre of uncoupler that is necessary to inhibit residual rates of photophosphorylation after partial inhibition by an inhibitor either of the electron-transfer chain or of the ATP synthase. In each case it has been found that less uncoupler is required to abolish the residual phosphorylation after the treatment with an inhibitor. Such a result is at least qualitatively reasonable in a chemiosmotic context for an experiment in which an electron transfer inhibitor is used. A slower rate of proton translocation by the electron transfer chain would be accompanied by lower requirement for uncoupler-mediated dissipative proton flow across the membrane to collapse Ap. Hence, in the presence of an electron transport inhibitor a lower concentration of uncoupler is needed to inhibit ATP synthesis. An explanation for the requirement for less uncoupler when ATP synthesis is partially blocked by either DCCD [139], oligomycin [140] or venturicidin [141] is not immediately apparent, especially as the experimental results followed a similar pattern irrespective of whether SF 6847, nigericin plus valinomycin or gramicidin D was the added uncoupler [141]. The interpretation that Hitchens and Kell [139-141] have chosen is that uncouplers rapidly diffuse within the membrane of a chromatophore and act by uncoupling at localised energy-coupling sites. When a fraction of the ATP synthases is inhibited, the uncoupler is not required at sites associated with these synthases and thus a lower titre of uncoupler suffices to uncouple at the remaining localised sites of energy coupling. Such an explanation supposes that the uncoupler spends a significant part of its time at an uninhibited energy coupling site and in the process of transporting a proton from the site to where it came from

(presumably one of the bulk aqueous phases surrounding the chromatophore membrane). Can an explanation of the data of hitchens and Kell [139-141] be formulated that would be consistent with the delocalised chemiosmotic hypothesis and generally held views of uncoupler action? The first problem in analysing a possible explanation is that, as outlined in subsection IV G, the effects of adding a partial titre of an inhibitor of the ATP synthase on both the magnitude of the protonmotive force and the turnover rate of the remaining uninhibited enzyme molecules are not fully understood. But for illustrative purposes it is assumed here that any increase in the protonmotive force that follows addition of an ATP synthase inhibitor is not reflected by an increased turnover rate of the uninhibited molecules. This description would seem to fit with observations made with titrations with inhibition of ATP synthase (subsection IVG). Hence, in the experiments of Hitchens and Kell [139-141] we must assume that in a preparation of chromatophores with, say 50% of the ATP synthases inhibited by DCCD, the magnitude of Ap is at least slightly larger than in the uninhibited preparation which catalyses twice the rate of ATP synthesis. Next we consider the consequences of adding the same concentration of uncoupler to identical quantities of the two preparations, against the observation that the percentage decrease in the rate of ATP synthesis is greater in the preparation that is partially inhibited at the level of the ATP synthase. Following our present line of argument we have to say that under these conditions (i.e., limited titre of uncoupler added) the protonmotive force is smaller in the inhibited preparation so as to account for the greater decrease in the rate of ATP synthesis catalysed by the individual ATP synthases of the partially inhibited preparation compared with the uninhibited preparation. Clearly, to confirm or deny this possibility measurements of Ap are required. It is not obvious how the postulated lowering of Ap could occur, although it is worth considering that titration with uncouplers might be difficult to analyse if the flux of uncoupler-mediated proton flow across the membrane is dependent on Ap. The latter behaviour can arise because the transport kinetics of the anionic form of an uncoupler may depend on the membrane potential. The ex-

80 periments of Hitchens and Kell [139-141] have provided some interesting new data which certainly require an explanation, although Cotton and Jackson [142] have suggested that the shape of titrations with an uncoupler following addition of ATP synthase inhibitors might critically depend on the experimental design. They report that alternative experimental protocols give results in which the shape of a titration with uncoupler is not altered by the partial inhibition of ATP synthases. If this is the case it will be necessary to have independent confirmation of this type of experiment, perhaps using incorporation of 32Pi into ATP as the assay method for ATP synthesis rather than proton uptake measurements used to date [139-142].

IVJ. The combined effects of restriction of electron transport and inhibitors of the A TP synthase," the double-inhibitor experiments In a sequence of two separate enzyme-catalysed reactions A ~ B ~ C, conditions are often found in which the second reaction has excess catalytic capacity. For example, in coupled enzyme assays it is usual to arrange conditions such that for the second reaction [B] < K m a, so that the rate of the second reaction reduces to a simple first-order dependence on [B]. In fact the second reaction can still match the first even when the number of units of the second enzyme is such that the steady-state concentration of B increases to the point where first order conditions no longer apply [143]. Thus a reduction in the rate of flux from A to B will usually render spare catalytic capacity for the reaction B to C. Therefore an inhibitor of the reaction B to C should have little or no effect on the overall rate of conversion of A to C until sufficient inhibitor has been added to cause a considerable reduction in the catalytic capacity for the conversion B to C. This behaviour is a basis for testing whether in a sequence of enzyme-catalysed reactions D E ~ F, E is a freely diffusable intermediate between two independent enzymes. A good example of this type of behaviour is found in the mitochondrial electron-transfer chain. Ubiquinone is considered to act as a mobile pool between the dehydrogenases and the cytochrome part of the chains. The catalytic capacity of the

