Photosynthetic cytochromes of oxygenic organisms

Biochimica etBiophysicaActa, 683 (1982) 119-151

119

Elsevier Biomedical Press

BBA 86088

PHOTOSYNTHETIC CYTOCHROMES OF OXYGENIC ORGANISMS DEREK S. BENDALL

Department of Biochemistry, University of Cambridge, Cambridge CB2 1Q W (U.K.) (Received February 4th, 1982)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119

1I, Characterization of chloroplast cytochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

120

III. Molecular properties o(chloroplast cytochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cytochromes e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cytochromes b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122 122 124

IV. Cytochrome complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

126

V.

Electron-transport pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. H + pumping by cyclic and non-cyclic electron flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mechanisms of reduction of cytochromes by quinols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The role of protein-bound quinone molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Electron flow through the cytochrome b-f complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Possible functions for cytochromes b-559 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

126 126 129 133 134 142

Vl. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145

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

146

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

146

I. Introduction The cytochrome complement of the photosynthetic apparatus of oxygenic organisms follows a remarkably consistent pattern in which the amounts of the individual components are comparable with the amounts of the photochemical reaction centres. The pattern already seems to be

Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; Chl, chlorophyll; PS, photosystem; ADRY, acceleration of the deactivation reactions of the water-splitting enzyme system Y. 0304-4173/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

established in the prokaryotic blue-green algae and the eukaryotic groups of algae and persists throughout the development of the plant kingdom. Furthermore, the relative amounts of the different components tend to remain constant under changing physiological conditions, for example, the ratio of total cytochrome b components to cytochrome f is remarkably similar in sun and shade plants [1]. One major exception is that many algae contain a soluble cytochrome c of high potential (Era, 7 +350 mV); but as will be discussed below, this component should be seen as a functional alternative to plastocyanin, and is therefore a variable addition to the normal complement. Blue-green

120

algae also contain a low-potential cytochrome c mV) [2-5] which appears to be associated with the photosynthetic membrane in vivo, although it can be removed by washing the membranes in vitro; this component is of doubtful function and may well have a respiratory rather than a photosynthetic role. An additional cytochrome b component, cytochrome b-560 (subsection IIIB), has recently been discovered in Chlamydomonas reinhardii (Bendall, D.S. and Sanguansermsri, M., unpublished observations) and there is evidence for its presence in higher plants, but its general occurrence is not yet firmly established. The universality and quantitative importance of the four membrane-bound components, i.e., cytochrome f (belonging to the cytochrome c group and occasionally referred to as cytochrome c 6) and cytochromes b-559Hp (high-potential component), b-559ep (low-potential component) and b-563, suggest that they all have fundamental roles to play in photosynthesis, but these functions have been remarkably difficult to establish. The discussion of electron-transport pathways in this review, therefore, must inevitably be tentative, but nevertheless two general formative ideas have emerged in recent years. The first is that, as with mitochondrial cytochromes, photosynthetic cytochromes occur in the membrane complexed with other proteins, and that these complexes are the functional units. A new procedure for isolation of an active cytochrome b-f complex has become available, but much remains to be done, especially by comparison with studies on the cytochrome b-c~ complex from mitochondria. The second concerns the mechanisms by which the protein complexes react with quinones and the possible involvements of a proton-motive Q-cycle, or variants thereof, in chloroplast electron transport; this aspect will feature largely in the discussion of cytochrome function which follows. All four components are rather firmly bound to the thylakoid membrane, although not all should necessarily be regarded as integral membrane proteins. Cytochrome f can be solubilized by extraction with organic solvents [6]. Further purification has in the past been hampered by a marked tendency to aggregate, but successful procedures have now been published [7-9] and, in particular, the (Era, 7 ~ - - 2 6 0

discovery that the protein can be extracted from certain species in monomeric form has effectively opened the way to detailed chemical and physical studies of the molecule [10-13]. The cytochromes b are exceptionally difficult to bring into solution as single proteins and often require extraction with a mixture of Triton X-100 and urea [14-17]; there is still considerable uncertainty about their properties. The first matter that requires discussion is the method of distinguishing between individual components, as this cannot yet be done unequivocally on the basis of purification studies and the claim made above that at least four components can be distinguished has not met with universal acceptance.

II. Characterization of chloroplast cytochromes The individual components are mainly distinguished by their absorption spectra, especially the positions of the a-peaks of the reduced form, and by their oxidation-reduction potentials, but neither criterion is adequate by itself. Redox poising can be used to characterize two groups of component with mid-point potentials separated by 300 mV or more. Cytochromes f and b-559Hp both have mid-point potentials close to + 370 mV, but may be distinguished by their a-peaks at 554 and 559 nm, respectively, and by the fact that various treatments, such as exposure to detergents, selectively lower the potential of cytochrome b-559Hp. The difficulties surround the low-potential group of cytochromes b. First there is a problem of nomenclature. In early work only one cytochrome b component, termed cytochrome b6, was thought to occur in chloroplasts [18]. As there is now general agreement that fresh chloroplast preparations contain two or more, it seems best to regard cytochrome b6 as an obsolete term referring to a mixture, although the literature on light-induced redox changes can usually be understood by reading b-563 for b6. Doubts about the nature of the low-potential components expressed in the literature concern two questions in particular: (a) is there a low-potential cytochrome b-559 in chloroplasts in vivo, and (b) does cytochrome b-563 possess an ~x-band that splits into peaks at 561 and 557 nm at the temperature of liquid nitrogen (77 K)

121

[19]? A further factor has now been introduced with the discovery of cytochrome b-560, which has a mid-point potential (Era, 7 = - 7 0 mV) close to that of cytochrome b-563. The latter remains the dominant component, however, as it is present at about twice the concentration of the former. According to some reports [20] the proportion of the total cytochrome b-559 in the high-potential form ( + 370 mV) may be as high as 90%, but this figure is probably an overestimate as it is based mainly on reduction by ascorbate, which gives incomplete reduction of the low-potential component. Although the latter can be observed without interference from other cytochromes in a difference spectrum of samples reduced under anaerobic conditions at pH 6, one with menadiol (giving E h -~ - - l 0 mV) and the other with duroquinol (giving E h ~ + 6 0 mV) [21], its complete reduction cannot be achieved without significant reduction of cytochromes b-563 and b-560, so redox poising is not the ideal analytical technique. For analytical purposes it is often preferable to take advantage of the different rates of reduction by hydrophilic reagents. One scarcely used possibility might be to use ferro-oxalate which can generate a lower potential (Era, 7 = 0 to +60 mV, depending on phosphate concentration [22,23]) than with menadiol without rapid reduction of cytochrome b-563 [24]. The alternative reductant is dithionite. Cytochrome b-559Lp is completely reduced within 20 s whereas cytochrome b-563 is reduced much more slowly (as is cytochrome b560), the rate depending on the dithionite concentration. A difference spectrum between samples reduced by dithionite for 20s and by hydroquinone shows cytochrome b-559Lp quantitatively with a little contamination from the cytochrome b-563 and b-560 components which can be corrected by following the time course of reduction of the latter and extrapolation to zero time [25]. All normal chloroplast preparations, including preparations that are well coupled or are capable of fixing CO 2, h a v e shown substantial amounts of cytochrome b-559Lp when examined by the above methods. These results support the view that chloroplasts in vivo normally contain a low-potential cytochrome b-559 (Em ~- + 2 0 mV) in addition to the high-potential component. Some authors have viewed the low-potential

component as a modified form of cytochrome b-559Hp, arising either as a result of damage during isolation or as a normal physiological variant [26,27]. Damage during isolation doubtless can occur, apparently increasing the complement of cytochrome b-559Lp, but probably not in chloroplasts freshly prepared by gentle techniques from leaves such as spinach, pea or lettuce, and once the chloroplasts have been isolated cytochrome b559Hp declines only slightly over a period of several hours at 0°C. Some of the modified forms have potentials in the range +55 to + I l0 mV [21,2830], distinctly higher than that of cytochrome b559Lp of fresh chloroplasts [21], but these may vary depending on the treatment and even on the species. If the two cytochromes are natural variants of each other they must both be products of the same gene. Evidence has recently been obtained that their synthesis is under separate genetic control in Chlamydomonas (Sanguansermsri, M. and Bendall, D.S., unpublished observations), but this is not conclusive unless the structural genes, rather than separate genes controlling their expression, can be shown to be concerned. Only one cytochrome b-559 has so far been purified [15,17,31,32]; although it is obtained in a low-potential form it may well have been derived from the high-potential component modified by the drastic conditions necessary for extraction [32]. Separate origins for the two cytochromes b-559 of intact chloroplasts are favoured by their apparent functional diversity, and is strongly suggested by differences in response to detergent or mechanical fractionation. For example, when chloroplasts are fractionated with digitonin, cytochrome b-559Hp remains associated with Photosystem (PS) II, whereas a lowpotential cytochrome b-559 fractionates with cytochrome f, either following P S I (D-144 particles) [33], or being obtained in a distinct cytochrome bf particle [24,34]. The possibility that the cytochrome b-559 of the digitonin-cytochrome complex is derived not from the cytochrome b-559ep of intact chloroplasts but by modification of cytochrome b-563 by the detergent is rendered unlikely by the fact that the proportions b-563 : f: b-559Lp are similar in the two preparations [34]. The association between cytochrome b-559Lp and cytochrome f is confirmed by their similar behaviour when chloroplasts are fragmented

122

mechanically [35,35a]. It appears to be loose, however, because solubilisation of the cytochrome b-f complex by a cholate/octylglucoside mixture leaves cytochrome b-559Lp behind in the membrane residue [36]. The solubilized complex retains plastoquinol-plastocyanin oxidoreductase activity. The possibility that cytochrome b-563 in its native state might possess a complex a-band would be adequate, a priori, to explain the occurrence of double peaks at 561 and 557 nm at 77K in samples reduced with dithionite [33], but the 557 nm peak might alternatively represent either cytochrome b-559Lp or cytochrome b-560. The best available low-temperature spectra of cytochrome b-563 have been obtained with detergent-solubilized preparations of the cytochrome b-f complex under conditions which extract little or no cytochrome b-560. With digitonin preparations, which 563

A

?

I

4:005

554

IA=0005

contain cytochrome b-559[,p, the difference spectrum of the component slowly reduced by dithionite was taken to represent cytochrome b-563 [24,37]; with p r e p a r a t i o n s solubilized by cholate/octylglucoside the difference spectrum of dithionite against ascorbate was taken [36] (Fig. 1). Both spectra showed a single peak at 561 nm with a slight shoulder in the region of 557 nm. Such preparations are catalytically active and the cytochrome does not appear to have undergone significant spectral modification as a result of the detergent treatment. The report of a double a-band in a purified preparation of cytochrome 'b 6' [14] must presumably be taken to mean that the preparation contains two components or that it is contaminated with a modified cytochrome b. A satisfactory analytical procedure that distinguishes cytochromes b-560 and b-563 has still to be devised, but will become possible when definitive spectra of the separate components are available. Qualitatively the presence of cytochrome b-560 may be detected by a slight shift in the peak of cytochrome b-563 towards shorter wavelengths and a broadening of its a-band.

111. Molecular properties of chloroplast cytochromcs

1 550

I

400

450

500

600

650

700

Wavelength (nm] 5608 S~/Asc

Asc/FeCy 5514

I

I

A=O02

f

500

i i , i I , , , lJ, 500 550 Wavelength (nm)

,AI,~,

550

Fig. 1. Spectra of the purified c y t o c h r o m e b-f complex. (A) A b s o l u t e s p e c t r u m in the presence of dithionite (lower curve) a n d difference spectra ( u p p e r curves) as indicated, r o o m temp e r a t u r e ; (B) difference spectra, 77 K. R e p r o d u c e d f r o m Ref. 36 with permission.

IliA. Cytochromes c The only cytochrome c-type component known to occur in higher-plant chloroplasts is cytochrome f. Hill and Scarisbrick [38] first described it and pointed out that it differed from mitochondrial cytochrome c in having a markedly asymmetric c~-band shifted 4-5 nm towards the red, and a characteristic oxidation-reduction potential 100 mV more positive. Davenport and Hill [6] extracted c y t o c h r o m e f from parsley leaves by grinding them with ethanol and Forti et al. [39] recommended the inclusion of 15 Triton X-100 in the solvent to improve the reproducibility of extraction, especially when a blender is used for homogenization. This method failed when applied to isolated chloroplast preparations and gave poor yields with leaves of other species. Davenport [25] developed a method of extraction with a mixture of ethyl acetate, ethanol and ammonia that could be applied to chloroplast preparations from a wide variety of species. It gives variable and often poor

123 yields, but Ho and Krogmann [40] have used a modified form of it to purify cytochrome f to homogeneity as an octamer from spinach. Singh and Wasserman [7] developed a method in which spinach chloroplasts were extracted with butanol and then sonicated in the presence of Triton X-100; Gray [9] extracted cytochrome f from ethanolwashed tobacco chloroplasts with a mixture of Triton X-100 and urea [15]. In both cases a colourless polypeptide copurified with cytochrome f, although in the latter case it could be separated after denaturation of the cytochrome with sodium dodecyl sulphate (SDS). Following its introduction by Matsuzaki et al. [11], the most general method of extraction, which may be applied to a wide variety of species, is to homogenize leaves in the presence of butan-2-one [13]. A major difficulty in purification of the cytochrome arises from its tendency to aggregate. The oligomeric protein (usually an octamer) is not readily purified in the absence of detergent. The discovery that cytochrome f could be extracted in monomeric form from Brassica komatsuna [11] and Raphanus sativus [10], and then purified by standard methods with little loss, represented a significant advance. Subsequently, a survey of butanone extracts of leaves from a variety of plants (Gray, J.C., personal communication) showed that cytochrome f was obtained in monomeric form from several members of the family Cruciferae. In addition, the common cereals, oats (Avena sativa), barley ( Hordeum vulgare) and wheat (Triticum aestivum), and also to a lesser extent parsley (Petroselinum crispum), gave fairly high proportions of monomers, but the cytochrome from spinach (Spinacea oleracea), beet (Beta vulgaris), pea ( Pisum sativum), tobacco ( Nicotiana tabacum ) and maize (Zea mays) is normally extracted entirely in aggregated form. These variations seem to depend partly on the properties of the proteins themselves and partly on the effects of polyphenols. A remarkable feature is that the relative molecular masses vary over quite a wide range. Measurements on purified samples from a number of species by polyacrylamide gel electrophoresis in the presence of SDS under identical conditions (Gray, J.C., personal communication) have given values ranging from 28500 for charlock (Sinapis arvensis), rape (Brassica napus) and turnip (Bras-

sica rapa ssp. rapa) to 37300 for pea ( P. sativum). In the case of charlock similar values are obtained by sedimentation equilibrium (27700) and amino acid analysis (27000) but gel filtration gives a significantly higher value (33900) [13]. The proteins with the higher relative molecular masses are not necessarily those that aggregate, e.g., barley (34800) is largely monomeric but tobacco and spinach (32700) are almost entirely oligomeric. The above variations are unlikely to be explicable solely as the result of proteolysis but proteolytic activity nevertheless remains a serious danger and in some cases seems to occur very rapidly during the initial extraction with organic solvent or detergent. There is evidence that in several species a small peptide of M r 1000-2000 may be removed by proteolysis (Gray, J.C., personal communication); phenylmethylsulphonyl fluoride is not an effective inhibitor. The oxidation-reduction potential of cytochrome f is independent of pH between pH 6 and 8 with a mid-point potential of +365 mV [6]. At higher pH values (pK 8.4) E m declines at the rate of 60 m V / p H unit. At physiological pH values the cytochrome therefore behaves formally as an electron acceptor while at alkaline pH it would be a hydrogen-atom acceptor in its predominant form. This is a description of the overall reaction and not necessarily of the actual mechanism (see subsection VB). The mid-point potential of mammalian cytochrome c shows a qualitatively similar pH profile with a pK at about 8.9 [41]. The same pK is shown by a weak absorbance band of the oxidized form at 695 nm, which disappears as the methionine ligand is displaced at alkaline pH [42]. Kinetic studies have shown that the pK is only apparent and represents the product of a true pK at 11 and the equilibrium constant of a slow conformational change of the deprotonated form [43,43a]. The redox pK of cytochrome f has not been interpreted but there is no 695 nm band and so presumably methionine is not a ligand [13,44]. Values of the redox potential of c y t o c h r o m e f in chloroplasts [28,45-48] and in isolated protein complexes [21,49,50] do not differ greatly from those originally reported [6] for the soluble component, although good values are difficult to obtain with thylakoid preparations because of the dominating presence of cytochrome b-559np. Cy-

124 tochrome f in a catalytically active preparation of the cytochrome b-f complex has a pH profile similar to that of the soluble component [21]. These observations lead to the important general conclusion that the equilibrium redox properties of the cytochrome are not significantly different whether it is bound to the membrane or free in solution. Cytochrome f is an acidic protein with an isoelectric point in the region of pH 5 [6,13,44]. The a m i n o acid c o m p o s i t i o n of the protein [9,13,15,40,51,52] refects this fact, and also shows, a little surprisingly, no marked degree of hydrophobicity: the sum of proline, alanine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan and cysteine varies from 41 to 45% in different higher-plant species. The sequence of an N-terminal decapeptide has recently been reported [40] and rapid progress can now be expected in sequencing both the protein and the gene. Prokaryotic and eukaryotic algae, and also Euglena, frequently contain a soluble acidic c-type cytochrome similar to higher-plant cytochrome f in spectrum and redox potential. This component was discovered nearly 50 years ago by Yakushiji [53], but only recently has its role become clear. It was often referred to as cytochrome f although its relative molecular mass is in the range 1000013500 and it is freely soluble. On the other hand, it seemed functionally similar to plastocyanin, donating directly to P-700 [54-57]. A membranebound c-type cytochrome had been reported in several green algae [58-60] and in the blue-green alga Phormidium [2]. Wood [61,62] demonstrated the presence in a wide range of algal species (Chlamydomonas, Euglena, Anacystis) of a membrane-bound cytochrome analogous in molecular properties and function to higher-plant cytochrome f and formulated clearly the distinction between the true algal cytochrome f and the soluble component, which is more appropriately named cytochrome c-552 or c-553 according to the position of its a-band. Cytochrome c-553 was shown to be functionally equivalent to plastocyanin and in green algae the two proteins are interchangeable, plastocyanin synthesis occurring preferentially unless copper is unavailable in the growth medium [63-66]. A similar exchangeability of plastocyanin and cytochrome c-553 occurs in the blue-green algae Anabaena variabilis and Plec-

tonema boryanum [67]. Wood's recommendation will be followed here and the term cytochrome f reserved for the membrane-bound component. Cytochrome f purified from the green algae, C. reinhardii [62], Bryopsis maxima [68] and Scenedesmus acutus [44], and also from Euglena gracilis [62], has been shown to have properties closely similar to those of the higher-plant protein. The protein from blue-green algae is significantly different ]40,62,69,70], with an a-band shifted to about 557 nm and an appreciably lower redox potential (reported values are +318 mV [70] or + 339 mV [40]) which is pH independent, at least up to pH 10 [70]. The protein is nevertheless of similar size and amino acid composition with an N-terminal sequence remarkably similar to that of spinach [40]. Cytochrome c-553 has been purified from a large number of algal species and its general properties are well known [71,72]. Although in spectrum and redox potential it is remarkably similar to cytochrome f, there are differences in detail, as recorded in Tablel, and the differences in size, reactivity with plastocyanin and nature of the haem ligand make it clear that we are dealing with two quite distinct classes of protein. Amino acid sequences of cytochromes c-553 [73-77a] show just enough similarity with those of mitochondrial cytochrome c and cytochrome c 2 of purple photosynthetic bacteria to suggest a common evolutionary origin [73,74,76-78]. The latter two cytochromes show the same characteristic 'cytochrome fold' [78] but the tertiary structure of cytochrome c-553 has yet to be determined. Homology would be expected between cytochromes f and c~, but not a close evolutionary relationship. They have about the same molecular weights but the amino acid compositions of charlock cytochrome f [13] and yeast cytochrome c t [79] show no significant homology when analysed by the method of CornishBowden [80]. HIB. Cytochromes b Characterization of the molecular properties of chloroplast b-type cytochromes still presents considerable difficulties and an unambiguous account cannot yet be given of any single component. Cytochromes b-559up and b-563, at least, occur in the membrane as parts of multi-component pro-

125 TABLE I C O M P A R I S O N OF C Y T O C H R O M E f A N D SOLUBLE C Y T O C H R O M E c-553 F R O M G R E E N A L G A E Data from Refs. 44, 58 and 62.