latter generally considerably exceeds that of the former. Consequently, a fraction of the cytochrome chains can be inactivated by the addition of antimycin without appreciable inhibitory effect on the overall respiration rate. Thus, whereas the binding curve for antimycin is hyperbolic its titration of overall activity is sigmoidal [144]. By analogy to the arguments and example just cited, a number of investigators have sought to test whether, in line with chemiosmotic theory, oxidative phosphorylation or photophosphorylation is catalysed by two independent and sequential reactions, electron transport and ATP synthesis, that are linked by Ap, which is by definition a delocalised intermediate. Probably the first study of this kind was made by Kahn [135]. He showed that a titration with tributyl tin, an inhibitor of ATP synthase, of the rate of ATP synthesis linked to cyclic electron flow in thylakoids from Euglena gracilis paralleled exactly the specific binding of the inhibitor to the thylakoids when a saturating light intensity was used. (Note that this finding apparently contradicts the predictions made in subsection IVG concerning the anticipated relation between the binding of an ATP synthase inhibitor and the inhibition of ATP synthesis). At limiting light intensities, or under conditions of non-cyclic electron flow, it was found that the initial titres of tributyl tin were less effective at inhibiting ATP synthesis than at saturating light intensities. Using a quantitative analytical procedure it was concluded that the results were consistent with a model in which each discrete electron-transfer chain was linked specifically to three ATP synthases. This accounted for the extent of the flattening of the titration profile. It was argued that only a much greater degree of insensitivity to tributyl tin would have been consistent with the notion that a delocalised pool connected the electron-transfer reactions to the ATP synthases [135]. A subsequently recognised difficulty with Kahn's work has been the discovery that tributyl tin can also act as an uncoupler with thylakoids as a consequence of its chloride/hydroxyl exchange activity. This property should have influenced the experiments of Kahn. However, any uncoupling effect, and therefore inhibition of ATP synthesis, should have been more dramatic at the lower light intensities (see subsection IVH).

81 In 1971 Baum and colleagues [145] reported studies using submitochondrial particles of the effects of rotenone and oligomycin on the reverse of oxidative phosphorylation, ATP-dependent reversed electron transport from succinate to NAD. They found that the shape of a titration of this reaction with rotenone was independent of the presence of selected titres of oligomycin to inhibit partially the ATPase. They argued that if a delocalised intermediate (Ap) was involved, then in the presence of partial titres of oligomycin there would be excess capacity in the electron-transfer chain so that the overall reaction should be relatively insensitive to the initial titres of rotenone [145]. On this b~isis it was suggested that such experiments gave an indication that perhaps electron transfer and ATP synthesis were not connected just by a delocalised Ap but that there could be also a direct interaction of some kind between electron transport proteins and the ATP synthase. However, it was also recognised that a delocalised model might be compatible with the data if certain properties were assigned to routes whereby Ap might be dissipated as heat (leaks) [145,146]. Such an explanation was, however, considered difficult to reconcile with the findings from experiments in which malonate rather than rotenone was used as the inhibitor of electron transport [147]. In this case the overall reaction of ATP-driven reversed electron transport from succinate to NAD became less sensitive to malonate when partially inhibiting amounts of oligomycin were present. It was thought that this might rule out an explanation based on the properties of leak pathways because these were anticipated to be the same irrespective of whether rotenone or malonate was used as inhibitor. By inhibiting electron transport at succinate dehydrogenase, and thus for reversed electron transport before the ubiquinone pool, the sensitivity of the reaction to malonate was expected to decrease in the presence of partial titres of oligomycin regardless of whether there was any localised interaction between electron transport and ATPase [147]. Thus the difference between titration behaviour of malonate and rotenone could be taken as evidence that inhibition of individual NADH dehydrogenases by rotenone abolishes the activity of discrete units containing both the dehydrogenase and the ATP

synthase. But another difference between malonate and rotenone as inhibitors is that only addition of the latter will reduce the number of functional energy-transducing NADH dehydrogenase complexes. If the rate of reversed electron transfer is a critical function of the number of active NADH dehydrogenase complexes then it could be that addition of rotenone will under all conditions have an identical inhibitory effect. Subsequently the original experiment of Baum et al. [145] has been rediscovered at least twice with much the same result [148,149]. This perhaps indicates that, however, controversial, the original work should have been reported in a journal. In their analysis Westerhoff et al. [149] have discussed the reliability of this double inhibitor approach. They argue that strictly only when the sensitivity to rotenone of the rate of ATP-dependent reversed electron transport actually increases after partial inhibition by oligomycin can this type of experiment provide unequivocal evidence in favour of a localised mode of energy transduction. Exactly this result was obtained by Westerhoff et al. [149] and there is evidence of this behaviour in the work of Baum et al. [145]. On the other hand, Ferguson [148] found that the sensitivity to rotenone was neither decreased nor increased by the presence of a partial titre of oligomycin. There have also been studies of the effects of two inhibitors, one for electron transport and one for ATP synthase, on the kinetics of steady-state photophosphorylation by chromatophores from photosynthetic bacteria [150] and of oxidative phosphorylation by vesicles from P. denitrificans [151]. In each case an identical titration curve for the inhibitor of the second enzyme in the sequence (i.e., the ATP synthase) has been obtained irrespective of the extent of inhibition of electron transport. Thus the general result from studies of the effects of combinations of inhibitors for electron transport and ATP synthase has been to produce data that at first sight are not inconsistent with a localised intermediate acting between the two reactions. The additive effects of the two types of inhibitor provide a temptation to conclude that electron-transport enzymes and ATP synthases are associated in discrete units that form at least transiently. Such a model would clearly be at variance

82 with views of the nature of energy-transducing membranes in which electron-transport components and ATP synthases are independent diffusing entities in the membrane. Furthermore, the operation of such discrete units is not envisaged by the chemiosmotic theory. It is also worth noting that the indiscriminate application of the double inhibitor technique can suggest that the adenine nucleotide translocator forms discrete complexes with both the respiratory chain and the ATP synthase [152]. There is little support for such a notion. However, experiments with malonate and carboxyatractyloside have shown recently that these inhibitors act additively at low osmolarity but not at high osmolarity [153]. Thus indications for a localisation of energy transduction were obtained at low but not high osmolarity [153]. Earlier in this section on kinetics (subsection IVB and G), the effects on Ap and rate of ATP synthesis of a single inhibitor, for either electron transport or ATP synthase, are explored and shown to be not fully understood. Therefore it is difficult to interpret with any certainty the results of the double inhibitor titrations. However, keeping in mind the arguments discussed in subsections IVB and G, the following pattern of behaviour might be expected on a strict chemiosmotic basis for a double inhibitor titration of oxidative phosphorylation. As indicated earlier, the effect of an electron transport inhibitor may be to cause a very small reduction in the steady-state value of Ap which in turn will cause the rate of ATP synthesis to decrease in proportion to the extent of attenuation of the rate of electron transport. Given that Ap is known to decrease during net ATP synthesis owing to the load put upon Ap by the operation of the ATP synthase enzyme, it is predicted that the effect of titrating with an inhibitor of the ATP synthase will be to cause a progressive increase in Ap. As it is postulated that a small drop in the Ap upon addition of electron transport inhibitor causes the ATP synthases to turn over at a significantly smaller rate, it follows that rises in Ap following addition of an ATP synthase inhibitor to a system in which electron transport is partially inhibited would cause those A T P synthase molecules that remain unbound by inhibitor to turn over at a higher rate. Hence, on a strict chemiosmotic basis it is hard to escape the conclusion that the rate of

A T P synthesis should become less sensitive to the initial titres of an inhibitor of the ATP synthase as the rate of electron transport is progressively reduced by titration with an inhibitor or by restriction of substrate supply. As this pattern of behaviour is generally not observed [145-151], there would appear to be a need for more attention to be paid to these types of observation. The additivity of the effects of electron transport and ATP synthase inhibitors certainly will continue to p r o m p t consideration of the possibility of some type of direct interaction between electron transport and ATP synthesis unless compatibility with the chemiosmotic hypothesis can be definitely established. It is also important to recognise that there is a possibility that the interaction of some inhibitors of ATP synthase with the enzyme may vary with changes in Ap. Such behaviour would complicate the analysis very greatly and could only be checked by direct binding studies or use of well-defined covalent inhibitors. The strategy of the dual-inhibitor type of experiment obviously supposes that variation is possible in the extent to which either electron transport or the ATP synthase is rate limiting in oxidative phosphorylation. It is not necessary that either reaction should be decisively rate limiting, but rather in the terminology of Kacser and Burns [93,94] the control strength of each of the two reactions should be variable according to the particular reaction conditions. Although such a situation might intuitively seem reasonable it has been argued elsewhere, for instance by Clark et al. [99], that the control strength of the ATP synthase, or the extent to which it is rate limiting, may not be altered by changes in the rate of electron transport. The argument is that the magnitude of the steady state Ap controls the fraction of the ATP synthases that are in the active state. The active state ATP synthases themselves are supposed to have a zero order dependence on Ap [99]. According to such a scheme the rate of ATP synthesis is adjusted to be proportional to the rate of electron transport by the precisely corresponding activation of A T P synthases. An implication of this scheme is that it is not possible to alter the control strength or extent of rate limitation, due to the ATP synthase. It has been suggested that in these circumstances the results of double inhibitor experi-

83 ments would not be inconsistent with a delocalized intermediate, Ap, between electron transport and ATP synthesis [99]. Such a proposal is not easily reconciled with the arguments discussed earlier (see above and see also subsection IVG) concerning the expectation that titrations of rates of ATP synthesis by ATP synthase inhibitors will be accompanied by increases in Ap. Recall that this means that of the ATP synthases that are not bound by such an inhibitor a higher fraction should be activated. Thus it is reasonable to suggest that the extent of any rate limitation by, or the control strength of, the ATP synthase would decrease. Hence titres of a synthase inhibitor for a given percentage reduction of the rate of ATP synthesis are again predicted to be increased by the presence of an electron transport inhibitor. But putting this aspect aside, we are left with the important question of whether indeed the control strength of the ATP synthase can alter. As mentioned earlier, the decreases in apparent g m (ADP) when the maximum rate of ATP synthesis is attenuated by restriction of the rate of electron transport could be taken as evidence that the ATP synthase does contribute less to the overall control of the rate of the reaction under such circumstances. The issue of whether the control strength of electron transport or ATP synthase towards the overall process of oxidative phosphorylation can vary has wide implications for understanding the kinetics of oxidative phosphorylation and requires further study. Although not strictly involving the double-inhibitor type of experiment, the work of Venturoli and Melandri [154] is conveniently discussed here. They measured the yield of ATP obtained from each very brief flash of light to which a suspension of chromatophores from Rhodopseudomonas sphaeroides was exposed. The yield of ATP varied according to the dark time between flashes, rising to a maximum at approx. 200 ms. Interesting results were obtained when the effect of antimycin, an inhibitor of the light-dependent cyclic electron-transport chain, was studied. The striking observations were that the inhibition of ATP synthesis was very closely related to the extent of inhibition of the electron-transfer chains irrespective of the dark times between flashes. Simultaneous measurement of the carotenoid band shift

showed that 40% inhibition of ATP synthesis was observed without any decrease in the size of the membrane potential. A second set of experiments involved studying the effects of DCCD, a specific inhibitor of the ATP synthase. In this case it was found that the decrease in the yield of ATP was directly proportional to the fraction of the ATP synthase enzymes that had been inhibited, irrespective of the dark times between flashes. These experimental findings using single turnover conditions were taken to mean that there must exist a functional interaction between electron-transport chains and ATP synthases, in agreement with earlier conclusions from steady-state kinetic experiments [68,155-157]. Such an interaction may be a requirement for photophosphorylation that is additional to the protonmotive force. It is interesting that in their discussion Venturoli and Melandri [154] express the view that in continuous light the turnover of the ATP synthase is a kinetic limiting step, but that this should not be the case when electron transport is restricted by firing a train of flashes at low frequency. In this context it was considered highly significant that the degree of inhibition of ATP synthesis by either antimycin or DCCD was practically independent of the flash frequency. However, as discussed earlier in this section it has been argued by Clark et al. [99] that it might be misleading to think in terms of either electron transport or ATP synthase being rate limiting. If this were true then the argument of Venturoli and Melandri [154] loses some of its force. Further work is needed on this aspect. The general conclusion from this discussion of double inhibitor experiments is that some of the data appear highly persuasive in favour of a role for localised interactions between electron-transport reactions and the ATP synthase. On the other hand explanations that would make the observations consistent with a delocalised intermediate have not been completely eliminated. Justification of the latter type of explanation will require the demonstration that the kinetics of the Ap-linked processes show certain very well-defined dependencies on Ap. One should also recall the possibility that the kinetics of energy transduction might be regulated by a localised interaction (cf. subsection IVC). This would not contradict the role of Ap as the intermediate.