Reduced spectrum (peak positions, nm) R o o m temperature a

77 K

Y 8 a

Chlam.vdomonas reinhardii

Scenedesrnus acutus

Cytochrome f

Cytochrome f

554 523 421

Cytochrome c-553

552.5 522.5 416.5

553 531.5 421.5 331 551,549

Cytochrome c-553

553 523 416 318 552, 548

absent

present

31000

12000

33000

10000

Redox potential, Era,7 (mV) pH dependence of E m

+ 350

+ 370

+ 380 - 6 0 m V / p H unit above pH 8 (pK 9.2)

Reactivity with parsley plastocyanin ( M - l . s 1)

k > 9-106

k:9.104

Mutual reactivity (M l,s i)

k > 107

Oxidized spectrum, 692 n m peak Relative molecular mass

5.1

Isoelectric pH

tein complexes and they can only be separated by drastic procedures that may denature them or significantly modify their properties. There are indications that cytochromes b-559Le and b-560 might be more easily obtained as discrete polypeptides. Purified preparations of both cytochrome b-563 [14,16,81] and b-559 [15,17,31,32,82] have been reported. The cytochrome b-559 was obtained in a low-potential form; the usual assumption that it originates in the high-potential component of the membrane remains a speculation, although Lach and B6ger [17] reported that a high-potential form persisted for a short time after solubilisation. The most serious difficulty with the purification of these cytochromes is probably proteolysis, for which a satisfactory means of control has not yet been found. Stuart and Wasserman [14,81] purified cytochrome b-563 after extracting ethanol-washed spinach chloroplasts with 2% Triton X-100 containing 4 M urea. Their final preparation ran as a single band on analytical gel electrophoresis in the presence of Triton X-100 and contained 1 mol haem/40000 g protein, but electrophoresis in the

3.7 + 380 - 6 0 m V / p H unit above pH 8 (pK 8.6)

presence of SDS showed that it was a complex of a large polypeptide of 20 kDa with two smaller polypeptides (9.6 and 6.6 kDa) in the proportion 1 : 1:2. The large polypeptide probably contained the haem because a form of the cytochrome containing a single polypeptide of 18 kDa has been obtained by a modified procedure [16] and preparations of the cytochrome b-f complex contain a polypeptide of about 20 kDa that stains with tetramethylbenzidine and H202 and which has been identified with cytochrome b-563 [36,83]. The redox potential of Stuart and Wasserman's preparation ( E m , 7 z --80 to - - 1 2 0 mV) is similar to that reported more recently for cytochrome b-563 in chloroplasts [21], but the purified material has a broad a-band at room temperature which splits into two well defined peaks at 557 and 561 nm at the temperature of liquid nitrogen whereas it is now clear that the membrane component has a sharp c~-band giving only a single 561 nm peak at low temperature. Cytochrome b-559 has been purified by a procedure [15,17,31,32,82] similar to that developed for cytochrome b-563 except that stability required the

126 inclusion in all media of dithiothreitol, which led to the destruction of cytochrome b-563. The purified cytochrome b-559 obtained by Garewal and Wasserman contained l mol h a e m / 4 5 9 0 0 g protein and on gel electrophoresis it ran as a single band of high M r in the presence of Triton X-100 and a single band of 5.6 kDa in SDS. These results suggest the possibility of haem loss or proteolysis, or both, during purification. Lach and B6ger's preparations from spinach and the alga Bumilleriopsis filiformis contained single polypeptides of 37 and 17 kDa, respectively, in SDS; these results are in marked contrast to those of Garewal and Wasserman and such a large difference between a higher-plant and an algal protein would not be expected. Some of the discrepancies might be explained by the purification of different proteins. Obviously more work must be done. Cytochrome b-560 has not yet been purified.

IV. Cytochrome complexes Cytochromes f, b-563 and b-559Hp occur in the thylakoid membrane as parts of larger protein complexes which are likely to be the true functional units. This concept has long been accepted for mitochondria and the work of Wessels [84,85] and Nelson and Neumann [49] established the existence of a cytochrome b-f complex in chloroplasts, but only recently has its detailed characterization been undertaken. Cytochrome b-559Hp is present in reaction centre preparations of PS II [86-88] which will not be discussed here. The crucial steps that have facilitated recent progress with the cytochrome b-f complex were firstly the discovery of a catalytic activity (plastoquinol-1plastocyanin oxidoreductase) characteristic of the complex [34] and secondly solubilization of the particle with a mixture of cholate and octylglucoside, which gives a much cleaner preparation than the original digitonin method [36]. The purified cytochrome b-f complex of Hurt and Hauska [36] appears to contain 1 mol cytochrome f, 2 mol cytochrome b-563, 1 tool Rieske Fe-S centre and 142000g protein per monomer. Polyacrylamide gel electrophoresis with SDS revealed five major polypeptides of 34, 33, 23.5, 20 and 17.5 kDa, the first three of which stained for haem by the method of Thomas et al. [89]. The

two largest polypeptides can probably be identified with cytochrome f, and the presence of a doublet suggests that the particle has been exposed to proteolytic action. The 23.5 kDa polypeptide probably represents cytochrome b-563. The complex tends to disintegrate with prolonged exposure to a high concentration of Triton X-100, leading to splitting out of the Rieske protein [90] and a lowering of the cytochrome b / f ratio. The Rieske protein has been purified from the complex in this way [90] and can be unambiguously identified with the 20 kDa polypeptide. The 17.5 kDa polypeptide is unidentified. Thus, the cytochrome b-f complex, when compared with mitochondrial Complex Ill, possesses a similar complement of redox components but lacks the large core proteins [91,92]. The plastoquinol-plastocyanin oxidoreductase activity of the cytochrome b-f complex is destroyed by Triton X-100 [34]. The primary effect is probably due to removal of lipid rather than loss of the Rieske protein because addition of lipid can protect to some extent [36]. The purified preparation may retain up to 30-50% of the original activity on a cytochrome f basis. Cruder preparations that have not been exposed to Triton X-100 usually retain full activity and can be used to study functional behaviour of the cytochromes (Section V) and to characterize redox components by EPR [93-95]. The following identifications have been made by EPR with concentrated digitonin preparations: (a) ferricytochrome f as g= 3.5; (b) the reduced Rieske centre as g~. 1.89; and more tentatively, (c) ferricytochrome b-563 with a high-spin haem signal around g6; and (d) ferricytochrome b-559Lp with g= 2.95. The assignment of the highspin signal is particularly uncertain because a considerably larger signal would be expected from the amount of cytochrome b-563 determined optically [95] (possibly because of spin coupling or the existence of an equilibrium with an unobserved low-spin form) and the unsatisfactory redox titration [931.

V. Electron-transport pathways VA. H + pumping b)' cyclic and non
127 electron carriers have been pursued with renewed vigour of late, the overall pattern of pathways in which these components participate requires some clarification. For this purpose measurements of photophosphorylation and of light-induced H ~ pumping by chloroplasts have an important part to play. To drive the Calvin cycle the electrontransport system must be able to generate ATP and N A D P H in the ratio 1.5:1, but there are additional requirements for ATP and probably for N A D P H also. Moreover, higher A T P / N A D P H ratios are required for CO 2 fixation in C 4 plants. The photosynthetic electron-transport system must therefore generate ATP and N A D P H in variable proportions, depending on the tissue and physiological conditions. Some comment on the way this might be achieved is necessary here. Although lower ratios have sometimes been reported [96], the most probable whole-number value of the H + / A T P ratio is 3 [97,98], and the widely accepted value of 2 for H + / e would give A T P / N A D P H = 1.33. So the problem is one of under-production rather than over-production of ATP. There is considerable experimental support for each of three distinct mechanisms for boosting ATP production, i.e., cyclic photophosphorylation, pseudocyclic photophosphorylation and an additional H+-translocating step linked to electron transport. Clear-cut evidence for a role of cyclic phosphorylation in normal CO 2 fixation has been difficult to obtain. Although there is good evidence that cyclic phosphorylation can provide ATP for use outside the chloroplast [99], the maximum rate of such a process is probably low and may not be commensurate with the additional ATP needed to drive CO 2 fixation. If we take a figure of 1.33 for the P / 2 e ratio in non-cyclic phosphorylation and a rate of CO 2 fixation of 300/~mol/mg Chl per h, an additional ATP requirement of 100 ~ m o l / m g Chl per h can be calculated. Much lower rates of ferredoxin-dependent cyclic phosphorylation by isolated chloroplasts have frequently been reported. However, two groups have recently reported rates as high as 178 [100] and 300 [101] ~mol A T P / m g Chl per h in the presence of concentrations of DCMU that would seem to preclude any non-cyclic or pseudocyclic contribution. Huber and Edwards [102-104] have used pyru-