84

IVK. The kinetics of the release of protons into the internal bulk aqueous phase of thylakoids Any doubts as to whether electron transport is linked to the simultaneous translocation of protons into a bulk aqueous phase can in principle be settled by suitable kinetic experiments. Such experiments have been especially the province of Jung and co-workers. They have used the pH-indicating dye neutral red as a spectroscopic probe of proton release into the internal aqueous phase of thylakoids following short flashes of light under conditions in which the external aqueous phase is buffered with bovine serum albumin. In their original work they reported that protons released by the water oxidation reaction were detected by internal neutral red within 0.1 to 1 ms and that protons released from plastohydroquinone oxidation appeared only 20 ms after excitation of the photosystems [15]. The evidence that neutral red detected protons released into an internal aqueous bulk phase was based especially upon the observation that hydrophilic buffers, including phosphate, reduced the extent of the changes in neutral red absorbance as a presumed consequence of increases in the internal buffering capacity. From these studies it was concluded that even the protons rapidly released in the water oxidation reaction equilibrated with internal bulk aqueous phase of thylakoids before passing to the ATP synthase [15]. Thus the results could be taken as strong evidence against special pathways for proteins or kinetic barriers preventing access of protons to the bulk aqueous phase. In more recent work Hong and Junge [158] have recognised that the results from experiments with neutral red are at variance with the claims of Dilley and co-workers, and also of Theg and Homann [159,160], that protons released by reactions water oxidation and plastohydroquinone oxidation were not equivalent .(see subsection liB). Upon reconsideration of the work with neutral red, Hong and Junge [158] have now found that use of fresh preparations of thylakoids gives different results from those previously reported which were obtained with preparations that had been stored at low temperatures in the presence of dimethylsulphoxide as a cryoprotective. In the new experiments with freshly prepared thylakoids it has been

found that changes in neutral red absorbance were increased rather than suppressed by the addition of small hydrophilic buffer molecules. A further difference between fresh and stored thylakoids was that salts did not influence the apparent pK of neutral red bound inside fresh thylakoids whereas they did in thylakoids that had been stored frozen in the presence of dimethylsulphoxide [158]. These results of experiments using neutral red with different types of preparations of thylakoids create a quandary. After storage and thawing thylakoids apparently catalyse reasonable rates of photophosphorylation [158] and the response of neutral red indicates orthodox chemiosmotic behaviour, thus providing, it has been argued [158], strong evidence against the involvement of localised protons in the link between electron flow and ATP synthesis. On the other hand, the new work with freshly prepared thylakoids has provided seemingly contradictory data. In an attempt to resolve this quandary, Hong and Jung [158] have suggested that the internal space of freshly prepared thylakoids cannot be regarded as a bulk aqueous phase and that there may exist a lateral diffusion limitation for protons [158]. The latter possibility is related to ideas of Haraux and Dekouchkovsky [161-163]. (ss subsection IVL). A possible consequence of the proposed properties of the internal phase of freshly prepared thylakoids is that the thermodynamics of photophosphorylation may be inadequately described in terms of a classical delocalised protonmotive force between two bulk aqueous phases. If the latter suggestion were valid then one might expect to find different relationships between apparent protonmotive force and phosphorylation potential in fresh and stored preparations of thylakoids. Such measurements are not available in the literature. In a continuation of studies using neutral red, Theg and Jung [141] have investigated the effect of low concentrations of uncouplers on the absorbance changes of neutral red following exposure of thylakoids to short flashes of light. It was found that in fresh preparations of thylakoids neutral red no longer detected the release of protons associated with oxidation of water after treatment of the thylakoids with low concentrations (sufficient to uncouple only partially) of gramicidin, FCCP or nigericin. In contrast, proton release

85 from either plastohydroquinone oxidation or PMS-mediated cyclic electron flow was still detected by neutral red under the same conditions. A titre of gramicidin that was sufficient to abolish the signal from neutral red associated with oxidation of water did not alter the extent of proton uptake at the external surface of the thylakoids that was required, together with electrons derived from the oxidation of water, for reduction of benzyl viologen. This last observation showed that the protons released in the water-oxidation reaction were not transferred to the external aqueous phase under these conditions. Thus, apparently the protons released by H20 oxidation in the presence of low concentrations of uncouplers or ionophores were to be found neither in the internal nor in the external bulk aqueous phases, and therefore were suggested to be deposited into a domain located within the thylakoid membrane. This proposal is clearly in accord with the suggestions of Dilley and coworkers that were derived from the entirely different experimental approach described in subsection liB. The proposal of a domain for protons that is kinetically separated from the internal bulk aqueous phase must be reconciled with the observation that neutral red can detect protons released upon oxidation of water in the absence of an uncoupler. Theg and Junge [164] have suggested that usually these domains are saturated with protons following a previous exposure to light. It is envisaged that these protons are retained within the domains until the thylakoids are treated with an uncoupler, especially at alkaline pH where the effect of low concentrations of gramicidin at suppressing detection of internal protons by neutral red is most pronounced. In agreement with this model it was found that in the presence of suitably low concentrations of gramicidin neutral red began to detect protons released by oxidation of water after several flashes, spaced at intervals of 150 ms. This is consistent with the requirement to fill up the proton binding sites within the domain before protons overflow into the bulk phase where they are detected by neutral red. At present it is difficult to know what to conclude from the experiments with neutral red. In thylakoids that have been stored frozen in the presence of dimethylsulphoxide the data from experiments with neutral red strongly support a pure

chemiosmotic view, and yet in experiments with fresh preparations the data are more consistent with an alternative view. These matters could be taken as evidence that neutral red is not a reliable probe for pH changes inside thylakoids, but Junge has strenuously argued that for conditions of flash excitation of thylakoids neutral red is an excellent probe for its intended purpose [158,165,166]. For the moment it seems advisable to wait for further experimental data and not to use the results of experiments with neutral red as particularly strong evidence for or against a particular pathway of proton flow from electron-transfer reactions to the ATP synthase. It must be said that in some respects the original contention that hydrophilic buffers, including phosphate and pyrophosphate, could readily permeate the thylakoid membrane [165-166] is surprising. If molecules such as these can readily pass through the membrane it is a little difficult to envisage how proton permeability can be restricted to permit generation of a sizeable protonmotive force (cf. Ref. 167). Hong and Junge [158] did not detect any difference in ionic permeability of the two types of membrane (fresh or stored thylakoids), and given the reported sharp dependence of the rate of ATP synthesis upon protonmotive force [38,103,104], the freeze-thawed thylakoids cannot be significantly more ion-permeable than the fresh ones. Thus if hydrophilic buffers are permeable the evidence seems to point to no difference in this respect between fresh and stored thylakoids. This is perhaps the place to add that in addition to the evidence deduced from work with neutral red and that obtained by Dilley and colleagues (subsection liB), experiments on chloride retention by darkened thylakoids have also given some indications for a proton domain in the thylakoid membrane. Theg and Homann [159] reported that a limited quantity of chloride ions could only be released after addition of uncouplers which were suggested to permit equilibration of protons within the domain with those in the bulk aqueous phases. Thus chloride is released upon raising the pH in these special domains. Although Theg and Homann [159] noted the parallel between their work and that of Dilley et al. (subsection IIB), they also recognised the problem in envisaging a proton domain that could stretch approx. 0.5/tm from the

86 Photosystem II reaction centres at the stacked granal regions to the ATP synthase at the unstacked regions and at the margins of the grana. In further work [160] additional evidence has been obtained for a large quantity of protons that are associated with thylakoids and yet equilibrate only slowly with the bulk aqueous phase, unless uncouplers, including nigericin as well as protonophores, were added. These protons were shown not to be associated with the surfaces of the membrane nor with added buffers. At the very least these experiments have generated data that requires an explanation, especially in the context of the role of chloride ions in the functioning of Photosystem II. It is also convenient to mention here the work of Ort and colleagues [167,191]. They have investigated the effect of increasing the buffering capacity of the intrathylakoid space on the initiation of ATP synthesis by thylakoids. The rationale has been that under conditions in which the A~b component of Ap is collapsed, the illumination time required to build up a sufficient ApH to activate ATP synthesis should increase with increasing buffering power. Ort et al., e.g. Ref. 191, have observed that the increased buffering power does not have this effect, and have therefore concluded that the pathway of protons from sites of production at redox reactions to sites of utilisation at the ATP synthase does not include the whole of the inner aqueous phase that they have shown to be occupied by the added buffers. Other workers [192,193] have disagreed with this conclusion, in part because it is difficult to check directly whether the measures taken to prevent the development of A~, and ApH have been effective.

IVL. Deductions from the use of 211,0 in place of 1420 in measurements of photophosphorylation A series of papers [161-163] has reported the effects of substituting 2H20 for H20 upon electron transport control, a qualitative estimate of the pH gradient (from 9-amino acridine fluorescence quenching), and the rate of ATP synthesis by thylakoids. The principal findings are that control of electron transport is enhanced following substitution of 2H20 despite a decrease in the pH gradient in this solvent [161-163]. These observations have been taken to mean that in 2H20 the

local effective pH at the site of proton production in redox reactions is lower than the bulk aqueous phase pH and thus control of electron transfer is greater in 2H20. During light-driven ATP synthesis it is suggested that the effective internal pH felt by the ATP synthase is higher than is found at the sites of proton production (i.e., water oxidation or plastohydroquinone oxidation), and hence a lowered rate of ATP synthesis is observed in 2 H 2° even when the pH gradient between the two aqueous phases is adjusted to be equal in both H 2 0 and 2H20. Taken together these proposals mean that there might be a lateral heterogeneity of pH along the inner surface of the thylakoid membrane. This heterogeneity is proposed to be more significant in 2H20 than in H 2 0 because the lateral mobility of 2H+ is less than that of H + However, according to information given by Kunst and Warman [168] this difference is only a factor of 4, at least for ice-like structures that are often taken as models for proton conduction along biological membranes. Thus given the high mobility of H + it is difficult to accept that the mobility of 2H+ is sufficiently lower than that of H + to generate the suggested lateral heterogeneity in pH. The difficulty with substituting 2H20 for H 2 0 is clearly that many factors can change, owing to, for instance, conformational changes brought about by solvent substitution. Although it has been reasonably argued that the extents of the changes observed in rates of ATP synthesis and ApH with thylakoids are greater than those usually seen as a consequence of structural perturbations, it nonetheless remains true that in a system such as a thylakoid, in which many proteins and many catalytic events contribute to photophosphorylation, it is probably difficult to make any unequivocal conclusions

I VM. Instances of failure to detect translocated protons following pulsed turnovers of mitochondrial and bacterial electron-transfer chains The direct measurement of protons translocated by an electron transfer chain is certain to be difficult. In the steady-state there will be no net changes in proton concentration to detect, whilst considerations of the probable magnitude of the electrical capacitance of membranes indicate that