vate-dependent CO 2 fixation by intact mesophyll chloroplasts from the C4 plant Digitaria under anaerobic conditions as a measure of cyclic phosphorylation. In this system ATP is required for phosphorylation of pyruvate by pyruvate, P~ dikinase and CO 2 is fixed by added phosphoenolpyruvate carboxylase. A maximum rate of ATP synthesis of 160 /~mol A T P / m g Chl per h was calculated [104] although there may have been some contribution from non-cyclic phosphorylation coupled to oxaloacetate reduction. A much used criterion for the presence of ferredoxin-dependent cyclic phosphorylation is inhibition by antimycin, but caution is necessary since antimycin uncouples at higher concentrations. Drechsler et al. [105] showed that the uncoupling effect on chloroplasts increases as the pH is lowered below 7.5, as would be expected from the pK of 5.1 for antimycin's weak acid function [105a]. Hind and colleagues [106] have considered 1 #M antimycin to act as a selective inhibitor of cyclic electron flow, such a concentration having no apparent uncoupling effect on non-cyclic phosphorylation in broken spinach chloroplasts. On this basis a contribution of cyclic phosphorylation to CO 2 fixation at high light intensities has been deduced [107]. In the case of site I phosphorylation by submitochondrial particles, Haas [108] observed the uncoupling effect to be already significant at 1/~M. Of course, the actual concentration of antimycin required for any given effect will depend on the nature and concentration of the particle preparation being used and on other conditions, particularly pH. Huber and Edwards [104] suggested that, in contrast to the situation with mitochondria and purple bacteria, the antimycin sensitivity of chloroplasts depends on redox poise. For example, they found that pyruvate-dependent CO 2 fixation by intact mesophyll chloroplasts from Dig#aria under far-red light was inhibited strongly by DCMU but only weakly by antimycin, whereas when ascorbate was present the effects of the two inhibitors were reversed. A further complication is that a substantial rate of electron flow might still occur when all inhibitor-binding sites are occupied. A slow antimycin leak occurs in mitochondria and a larger leak in chloroplasts would help to explain the variability in the reported sensitivity of cyclic phosphorylation. While

128 the mode of action of antimycin on chloroplasts is as poorly understood as it is at present it may be unwise to rely too heavily on its action being completely selective. Certainly, at concentrations of 10 I~M and above antimycin can inhibit noncyclic electron transport, especially at p H > 7 . 5 [105]. Less ambiguous information is available about pseudocyclic photophosphorylation. We assume that the electron-transport pathway and the P / 2 e ratio are the same as under normal non-cyclic conditions except that O z has replaced N A D P as the terminal electron acceptor (Mehler reaction). 02 uptake can be measured as ~80z with a mass spectrometer [107,109]. Additional criteria are the sensitivity of the pseudocyclic system to 02 concentration [110] and with isolated intact chloroplasts, which lack catalase, formation of H202 [109]. Light-induced 02 uptake as a result of the oxygenase activity of ribulosebisph'osphate carboxylase and glycollic oxidase can be minimised at high CO z concentrations. Pseudocyclic phosphorylation can occur at a high rate. Radmer et al. [111,112] reported that during the induction phase of whole algal cells after a period of darkness, O z uptake was only a little slower than the steady-state rate of O z evolution; the rate then fell as CO z fixation got under way so that 02 and CO 2 seemed to be in direct competition for reducing power. Mitchell [113-115] was quick to suggest that his idea of a proton-motive Q-cycle for the cytochrome bc t region of the mitochondrial respiratory chain might be extended to the cytochrome b f region of the chloroplast system. The key feature of the Q-cycle is that an additional electrogenic reaction (possibly transmembrane electron transfer through b-type cytochromes) allows the uptake of one extra proton by plastoquinone for each electron transferred to P-700. The H + / e - ratio for non-cyclic electron flow would then become 3. Most direct measurements of this ratio have in fact yielded a value of 2, but Fowler and Kok [1161 reported a ratio of 3 during flash excitation with ferricyanide as acceptor while under continuous illumination the ratio fell from 3 at low intensity to 2 at high intensity. Although Saphon and Crofts [117] raised serious doubts about some of Fowler and Kok's assumptions, ratios higher than 2 have

recently been confirmed [98,118,119]. The experiments of Fowler and Kok raised the interesting possibility of a facultative Q-cycle, i.e., the additional electrogenic step might be switched on or off, depending on conditions. Enhanced H + / e ratios might result not only from a Q-cycle but also from a conformational H ~ pump, as suggested for mitochondria by Papa [120] and more recently in the 'b-cycle' of WikstrOm et al. [121,122], or indeed from an additional vectorial redox loop such as has been favoured for the purple photosynthetic bacteria by Crofts et al. [123,124]. Direct evidence for a conformational H + pump is difficult to obtain and there is no clear experimental support for the insertion of an extra redox loop into a linear sequence between the two photosystems. On the other hand, it has been argued that the electrogenic reaction of a Q-cycle can be detected directly by observation of the electrochromic effect on the thylakoid membrane pigments. Witt [125] developed the idea that the light-induced absorption changes with a peak in the region 515-520 nm and a trough at about 480 nm, which Duysens [126] observed in Chlorella and green plants, could be explained as an electrochromic shift in the absorption bands of thylakoid pigments in response to the charge separation of the primary photochemical reactions. The effect had a rise time of less than 20 ns [127] and received contributions from both photosystems. Joliot and Delosme [128] discovered an additional slow phase (previously reported also by Witt and Moraw [129]) in Chlorella with a rise time of 20-30 ms depending on the degree of dark adaptation. The spectrum of the slow phase (which Joliot and Delosme termed 'phase b') was very similar to that of the rapid rise (phase a), but differed in that it seemed to be associated with P S I only. Joliot and Delosme considered two types of explanation for phase b. In the first the full amplitude of the electrochromic effect is not developed as a result of the charge separations in the primary photochemical reactions but depends on the delocalisation of the separated charges as free ions in the two aqueous phases; this would be associated with electron transfer between the two photosysterns and H * release to the inner thylakoid space. The second kind of explanation, favoured by most authors, invokes the transfer of an additional

129

charge through the membrane, either an electron moving outwards or a cation inwards. In a Q-cycle the onset of phase b would be associated with the outward transfer of an electron which would be followed by the inward transfer of an additional H + via plastoquinol. According to this interpretation phase b would indicate an H + / e - ratio of 3 for a non-cyclic system or 2 for a cyclic one. An interesting feature, at least in algal cells, is that phase b disappears after about six actinic flashes spaced not too far apart [128,130], possibly because the electrogenic process cannot occur against a membrane potential. This suggests that the extra redox loop of the Q-cycle would only operate very briefly at the onset of illumination or during the steady state at low light intensity. The latter would be consistent with the measurements of Fowler and Kok referred to above, but would be in contrast to the situation in mitochondria in which a Q-cycle has been considered to function in the steady state. Hind et al. [131-133] have taken the view that in chloroplasts phase b can only be detected under conditions in which cyclic electron flow around P S I is occurring, even though it appears to be rather insensitive to antimycin. Velthuys [118,119] has observed phase b, which tends to be masked by the decay of phase a, in the presence of ferricyanide or methyl viologen, which do not favour cyclic flow, but a compete cycle may not be necessary for phase b to be seen on the first one or two flashes of a series [134]. Bouges-Bocquet [135] has reported binary oscillations in the amplitude of phase b in Chlorella which are suppressed by D C M U , suggesting that phase b is powered by electrons from PS II via the two-electron gate which governs the reduction of the plastoquinone pool. In contrast to the above, Olsen et al. [136,137] have questioned whether phase b is symptomatic of an additional electrogenic process. A large phase b was detected with similar kinetics to those of H + release to the thylakoid interior, but clear evidence for involvement of an extra H + was not obtained. They developed a model of phase b which in general terms was consistent with the first type of explanation offered by Joliot and Delosme. Olsen et al. [136,137] have raised questions about the interpretation of the slow electrochromic effect that are worthy of serious consideration.

Nevertheless, their experimental evidence against the extra H + is negative in character and stoicheiometries are notoriously difficult to establish. I consider that on the whole the experimental evidence, though patchy, favours the existence in chloroplasts under some circumstances of an electrogenic reaction associated with movement of an extra H + . Such a position allows interesting comparisons to be made (although space does not allow it here) with the cytochrome bq region of mitochondria and chromatophores for which the stoicheiometries are more firmly established. I will assume that phase b forms a useful indicator in chloroplasts of this additional electrogenic process.

VB. Mechanisms of reduction of cytochromes by quinols There is general agreement that quinols play a crucial role in the reduction of cytochromes in vivo. We now consider the physical chemistry of such reactions in simple model systems: such considerations are essential for understanding what happens in the thylakoid membrane, but in this case additional complicating factors must be taken into account. It is worth pointing out that the reduction of cytochrome f in chloroplasts is first order with respect to both [ferricytochrome f ] and [plastoquinol] [138-140] and hence model reactions that are treated as bimolecular collision processes have direct relevance. The most immediately obvious problem is that the haem group normally behaves as a one-equivalent redox system whereas quinols in solution are

Protonotlon

)

O

~

OH*,

' OH~ ÷

Q:

- - ~

OH- .

' QH'~

Electron Reduction

"•-atom

'~eduction

0 2. . F i g . 2. P o s s i b l e

redo×

~ OH- . and protonation

' QH 2 states o f q u i n o n e s

in

solution. E a c h r e a c t i o n s h o w n involves a d d i t i o n or r e m o v a l of o n e electron, o n e p r o t o n or o n e h y d r o g e n a t o m . R e d r a w n f r o m Ref. 144.

130 TABLE II ESTIMATED E o VALUES FOR COUPLES OF PLASTOQ U I N O N E - I IN ETHANOL Sources of data are given in Ref. 141. Couple

n

stable, as shown by the value of 3.3 for the semiquinone formation constant Ki, i.e., the equilibrium constant of the following reaction: Q+Q2

~-Q; + Q -

(l)

E 0 (mV)

Electron transfers Q/QQ./Q2 Q/Q2 QH'/QH

1 1 2 1

- 165 - 196 - 181 +239

H-atom transfers Q/QH" QT/QH Q/QH QH'/QH 2 Q/QH 2

1 1 2 1 2

+ 190 +575 +383 + 870 + 530

two-equivalent donors. This inevitably n~eans that semiquinones are involved in the reaction mechanism. Formally, we may consider there to be nine possible species in the quinol/quinone system when both redox state and protonation state are taken into account (Fig. 2), but in practice the six contained within the triangle QQ2 QH 2 of Fig. 2 are the most important. The characteristic potentials of the redox couples are relevant not only because they define equilibrium positions but also because they often determine the rate of the redox reaction. The six species mentioned above involve three electron-transfer reactions and three hydrogen-atom transfers can be defined. An estimate (constructed by calculation from various data in the literature) of the E 0 values of these couples for plastoquinol in ethanol is shown in Table II. In the case of the H-transfer couples E m is dependent on pH, decreasing by 58 m V / p H unit at 20°C. The quinol has pK values of 10.8 and 12.9, so that QH 2 is the predominant species at physiological pH values. Conversely, the semiquinone has pK = 5.9, so that the semiquinone anion Q - tends to predominate, but Q H may become the major form in energized thylakoids, which have an internal pH below this pK. The effect of a more hydrophobic environment, such as may occur in the membrane, would be to raise all these pK values. At neutral pH the semiquinone is unstable and dismutates, but at alkaline pH it is moderately

Most c-type cytochromes have pH-independent mid-point potentials at neutral pH so that the overall process of reduction is electron transfer, but this does not preclude H-atom transfer as the actual mechanism [142]. Cytochrome f has a redox p K on the oxidized form at 8.4 [6]. The two possibilities could be written: H*cyt 3+ + e

~ H + c y t 2~

cyt 3+ + H ~ H + c y t 2+

(2) (3)

The most extensive studies of cytochrome reduction by quinols in solution have been carried out with mammalian cytochrome c, which has a p K on the oxidized form of about 8.9 [41] so that Eqns. 2 and 3 apply. Yamazaki and Ohnishi [143] showed that the reduction of cytochrome c by benzoquinol at p H 7 is autocatalytic and the reductant species is the semiquinone anion Q -. The rate is stimulated by small amounts of quinone and depends upon [H +] 2 ]141,143]. The latter fact indicates that the rate-limiting step is disproportionation according to Eqn. I. Stiehl and Witt [138] proposed a similar mechanism for reduction of c y t o c h r o m e f by plastoquinol in chloroplasts. At lower pH values the rate of reduction of cytochrome c by benzoquinol becomes proportional to [H + ] 1 rather than [H ~] 2 and ceases to be autocatalytic [141]. The kinetics may be explained by either of two mechanisms shown in Fig. 3 [141,144]. In both, the rate-limiting step is the transfer of the first reducing equivalent from the quinol to cytochrome c, with transfer of a second equivalent from the semiquinone following rapidly. In the electron-transfer mechanism the electron donor is QH and the dependence on [H +] r originates in dissociation of QH 2. In this case electron transfer from Q2 would also be possible, but the pH dependence shows that this pathway is quantitatively insignificant. In the Hatom-transfer mechanism the H donor is QH 2 and the dependence on [H +] 1 originates in the need

131

(a)

[b) H-t-

H

+ +

QH 2

> QH

"t"

QH- L ~

Q-

~,,.,~

cyt c 3 + <

QH 2

H cyt _c3

QH-

H+ cyt c 2+

~

H÷

cyt C3+

H + cyt £2+

y

cyt C3+<

" ~ Q

H+cyt c 2÷

H+cyt C_3 + H÷

Q

H÷cyt £3÷ H ÷ cyt _c2+

Overall

QH 2 + 2H÷ cyt c

3+

>Q-t-2H'cyt c2~2H +

Fig. 3. Possible mechanisms of reduction of cytochrome c by quinols at acid pH. (a) Electron-transfer mechanism. (b) H-atom-transfer mechanism. Mechanism a is preferred for reasons discussed in the text.

for H+cyt c 3+ to dissociate to cyt c 3+ + H + before the cytochrome can accept an H atom. In either case, the simple pH dependence is only possible because the pH range involved is far from the relevant pK values. A choice may be made between the two mechanisms by examining the effect of ionic strength on the reaction rate. The marked effect that was found [141] favours the electron-transfer mechanism because in the case of H-atom transfer one of the reactants is uncharged. This conclusion was confirmed by the demonstration that when the rates of reaction with a range of differently substituted benzoquinols were compared a linear relation was obtained between the logarithm of the second-order rate constant and the difference between the E 0 values for the coup l e s H + c y t c 3 + / H + c y t c 2+ a n d Q H / Q H .The reaction rate decreased by a factor of 10 for every 118 mV increase in E 0 ( Q H / Q H ), in agreement with Marcus's theory [145,146] for outer-sphere electron-transfer reactions. The reduction of purified cytochrome f by benzoquinols in solution also proceeds by electron transfer from Q H - (Ref. 147, and Rich, P.R. and Bendall, D.S., unpublished results). The rate is

again dependent on [H +] 1 and follows the Marcus prediction for the calculated values of E0(QH/QH ). Plastoquinol-1 reduces cytochrome f about 100-times faster than cytochrome c; only part of this difference can be ascribed to the higher mid-point potential of cytochrome f. Nevertheless, reduction of cytochrome f in solution is much slower than in vivo, but rates similar to those in vivo can be obtained with crude cytochrome bf particles prepared by digitonin extraction of thylakoid membranes. These particles catalyse the reduction of plastocyanin by plastoquinol-1 in a dibromothymoquinone-sensitive reaction and probably closely mimic the redox behaviour of cytochrome f in the membrane [148]. The rate of reduction of the particle-bound cytochrome f is proportional to [plastoquinol-1] up to the limit of solubility, so the reaction may be treated as a bimolecular collision process. The second-order rate constant is about 100-times higher than with purified cytochrome f, if the reductant is assumed to be Q H - . At low pH the rate is proportional to [H +] 1 consistent with electron transfer from QH , but as the pH is raised autocatalytic behaviour does not set in, as

132

C

4~ / C:

/,? (()

Fig. 4. Experimental and simulated data for the reduction of cytochrome f by plastoquinol-I in a crude preparation of cytochrome b-f complex. Ordinate: arbitrary units. Open circles, experimental points; continuous line, computer simulation. The computer simulation was based on the following reactions: PQH

+H+cytf 3+~PQH'+H+cytf

pQz

+H+cytf3+ ~pQ:+H+cytfZ+

2+

l; 2

The following constants were assumed: k 2 ~ kl; p K ( P Q H 2 / P Q H - ) = 1 0 . 8 ; pK(PQH / p Q 2 - ) = 1 2 . 9 ; acceptor redox pK =9; acceptor site pK =6.5.

in solution, and a bell-shaped pH profile is obtained (Fig. 4). The shape of this curve can be simulated rather closely by assuming that the rate-limiting step is electron transfer from QH (and also from Q2 at alkaline pH) and by taking into account the pK values for QH2, the redox pK on cytochrome f, and an additional pK on the acceptor at pH 6.5. This additional pK may be associated with the Rieske Fe-S centre, which is known to be present, rather than with cytochrome f itself. The alkaline side of this pH curve has not been studied in any reaction involving intact thylakoids. The acid side is closely similar to the pH curve for the rate-limiting step in chloroplast electron transport except for a shift of about one unit towards higher pH values. The second-order rate constant for reduction of cytochrome f in digitonin particles by the anionic form of plastoquinol-1 is close to the diffusionlimited value. The reaction is nevertheless rate limiting for chloroplast electron transport because of the low concentration of QH at physiological pH values. An earlier suggestion [34,149] that be-

cause of this the reaction might involve H-atom transfer no longer seems to be valid. Several factors may contribute to the high rate of electron transfer in the membrane. The Rieske centre may provide a better acceptor site for cytochrome f, and the departure from strict Marcus behaviour in digitonin particles suggests that hydrophobic binding may contribute. Further enhancement might come from an orienting effect of the membrane which could increase the proportion of productive collisions. The relative importance of these factors is unknown, but enhancement by as much as 100-fold as a result of their combined effect seems not unreasonable. An important conclusion from these experiments is that the reduction of c y t o c h r o m e f in vivo is essentially an ionic reaction, involving QH and a protonated acceptor group, which is most probably a histidine residue in view of its pK. A plausible interpretation is that the reaction takes place at the interface between the lipid phase of the membrane and the aqueous medium. A further argument that favours the accessibility of the haem group is the fact that the rate constant for the oxidation of cytochrome f by plastocyanin is the same for digitonin particles as for purified cytochrome f in solution [34]. The reduction of the b-type cytochromes of chloroplasts by quinols has been less thoroughly studied, but it is obvious that equilibrium considerations are more important. Although cytochrome b-559Hp is readily reduced by most benzoquinols, this is true of neither cytochrome b-559Lp nor b-563. Era, v for the plastoquinone pool of chloroplasts has been reported to be + 118 mV [150]; no physiologically attainable degree of reduction of the pool is likely to achieve a low enough potential to give significant reduction of cytochrome b-563 ( E m , 7 ~ - - - 100 mV), and only slight reduction of cytochrome b-5591, P ( E r a . 7 = + 2 0 mV) is likely. This is in agreement with the classic experiments of Amesz and co-workers [151,152] who observed no redox behaviour of b-type cytochromes when PS I and PS II were excited alternately. However, photoreduction of cytochrome b-563 has been observed [132,153 162], especially with flash excitation, and the implication is that in these cases the cytochrome is not in equilibrium with the quinone pool. Rich