87 the number of protons that must be translocated to establish membrane potentials of the order of 200 mV is very small, and possibly below the limits of detection. The latter factor has meant that translocated protons are usually detected in the presence of compensating ion movements, e.g., of K + via valinomycin, which dissipate A~k and permit a greater efflux of protons before a steady state pH gradient is established. Concern has been expressed in recent years as to whether the latter explanation is completely adequate to account for the extra protons that are detected in the presence of compensating ion movements [12]. Attempts have also been made to detect the smaller number of protons that should be translocated in the absence of compensating ion movements. For example, Archbold et al. [169] calculated the expected efflux of protons from mitochondria following an oxygen pulse on the basis of an earlier calculation by Mitchell of the specific capacitance of the inner mitochondrial membrane. They showed that their experimental procedures were sufficiently sensitive to detect the predicted number of moles of translocated protons. When an anaerobic suspension of mitochondria was pulsed with oxygen, no translocated protons were detected, and this result was taken to be inconsistent with the chemiosmotic theory [169]. A similar line of approach was taken by Conover and Azzone [170] who failed to detect translocated protons using phenol red as an indicator rather than with the glass electrode used by Archbold et al. [169]. A difficulty with both these sets of experiments is that they rely upon the estimate of the total capacitance of the inner membrane in a given preparation of mitochondria. Inevitably this is a rather approximate calculation and could plausibly be in error by a significant factor because it requires an estimate of the total surface area of the inner membrane. Whilst it has seemingly not proved possible to detect the primary electrogenic proton movement out of mitochondria, proton uptake in response to single turnovers of the cyclic electron-transfer chain of chromatophores has been detected using pH-indicating dyes [171]. Other unexpected aspects of proton translocation from bacterial cells have been reported by Gould and Cramer [172]. They found that in the

absence of charge-compensating systems the proton efflux from cells of E. coli was much slower (a factor of 10) than the rate of oxygen reduction. For this result it was suggested that the protons translocated by the electron-transport chain were not expelled into the external aqueous phase that contained the pH electrode but were sequestered in a reservoir of some kind. An important point about this argument is that it requires this reservoir to be also inaccessible from the aqueous external phase. Otherwise, this putative buffer reservoir for protons would be taken account of when the pH electrode response was calibrated by addition of known amounts of acid. In an extension of this work Gould reported that at very low concentrations (nanomolar) the protonophore FCCP actually enhanced the efflux of protons from E. coli following a burst of electron transport to oxygen [172]. Higher concentrations of FCCP, in the micromolar range, abolished the efflux of protons, just as has been observed in many other systems. Gould [173] interpreted the low concentrations of FCCP, as acting to equilibrate the postulated reservoir of protons with bulk aqueous phase. This effect of a very low concentration of FCCP was distinct from the effects of adding SCN- or K ÷ plus valinomycin, in that the latter enhanced the rates of H ÷ efflux and influx of H ÷ following return to anaerobiosis. Thus it is improbable that FCCP was catalysing a charge-compensating movement of another ion across the membrane. Certainly these observations on E. coli could be taken as evidence that protons are translocated by the respiratory chain into a special domain, and the comparison with the suggested effect of uncouplers in equilibrating such a domain in thylakoids with a bulk aqueous phase (subsection IIB). An alternative explanation for this result of Gould has been suggested (Jackson, J.B., personal communication). Measurements of membrane potential across bacterial cytoplasmic membranes have shown that in some instances supposedly anaerobic suspensions of cells are in fact receiving sufficient oxygen through gas leaks to sustain a very slow rate of respiration [174,175]. Because of the distinctly nonohmic properties of the cytoplasmic membrane [92,99] this low rate of respiration might sustain a sizeable membrane potential

88 that would nevertheless be collapsed upon titration with a very low concentration of uncoupler. If now a pulse of oxygen is added to induce a high rate of respiration then this low concentration of uncoupler will be ineffective in rapidly returning translocated protons to the cell cytoplasm. Thus an H + / O ratio can be measured. In the absence of the uncoupler the membrane potential or pH gradient can provide a driving force for rapid proton return to the cytoplasm thus giving estimates for H + / O that tend to zero. In discussing this proposal the question arises as to whether the S C N - often added to prevent build-up of membrane potential is effective. The consequence of a slow but unsuspected rate of respiration would be to drive S C N - to its electrochemical equilibrium across the plasma membrane. Hence, upon subsequently introducing a pulse of oxygen, extrusion of insufficient S C N - could result (depending on the extent, if any, of the change in membrane potential). H e n c e the membrane potential would not be fully collapsed and an underestimate for H + / O might ensue. In another type of experiment Gould and Cramer [172] measured, again in the absence of charge-compensating ions the efflux of protons from cells of E. coli at a range of milligram dry wt. of cells oxygen ratios. They reasoned that at the highest ratios of cells to oxygen the membrane potential developed should have been too small to prevent the full stoichiometric efflux of protons from the cells. But the H + / O ratio remained considerably below the limiting value of 4 that was observed in the presence of SCN-. Furthermore, the addition of a second 02 pulse immediately after the first one gave an identical stoichiometry of proton translocation. This result has been said to be contrary to expectation, because the membrane potential generated by the first burst of respiration should have prevented or at least restricted any proton translocation associated with the second pulse. Subsequently, Kell and Hitchens [176] have done similar experiments with cells of P. denitrificans and obtained results that agree with those obtained with E. coli by Gould and Cramer [172]. As discussed earlier, the problem of studying proton movement in the absence of compensating ion movements to collapse the membrane potential

is that it is necessary to estimate the electric capacitance of the entire area of cytoplasmic membrane in the sample. If this is seriously in error then the protons detected in such experiments might conceivably be unconnected with the activity of the electron transfer chain. For example, the pH changes observed could be connected with movements of substrates of other ions across the membrane. This is, of course, pure supposition, but it is of interest to compare the rate of disappearance of protons from the aqueous media surrounding bacteria following oxygen pulse experiments in the presence of absence of a high concentration of S C N - to allow charge neutralisation. The data given in Fig. 3 of the paper by Kell and Hitchens [176] serve as an example. In record b of that Figure it is shown that the protons disappear much faster following an oxygen pulse in the presence of S C N - that they do in the experiment from which SCN was omitted (record a). This is perhaps surprising because the effect of SCN should have been to dissipate A~p with an increase in A pH that would only partially compensate owing to the intra- and extracellular buffering capacities. Consequently, the total A p should have been similar or even less than its value in the absence of SCN-. Hence one might have predicted that the driving force for the return of protons from the extracellular medium to the cytoplasm was similar in the two experiments, and that therefore the rate of this return of protons would have been comparable in the two experiments. The considerably slower return in the absence of SCN thus poses a problem, and could be taken to mean that other ions are moving under these conditions. In other work Kell and Hitchens [176], in an extension of an analysis first put forward by Kell in 1979 [12], have gone on to study whether the additional proton efflux from cells of P. denitrificans that is seen in the presence of permeant ions can be quantitatively accounted for by the movement of the ion across the membrane. From studies with the tetraphenylboron anion they concluded that the molarity of the observed proton movement was as much as 20-fold greater than the movement of tetraphenylboron anions. Thus the role of tetraphenylboron was proposed to be not just simply to provide charge-compensating ion movements. It was suggested that tetraphenyl-