133

and Bendall [163] have described steady-state conditions under which reduction of cytochrome b-563 can be observed with digitonin particles. As expected, duroquinol does not reduce cytochrome b-563 in the dark, but upon illumination a slow flux through cytochrome f and plastocyanin is brought about by the photo-oxidation of the small residual amount of P-700 in the preparation. Under these conditions cytochromefremains reduced, despite the light, and cytochrome b-563 becomes largely reduced. The steady-state redox level of the cytochromes can be manipulated by adding dibromothymoquinone to slow down the rate of reduction of cytochrome f by duroquinol; as the concentration of dibromothymoquinone is increased the steady-state levels of oxidation of cytochromes f and b-563 increase in parallel fashion. Reduction by superoxide anion was ruled out, and so there are two-possible explanations of this experiment. Several arguments may be raised against the likelihood of cytochrome b-563 receiving electrons directly from P S I (for example, the addition of acceptors such as methyl viologen to divert electrons to O2 has no effect). The alternative is that cytochrome b-563 is reduced by semiquinone produced as a product of the reduction of cytochrome f by QH . In the experiments discussed above on the mechanism of reduction of cytochrome f by quinols there was always enough oxidized cytochromef present for the semiquinone to reduce rapidly a second molecule of cytochrome f. In the present case very little oxidized cytochrome f is present in the absence of dibromothymoquinone and the semiquinone is available for reduction of cytochrome b-563 (and b559Lp ). This is analogous to the oxidant-induced reduction of cytochrome b in mitochondria. With the chloroplast particles the process can be regulated by manipulating the relative rates of oxidation and reduction, and hence the steady-state redox level, of cytochrome f.

VC. The role of protein-bound quinone molecules In recent years strong evidence has accumulated that in chloroplasts, bacterial chromatophores and mitochondria, a proportion of the total quinone may be bound to protein rather than free in solution in the lipid phase of the membrane [164]. The properties of bound quinone molecules would be

significantly modified and demonstration of a stable semiquinone by optical or EPR spectroscopy is good evidence for them. The notion of bound quinones has been of particular value in attempting to interpret kinetics of electron transport activated by single-turnover flashes. The best attested case in chloroplasts is the acceptor complex of PS II which is thought to contain a pair of bound quinone molecules, the primary acceptor Q and the secondary acceptor labelled B or R [165169]. Recently, Bouges-Bocquet [ 135] has proposed the existence of a third bound form, U, that acts as the immediate donor to the Rieske iron-sulphur centre or cytochrome f in a fashion analogous to that of the component Z postulated in the cyclic electron-transport system of bacterial chromatophores [124,170-177]; evidence for this came from studies of phase b of the flash-induced electrochromic effect at 518 nm. Furthermore, it is difficult to explain the inhibitory effect of dibromothymoquinone, usually thought of as a plastoquinone analogue, without postulating a quinone-binding site on the acceptor [178-181]. Recent evidence for the indispensable role of protein-bound quinones, which has been obtained with a variety of electron-transport systems, might seem to be incompatible with the older idea, developed in most explicit form by Kr6ger and Klingenberg [182,183] for mitochondria, of quinones acting as mobile pools of electron carriers. It has been suggested, for instance, that the quinone pool

Quinone pool Donor

Acceptor Q

Donor ..... Q 2e-2H .I~

Q ...... A c c e p t o r A

i .2e- 2.;

I

Donor...... QH 2

QH2-.. Acceptor QH 2

Donor

Acceptor

Fig. 5. Model of quinone function in membranes of thylakoids and other bioenergetic systems. The pool contains a relatively large number of mobile diffusible molecules. The electron donor and acceptor proteins may possess quinone or quinol molecules loosely bound in the manner of enzyme-substrate intermediates; they equilibrate with the pool by association or dissociation reactions. Redrawn from Ref. 190.

134 may have merely a redox buffering function and may not be an essential intermediate in electron transport. Nevertheless, the view that the pool is a normal intermediate is supported by a variety of lines of evidence, e.g., pseudo-first-order kinetics [138-140,182], inhibitor studies [183-185], some extraction-reconstitution experiments [186-188] and substrate competition [189]; in other experiments second-order kinetics indicate relative mobility of components, but not necessarily that of the pool [124,173,175]. A way of reconciling these two extremes, suggested by Rich [144,190], is illustrated in outline in Fig. 5. Here, the protein-bound quinone and quinol are regarded as dissociable and able to mix with the pool so that they are transient intermediates analogous to enzyme-substrate complexes rather than prosthetic groups. First-order kinetics are likely to be observed if further reaction of the quinone/quinol-enzyme intermediate is very rapid compared with the binding reaction. The intermediate complex would not then be readily detectable in steady-state experiments but might be revealed in flash experiments under conditions in which the electron acceptor (in the case of quinol oxidation) is initially reduced in the dark. Experimental evidence that the secondary acceptor, B or R, of PS II behaves as a dissociable intermediate has been reported by Velthuys [180,191] and by Bowes and Crofts [192]. On the other hand, the primary acceptor Q probably remains firmly bound and has a highly stabilized semiquinone. The validity of the model outlined in Fig. 5 need not rule out the possibility of direct collisional interaction between two different proteinbound quinones. Whether or not the soluble pool played a significant part in electron transfer would depend on relative concentrations and relative rate constants (including diffusion). This question is discussed further for particular cases in the next subsection. An important question is whether quinones and quinols, either free in solution or protein bound, interact by H-atom or electron transfer. This is especially relevant to transfer across the membrane. Hauska et al. [193-195] demonstrated that plastoquinone and ubiquinone can catalyse the transfer of electrons and H + across liposome membranes. This must represent diffusion of indi-

vidual quinol molecules rather than transfer of H atoms along a chain [196] because Rich [197] has shown that quinones and quinols interact by electron transfer (attendant protonation/deprotonation reactions occurring very rapidly) in a polar medium but are quite inert in a non-polar solvent; H-atom-transfer reactions cannot occur between molecules free in solution and are inherently unlikely when proteins are involved.

VD. Electron flow through the cytochrome b-f complex The cytochrome complex of chloroplasts is involved in two distinct modes of electron flow, cyclic and non-cyclic. The cyclic system has no counterpart in mitochondria, but in reality may not be so very different from the non-cyclic pathway, especially if the plastoquinone pool is involved in both. The extra electrogenic loop associated with phase b of the electrochromic effect seems to be associated with both pathways, but under non-cyclic conditions it may behave as an additional mode of electron flow which is not essential to the catalytic function of the complex. We will consider first the mechanisms of reduction of the complex by PS II. The classical theory is based upon the presence of a diffusible pool of plastoquinone molecules which is commonly observed in fluorescence induction or 02 gush experiments. Bouges-Bocquet [135] has recently cast doubt on an obligatory role for the plastoquinone pool. In line with some current thinking on bacterial chromatophores and mitochondria, she suggests instead that direct electron transfer can occur more rapidly between two bound quinone molecules (B, the secondary acceptor of PS II, and U, the donor to the cytochrome bf particle) than via the diffusible plastoquinone pool. The argument was based on measurements of the recovery of the ability of a flash to generate phase b after one or two pre-illuminating flashes: its extent was taken as a measure of the availability of reduced U. A less convincing argument was based on the observation of period 2 oscillations when algal cells were excited with a series of closely spaced flashes. The oscillations would be expected to originate in B, and if the pool were involved it would damp out the oscillations. In fact, the pool

135 is remarkably small, corresponding to a total of only seven or eight molecules of plastoquinone when the bound forms are included, so the damping effect would be severely limited. Further experiments that suggest direct electron transfer from PS II to the cytochrome complex were recently reported by Joliot and Joliot [198,199]. Strong red illumination of chloroplasts in the presence of 10/zM DCMU gave reduction of cytochrome b-563. Far-red light did not have the same effect and addition of an electron acceptor such as methyl viologen or ferricyanide did not prevent cytochrome reduction. The extent of cytochrome reduction was small, however, probably representing not more than 0.1 cytochrome/reaction centre. A small but significant yield of 02 occurred with a sequence of rather long (several microseconds) saturating flashes in the presence of DCMU, but not with-single-turnover flashes. These observations were interpreted in terms of a proposal that a proportion of PS II reaction centres contain a quencher Q2 [200,201] (which may be equivalent to QL [169,202]) which is less efficient than Q1 and requires several flashes for complete reduction. Q2 tends to back-react rapidly with P-680 + unless the charge is stabilized by transfer to cytochrome b-563. Dissociation of the semiquinone, which in turn could reduce the cytochrome, would also stabilize the charge. Only QI is connected to the plastoquinone pool via the DCMU-sensitive B- (R-) protein. A direct association between at least a proportion of the cytochrome complexes and the PS II units is possible. It seems that Q2 and cytochrome b-563 are involved in a low leak rate which is D C M U insensitive and may occur in parallel with the 'normal' pathway involving Q~ and the pool. This proposal is rather different from that of Bouges-Bocquet mentioned above, which involves a rapid, DCMU-sensitive direct electron transfer from Q1 via B / R to U, by-passing the pool. Both the above types of observation nnght possibly be rationalized as examples of metabolite 'channelling' which would be dependent upon association, if only temporarily, between units of PS II and the cytochrome b-f complex. Channelling has been discussed quite extensively in connection with complexes formed by soluble enzymes [203,204]. It arises when the product of one en-