89 boron might cause an inhibition of the transition of hypothetical protoneural networks between their nonconducting and conducting states with the result that protons normally be confined to these networks could leak out into "the bulk aqueous phase [1761. Kell and Hitchens [177] have also studied proton uptake by chromatophores from photosynthetic bacteria following brief flashes of light. The flash frequency was varied to provide a comparable experiment to that in which the size of an oxygen pulse was varied with bacterial cells. The proton uptake into chromatophores increased monotonically with increasing flash number [177]. This observation was taken to be incompatible with chemiosmotic principles, for the same reason as suggested for the observations made upon adding variable sized oxygen pulses to anaerobic suspensions of bacteria.

IVN. Observations made with fluorescent probes Two classes of molecule have been especially used for detecting energy conservation associated with electron transport or ATP hydrolysis by submitochondrial particles or other membrane vesicles having the same orientation. 1-Anilinonaphthalene-8-sulphonate (ANS) was probably the first of these probes to be used. Its fluorescence is enhanced in an uncoupler-sensitive fashion by ATP hydrolysis or oxidation of respiratory chain substrates. The exact basis for the observed fluorescence enhancement, although most probably related to the generation of A~k, has been often disputed [178]. In the present context it is aspects of the kinetics of the fluorescence enhancement that warrant consideration. The kinetics of the ANS fluorescence enhancement in suspensions of submitochondrial particles do not depend on whether the respiratory substrate or ATP is added before or after the ANS [179]. This indicates that the observed rate of fluorescence enhancement is limited by the response of the probe and not by the rate at which the membrane potential, or other energised state, is established. The rate constant for the response of ANS fluorescence to ATP hydrolysis was not changed as the proportion of active ATPases was progressively reduced by titration with specific

covalent inhibitors, although the extent of the fluorescence enhancement was reduced in parallel [179]. This suggested that the kinetics of the ANS fluorescence enhancement were not influenced by the steady-state magnitude of A~k or other undefined delocalised membrane energisation. Yet when ITP, which was hydrolysed at approx. 50% of the rate for ATP, was used as substrate, the rate constant for the enhancement of ANS fluorescence decreased by a factor of approx. 2 [179]. But the extent of the fluorescence enhancement with ITP was the same as observed upon addition of ATP to submitochondrial particles in which 50% of the ATPases had been inactivated by covalent modification [179]. Hence the kinetics of the response of the fluorescence probe response were sensitive to local events, and not just to a delocalised membrane potential. The fluorescence of ANS can also be enhanced by application of potassium diffusion potentials to submitochondrial particles. The response in this case differs in two respects from that seen with substrates. First, the enhancement of the ANS fluorescence occurs much more rapidly than when ATP or respiratory chain substrates provide the energy source. The potassium diffusion potentials were estimated to be smaller than the membrane potentials that are generally considered to be established by respiration or ATP hydrolysis in submitochondrial particles. Thus the considerably faster rate of response of the probe to the diffusion potential cannot be correlated with a higher potential. The second feature of the response to a potassium diffusion potential that distinguishes it from the response to substrates is that whereas in the former case the fluorescence enhancement is a consequence of an increased quantum yield of ANS bound to submitochondrial particles, in the latter case the fluorescence enhancement arises from an increase in the quantity of A N S that is bound to the submitochondrial particles [179]. Thus on two counts the probe ANS does not respond to a potassium diffusion potential as if it were entirely equivalent to the potential that is accepted to be generated by electron transport or ATP hydrolysis. Although this might in principle reflect an unrecognised idiosyncrasy of the behaviour of ANS, which is certainly not fully understood, there is an interesting connection to be

90 made between these observations and the observation that ATP synthesis by alkalophilic bacteria responds differently to potassium diffusion potentials and electron transport at alkaline external pH values (Section III). Related to the kinetics of the ANS fluorescence changes might be the recent report of Skulskii et al. [180]. These authors have observed that the kinetics of efflux of permeant ions from mitochondria do not conform to their expectations for behaviour controlled by a bulk phase membrane potential. It was concluded that other factors including perhaps localised potentials had to be taken into account. Del6age et al. [181] have recently concluded that the protons involved in ATP synthesis are not delocalized into the bulk aqueous phases on the basis of the following experimental strategy. The FoF1-ATP synthase was incorporated into phospholipid vesicles. The fluorescent probe 9-amino6-chloro-2-methoxyacridine (ACMA) was used to detect the ATP-driven translocation of protons into the lumen of such vesicles. It was also shown that these vesicles would catalyse ATP ~ P~ exchange and that the higher the P~ concentration the greater the rate of incorporation of 32p~ into ATP relative to the rate of ATP hydrolysis [181]. The higher rate of resynthesis of ATP observed at higher Pi concentrations was accompanied by a smaller steady-state fluorescence quenching of ACMA fluorescence. By back extrapolation it was found that at a hypothetical point at which the rate of resynthesis of ATP equalled the rate of ATP hydrolysis the ACMA fluorescence quenching would be zero. Since ACMA fluorescence quenching was taken to be a measure of a bulk phase pH gradient, it was concluded that for maximal efficiency of ATP synthesis the bulk phase pH gradient could not be involved, and that therefore localised protons, held within the vesicle membrane, were involved in the energy transduction process [181]. However, one can suggest an alternative explanation for these findings. Conditions in which net ATP hydrolysis is exactly balanced by resynthesis of ATP cannot be achieved with vesicles containing just F0F v If the resynthesis of ATP exactly balances the hydrolysis of ATP, then no energy can be stored in any type of intermediate. Yet for molecules of FoFI-ATP synthase to synthesise ATP they must be poised by

the intermediate; otherwise thermodynamics dictates that net ATP hydrolysis must occur. Thus the arguments of Del6age et al. [181] cannot be construed as evidence for a role of localised protons. V. Conclusions