zyme does not freely equilibrate with the surrounding medium before reacting as the substrate of the next enzyme in the sequence. Several factors may contribute to such restricted diffusion of metabolic intermediates, but clearly the dominant one is the distance a metabolite has to travel between one enzyme and the next. In the present context some experiments of Ragan and colleagues [205-207] are of particular interest. They found that preparations of NADH-ubiquinone oxidoreductase (Complex I) and ubiquinol-cytochrome c oxidoreductase (Complex III) purified from bovine heart mitochondria formed a 1:1 complex when mixed together in aqueous solution. The small amount of endogenous ubiquinone allowed rapid electron transfer (NADH to cytochrome c) but there was no Q-pool behaviour according to the method of Kr6ger and Klingenberg [182]. The enzyme preparations were thought to contain only annular lipid. Addition of further phospholipid restored Q-pool behaviour and presumably dissociated the complex, but decreased activity unless ubiquinone was also added when a stimulation of the original rate was obtained. The fairly rapid electron transfer under conditions of stoicheiometric association of the two enzymes might be regarded as a case of channelling, and addition of extra phospholipid would restore normal 'solution kinetics'. The latter require the Q-pool behaviour that is observed in both mitochondria and chloroplasts under steadystate conditions. Heron et al. [206] proposed, however, that electron transfer from N A D H to cytochrome c takes place only through Complex IComplex III units that are formed and reformed at rates greater than that of electron transfer from Complex I to Complex III. Although this type of explanation can account for Q-pool behaviour it does not explain why in both submitochondrial particles and chloroplasts the total quinone pool undergoes oxidation and reduction at rates commensurate with overall steady-state electron flow [138,182], and also why the individual complexes (and PS II units) in isolation can react rapidly with quinones or quinols as appropriate. On the whole, therefore, the evidence remains strong that under steady-state conditions in chloroplasts most electron flow goes through the plastoquinone pool. According to the ideas about cytochrome f re-

136

duction developed in the two previous sections, U should be r e g a r d e d as an i n t e r m e d i a t e enzyme-substrate complex. The rate-limiting step in plastoquinol oxidation would be binding of plastoquinol to an acceptor site to form U (or possibly the dissociation of the bound quinone). Oxidation of U would be relatively rapid provided an oxidized acceptor was available. This model would be more easily reconciled with inhibition by dibromothymoquinone, regarded as a plastoquinone analogue, than the alternative in which U is treated as a firmly bound prosthetic group. If U is a dissociable plastoquinone-acceptor complex dibromothymoquinone should act as a competitive inhibitor. Addition of plastoquinone-9 does, in fact, reverse the inhibition [178], but such experiments are difficult to interpret because of the first-order oxidation of plastoquinol and the extreme insolubility of plastoquinone-9 in water. The behaviour of dibromothymoquinone as an inhibitor is in a way paradoxical. Dibromothymoquinone is a lipophilic quinone which can be reduced by PS II and from solution studies [141] one would expect it to catalyse rather than inhibit the reduction of cytochrome f. (It is also readily photoreduced in ethanolic solution, a fact which explains its apparent reducing properties when added to chloroplast suspension in the dark [208,209].) B~Shme [159] showed that with chloroplast suspensions dibromothymoquinone inhibits cytochrome f reduction in the light but not in the dark. Although it inhibits the ability of digitonin extracts of chloroplasts to catalyse the dark reduction of plastocyanin by plastoquinol-1 [34], the single-turnover reduction of cytochrome f is not inhibited [181]. These observations are consistent with dibromothymoquinol acting as a competitive substrate for the cytochrome b-f complex. The rate-limiting step which causes inhibition under steady-state conditions would most probably be the dissociation of the acceptor-dibromothymoquinone complex [ 181]. Cytochrome f now seems unlikely to be the quinol-binding site. A more plausible candidate is the high-potential iron-sulphur protein of the Rieske type which was discovered in chloroplasts by Malkin and Aparicio [210]. This component has been shown to be photo-oxidized by far-red light and photoreduced by red light. Its character-

istic potential of +290 mV would place it on the reducing side of cytochrome f, consistent with the inability of a Lernna mutant [211] and of the Chlamydomonas mutant ac-21 (Sanguansermsri, M. and Bendall, D.S., unpublished observations) to catalyse electron transfer between plastoquinol and plastocyanin, and with the properties of a comparable component in mitochondria and bacterial chromatophores [212]. Chain and Malkin [209] quoted a shift in the EPR spectrum of the Rieske centre from g 1.89 to g 1.94 in the presence of dibromothymoquinone as evidence that the Rieske protein is the quinol-binding protein, but no such shift could be observed with catalytically active digitonin extracts of chloroplasts [93]. A useful new inhibitor which should help to elucidate the nature of the quinol-binding site is the dinitrophenyl ether of iodonitrothymol (DNP-INT) [179,213]. This compound appears to act at the same site as dibromothymoquinone but has no redox properties. Another inhibitor with a similar site of action is bathophenanthroline [214,215]. On the other hand, undecylhydroxydioxybenzothiazol (UHDBT), in contrast to its effect on mitochondria [216] and chromatophores [217], acts primarily on the DCMU-binding protein rather than on quinol oxidation [218]. Although cytochrome b-563 has usually been regarded as being reduced rather directly by P S I (but see below), there is strong evidence that it can be reduced by PS II. Illumination by both flashes [160] and continuous light [158,159,161] can cause a DCMU-sensitive reduction of cytochrome b-563 even in the presence of an electron acceptor such as ferricyanide or methyl viologen which would preclude cyclic electron flow. Furthermore, red light can give more complete reduction than far-red light. At least when DCMU inhibits, reduction of cytochrome b-563 by PS II probably occurs via the plastoquinone pool (but see the discussion above). Velthuys [160] found heavily damped period 2 oscillations in the extent of cytochrome b-563 reduction in a flash sequence when the pool was initially oxidized, i.e., much greater reduction on the second flash than the first, followed by a smaller extent of reduction on the third and fourth flashes: no cytochrome reduction could occur until plastoquinol (PQH2) was liberated into the pool from PS II after the second flash. When the pool

137

was already partly reduced the oscillations disappeared and the rates of reduction and oxidation of cytochrome b-563 and of re-reduction of cytochrome f were much greater, as expected if the rate of reduction depends on [PQH2]. Cytochrome b563 cannot be reduced in equilibrium with the plastoquinone pool because of the large difference in characteristic potentials [21,150]. This implies that the reductant is semiquinone (see subsection VB), most probably the deprotonated form P Q - , so that reduction of cytochrome b-563 would be a process secondary to the one-electron oxidation of plastoquinol (probably PQH ) by the Rieske centre. A difficulty with this proposal is that cytochrome b reduction is surprisingly fast, considerably more rapid than the re-reduction of cytochrome f [157,160,162]. The situation is similar in purple bacteria [124]. However, the kinetics of cytochrome f turnovei" are complicated by its relations with the Rieske centre and plastocyanin, and are not completely understood. The reduction of the cytochromes could be formulated according to the following series of reactions: A H 2 .~ A H AH

+H +

(4)

+FeS + =U-FeS +

(5)

U - F e S + ~ A H " + FeS

(6)

FeS+cytf

(7)

3+ = F e S + + c y t f 2+

AH'~-A~+H

+

A~ + c y t b 3+ ~ A + c y t

(8) b 2+

(9)

where A is a plastoquinone molecule of the pool, FeS the Rieske iron-sulphur protein, and U the bound plastoquinol donor to the Rieske centre, interpreted as an enzyme-substrate intermediate. An important, but unresolved, question is whether the semiquinone must be released from its binding site in order to reduce cytochrome b-563. The presence of a positive charge at the binding site (subsection VB) would favour retention of the semiquinone, but P Q H - might complete successfully, depending on the degree of reduction of the pool. The fact that the negative charge is localized in P Q H - and delocalized in P Q - would favour binding of the former, but this would be opposed

by its low concentration. Of course, there may be other more subtle factors favouring semiquinone release. The behaviour of the semiquinone might differ significantly between flash and continuous illumination. On the whole the evidence is against an obligatory role of cytochrome b-563 in steady-state electron flow, and this is consistent with measured values of the H + / e ratio (subsection VA) and the failure of antimycin to inhibit under most conditions. Chloroplasts therefore seem to possess a switch, missing in mitochondria, which determines the fate of the semiquinone form of U. The redox state of the plastoquinone pool has been suggested to be an important determinant of b reduction [159,160]. A simple kinetic switch has been shown to operate in crude digitonin particles with duroquinol as the reductant (see subsection VB). Under these conditions the semiquinone preferentially reduces cytochrome f (or else undergoes dismutation) and only reduces the b cytochromes when cytochrome f remains largely reduced in the steady state. The relative rates of reduction may be reversed in the intact membrane with endogenous plastoquinol as reductant, and, indeed, both in chloroplasts and chromatophores cytochrome b is usually reduced more rapidly in response to a flash than the c-type cytochrome is re-reduced. Evidence that the switch may be operated by the membrane potential and ApH has also been mentioned above. Under cyclic conditions, with ferredoxin as cofactor, the semiquinone seems to be switched permanently towards reduction of cytochrome b-563, so ferredoxin may be able to exert control through a conformational effect on the cytochrome b-f complex. The rate of re-oxidation of cytochrome b-563 is often similar to the rate of re-reduction of cytochrome f, but cytochrome f reduction can be faster. Under strongly oxidizing conditions when cytochrome f is kept in the oxidized form, cytochrome b-563 re-oxidation is slowed down rather than accelerated [160]. A direct connection between the two cytochromes is therefore unlikely. Cytochrome b-563 appears to be fairly rapidly auto-oxidizable, but whether this occurs on a millisecond time scale is doubtful. The fact that dibromothymoquinone can inhibit the oxidation of cytochrome b-563 as well as the reduction of cyto-

138

chrome f [132[ indicates that plastoquinone is involved in the oxidation but does not necessarily indicate a second binding site. Three possible forms of oxidant can be considered, if one assumes that the pool is concerned. These are the quinone, A, by the reverse of reaction 9, the semiquinone anion, A - or the neutral semiquinone AH" by reactions 10 and 11: A ~ + c y t h 2+ ~ A 2 + c y t b 3+