If this article has only served as a compilation (albeit doubtless incomplete) of data and arguments that are suggested to implicate localised pathways for protons in energy-transduction processes, then at least it should have presented the reader with an impression of the extent of this subject. The reader could then go on to ask himself or herself whether all the authors who have published papers in which it is concluded that localised processes, either instead of or in addition to a role for the classical delocalised protonmotive force of the chemiosmotic theory, can be wrong. As stated at the outset, one of the aims of this article was to examine whether any of the conclusions in this subject area were based on inappropriate experimentation. In this respect the article discusses a number of possible explanations for some of the data that would not require any deviation from the 'pure' chemiosmotic viewpoint. Naturally many of these explanations are hypothetical and would require further experimental scrutiny before their validity could be confirmed or denied. In other examples no such explanations are offered. This does not necessarily mean that none could be devised; lack of space and the wish to avoid repetition was the reason for their omission in some cases. It is often a problem to know exactly what is meant by the chemiosmotic theory. In an interesting analysis of statements by practitioners of bioenergetics, two sociologists [182] have noticed that the term chemiosmotic theory does not mean the same thing to all scientists. To provide a reference point in this article, the chemiosmotic theory is taken to be as described by Nicholls [183], with the sole link between electron transfer chains and ATP synthases being the proton circuit through the aqueous bulk phases. It is sometimes suggested (e.g., Skulachev in Ref. 67) that quantitative discrepancies with the predictions of the chemiosmotic theory are not very compelling reasons for doubting the validity

91 of the theory. The argument [67] is that the membranes that catalyse energy transduction are so complex, and the methods for determining the magnitude of the protonmotive force so fraught with possible errors, that quantitative measurements might be misleading. This argument, although not without some justification, is dangerous, because ultimately only quantitative and not qualitative measurements can substantiate theory. The conclusion of the present writer is that the thermodynamic discrepancies discussed in Section III must not be ignored. There is a need for further work in this area particularly with a view to resolving the basis for the differences in size of membrane potential indicated by permeant ion uptake, carotenoid band shifts and microelectrodes. There is a special need for Tedeschi's observations with microelectrodes to be confirmed or denied in a second laboratory. Some biochemists outside the immediate field of bioenergetics are puzzled why the apparent absence of a membrane potential according to microelectrodes does not create more concern amongst those working in the field of bioenergetics. A greater part of this article is concerned with the discussion of kinetic observations. This is probably the area of bioenergetics in which the biggest gaps in knowledge occur. Yet perhaps because the fundamental chemiosmotic theory is essentially concerned with thermodynamics and stoichiometric matters [183], kinetic aspects which should be of physiological and mechanistic interest have not attracted as much attention. Some of the kinetic topics discussed in Section IV are reconcilable with our definition of the chemiosmotic theory [183] provided that very small changes in Ap are responsible for many of the changes in rate observed. The present writer concludes that it is possible, but certainly not definitely proven, that this phenomenon can account for many of the published data. There is no doubt that further experimental work is required in this area. This should lead to an answer to the question of whether the results of kinetic experiments do require a link, perhaps solely for control purposes rather than for energy transduction, other than the delocalised proton circuit [183] between electron transport and ATP synthesis. At this point it is salutary to recall that ap-

parent kinetic discrepancies have led to the questioning of other generally accepted principles of biochemistry. Krebs [184] has pointed out in 1982 that to postulate, as did Bronk and Fisher [184] in 1952, an alternative to the urea cycle on the basis of some kinetic discrepancies, was far more rash and fanciful than to accept kinetic abnormalities as due to other complications when dealing with very complex systems. On the other hand, the kinetic discrepancies were based on correct observations and ultimately found their explanation in terms of permeability barriers that were not recognised at the time that Bronk and Fisher did their experiments [184]. If we accept that the chemiosmotic theory does not provide a complete description of bioenergetics then we must enquire what we might put in its place. There are several suggestions in the literature starting from the early views of Williams [185,186], and followed for example by Skulachev [187] and Westerhoff et al. [105]. Discussion of the feasibility of these schemes is outside the scope of this article, but it must be remembered that many aspects of energy transduction, especially the synergistic action of nigericin and valinomycin plus K + as an uncoupler, cannot be readily accommodated within these localised schemes. This review was largely completed by early May 1984 and its contents reflect information then available to the author. Limitations on space precluded discussion of some papers for which apologies are rendered to their authors. There have naturally been many developments since May. One of these is so closely related to a topic of this review that it warrants mention here. Van Dam and coworkers have reexamined their work concerning the relationship between Ap and AGp generated by respiring rat liver mitochondria (subsection IIIC) with the conclusion that they have identified experimental difficulties with their earlier work. Their current work indicates that decreases in Ap are accompanied by proportional decreasees in AGp, thus conforming to the expectations of the chemiosmotic hypothesis and agreeing with data obtained from experiments with brown fat mitochondria. As a final conclusion, it has been this reviewer's contention for some time [92,148] that not all aspects of energy transduction are obviously

92

accounted for by the chemiosmotic theory and that these matters should not be ignored as merely inconvenient. On the other hand, much of the evidence in favour of the chemiosmotic theory is so persuasive that equally persuasive data is needed to prompt its revision. Perhaps the present article will direct consideration to some of these aspects and generate a resolution of the question of whether localised processes are important in proton-linked energy transduction by mitochondrial, bacterial and thylakoid energy transduction. We shall see.

Acknowledgments I thank many colleagues, in particular Baz Jackson, Douglas Kell, John McCarthy, Derek Parsonage and Catia Sorgato, for many helpful discussions. Experimental work from the author's own laboratory was supported by the U.K. Science and Engineering Research council and by N.A.T.O. (Grant 1771 held jointly with Dr. M.C. Sorgato).

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