(10)

A H ' + c y t b 2÷ ~ A H

(11)

+ c y t b 3+

The quinone would be a slightly stronger oxidant than the semiquinone anion in the free form, and would be present at higher concentration. However, the reaction has been observed to go faster as the pool becomes more reduced [160], suggesting semiquinone as the oxidant, although, according to Crowther and Hind [134], an ambient potential well below that of the pool is required. The rate constant for oxidation by AH" would be expected to be larger than for oxidation by A - , because of a difference of about 400 mV in the mid-point potentials of the relevant couples [141]. On the other hand, AH" may be present at lower concentration than A - because of a low p K value. The suggestion that semiquinone may be involved in both the reduction and the oxidation of cytochrome b is not new, and in particular is featured in the proton-motive Q-cycle proposed by Mitchell [113-115] and the analogous 'b-cycle' of Wikstr6m [121,122]. The real issue is how these processes are ordered and in particular where the protons come from for reduction of semiquinone to quinol. Unless they come specifically from the external medium (or stroma phase), the role of cytochrome b-563 is reduced to that of a 'semiquinone dismutase' [136] or a 'semiquinone buffer' [134,162]. On the other hand, if a Q-cycle is involved the cytochrome might be better regarded as stabilizing the semiquinone sufficiently to allow it to diffuse across the membrane, rather than as acting like an electric wire across the membrane (Mitchell's original formulation). The Q-cycle and b-cycle models are similar in that they both provide a ready explanation for oxidant-induced reduction of cytochrome b, but differ markedly in what they say about the locations of the b haems.

Consideration of cyclic pathways raises new problems. Several years ago, Trebst [219] concluded that a cofactor of cyclic electron flow is an acceptor of P S I and closes the electron cycle back to the donor site of PS I by reacting in one of three ways: (a) with plastoquinone (e.g., the natural cofactor ferredoxin), (b) with plastocyanin (permeant cofactors such as 2,3,5,6-tetramethyl-pphenylenediamine) and (c) with P-700 (phenazine methosulphate). We are only concerned here with class a, as b and c by-pass the cytochrome complex. More than one type of compound might be considered under class a, however. With the natural cofactor ferredoxin, phosphorylation and electron flow are characterised by being antimycin sensitive [ 100,101,220] and associated with the slow phase b of the electrochromic effect (P-518~) [131,221,222], as well as being inhibited by dibromothymoquinone which indicates the involvement of plastoquinone [223]. Anthraquinone-2sulphonate is an alternative cofactor in some ways similar to ferredoxin in that it is non-permeant and the reaction is sensitive to dibromothymoquinone, but a n t i m y c i n does not inhibit [100,101,223,224]; whether there is an associated P-518~ is unknown. A crucial question is how electrons pass back to a carrier within the membrane from non-permeant cofactors. Although plastoquinone is involved in the cyclic pathway the nature of the immediate acceptor, and the role of the pool, are uncertain. Arguments in favour of the involvement of the pool have usually relied heavily on analogy with the non-cyclic pathway, but a by-pass is a possibility for both the cyclic and non-cyclic pathways. Concrete evidence that ferredoxin can reduce the pool has recently been obtained. N A D P H and ferredoxin were shown to reduce both C-550 [225] and the fluorescence quencher Q [226], presumably indirectly as Q is generally inaccessible to external reagents. Analysis of fluorescence rise curves confirmed that the pool was reduced [227], but rather slowly (t /2-~20s), perhaps because N A D P H is a poor reductant for ferredoxin. A common assumption has been that the direct acceptor for electrons from ferredoxin is cytochrome b-563. Cramer and Butler [154] observed reduction in far-red light and Dolan and Hind [157] later showed it to be rapid, with t~j 2 -~ 1 ms.

139 bly works as a two-electron accumulator for which the B / R protein of PS II provides a ready model (the two proteins are not identical because D C M U does not inhibit ferredoxin cyclic phosphorylation). I n h i b i t o r studies involving disulphosalicylidenepropanediamine and maleimides have suggested that the flavoprotein ferredoxin-NADP oxidoreductase mediates electron flow between ferredoxin and the plastoquinone reductase [229] even though a flavoprotein antiserum does not inhibit cyclic electron flow [230]. The plastoquinone reductase need not necessarily be part of the cytochrome b-f complex, especially if the plastoquinone pool is involved. Bouges-Bocquet [222] has reported that a component C in Chlorella exhibits absorbance changes with kinetics consistent with its being the electron acceptor of the electrogenic reaction of a Q-cycle and therefore possibly identical with V. It has a very diffuse difference spectrum, roughly comparable with that of the Rieske iron-sulphur centre. An odd feature is that redox changes of cytochrome b-563 have not been observed in Chlorella under these conditions.

This interpretation is no longer tenable, however, in view of the demonstration by Cox [228] that ferredoxin does not accelerate the very slow reduction of cytochrome b-563 by dithionite. Non-permeant mediators (anthraquinone-2-sulphonate) are similarly ineffective whereas permeant mediators (anthraquinone, methyl viologen) do cause acceleration; thus the cytochrome seems to be buried in the hydrophobic phase of the membrane and inaccessible to hydrophilic reagents (reduction was also slow with digitonin preparations of the cytochrome b-f complex [228] and with inverted thylakoid vesicles [35]). A more plausible explanation for the behaviour of the cytochrome is oxidant-induced reduction, with P S I as the oxidant, as discussed above. Thermodynamically, ferredoxin would be expected to reduce plastoquinone at a reasonable rate but in practice the direct reaction is probably slow through lack of accessibility. It seems more likely that the membrane contains an unidentified ferredoxin-plastoquinone reductase, denoted 'V' by Crowther and Hind [134,162]. This enzyme proba-

1/2H20

2H +

1/402+H ÷

T PQuH2 //'/semiquinone

P680

L, mo°

,"

I

PC FeS

]

> cyt f P700

J

// ~QAH2 ] / " /

cyt b-563

Ii It~'~

cyt b-563 AA

~ ~\~-~

o o Q

~

B/R

x

2H+

~

AQS Fig. 6. Scheme for electron flow through the cytochromeb-f complex incorporating a Q-cycle. Q, B/R, PQu and V represent bound forms of plastoquinone; PQA, the plastoquinone pool; FeS, the Rieske iron-sulphur centre; PC, plastocyanin; Fd, ferredoxin; AQS, anthraquinone-2-sulphonate; AA, antimycin; solid arrows, electron or proton transfer; broken arrows, binding, dissociation and diffusion of plastoquinone. The scheme is complete with a single turnover of cytochromef.

140 against the pathway shown in Fig. 6 is the fact that reduction of cytochrome b-563 (tl/2~-1 ms) is considerably faster than the appearance of P-518~ (t~/2~'-10 ms); the latter usually correlates well with the rate of re-reduction of cytochrome f [ 130,162,231 ]. Re-reduction of PQ u is presumed to be an electroneutral process, i.e, the overall reaction involves H-atom transfer. The inability of quinones and quinols to interact at an appreciable rate by H-atom transfer (subsection VC) suggests that transmembrane diffusion of plastoquinol is involved, either via the pool or by channelling. The Q-cycle scheme shown in Fig. 6 offers an automatic explanation of the fact that cyclic phosphorylation with anthraquinone sulphonate as cofactor is antimycin insensitive, because it is assumed that antimycin is a specific inhibitor of the electrogenic reaction of the Q-cycle loop which cannot operate when V is by-passed by artificial cofactors. This sort of control is not a feature of the alternative b-cycle (Fig. 7). Both schemes suggest an additional H +-pumping loop which may operate in non-cyclic electron flow under some conditions. In the Q-cycle scheme this would be true if V could receive both of the electrons re-

It is by no means certain that cyclic electron flow with other non-permeant cofactors follows the same route as with ferredoxin, and if it did the different responses to antimycin would be difficult to explain. A small molecule such as anthraquinol sulphonate might penetrate the membrane surface sufficiently to reduce the plastoquinone pool directly, but plastosemiquinone would be expected as an intermediate and it may be significant that there is no rapid reduction of cytochrome b-563 under these circumstances [228]. Alternative schemes that may be able to explain cyclic electron flow are illustrated in Figs. 6 and 7. These schemes are based on the Q-cycle and the b-cycle, respectively, and should be treated as no more than working hypotheses. The basic assumption is made that P-518~ reflects the occurrence of a slow electrogenic reaction (an electron moving outwards in Fig. 6 and a proton moving inwards in Fig. 7) rather than the mere redistribution of charge at the membrane surfaces. In both schemes cytochrome b-563 is represented as being reduced by semiquinone as examples of oxidant-induced reduction, in contrast to some other formulations of the Q-cycle in chloroplasts [134,162]. However,

2H +

4'-

H20

2H +

;/202 + 2H

PC

,j-t PQuH 2 ,- " / semiquinone ," L, PQu

P680

f

i/ /

FeS---~ cyt f

l

P700

!

cyt b-563

/I //

PO A H 2 ] / " po

cyt b-56a

P-"J 9 e.

//

\\

Ii II tl/

Q ~

/\

'5

B/R

2H ÷

,H +

~

AQS Fig. 7. Scheme for electron flow through the cytochome b-f complex incorporating a b-cycle. Symbols as in Fig. 6. The scheme requires two turnovers of cytochrome f.

141

quired for its reduction from PQu or if one of them could come from PS II; this remains a controversial point [134]. It seems clear that the extra loop does not function in steady-state non-cyclic electron flow at high light intensity. A simple explanation of such a switch, as discussed earlier, might be that PQ~ preferentially reduces the Rieske centre and cytochrome f when these are maintained in highly oxidized states. In addition, development of P-518~ is slowed by both A~b and ApH [128,130-132]. The mechanism of oxidation of cytochrome f and plastoquinol by P S I remains controversial despite the strength of the evidence of various kinds [149,232] in favour of the simple linear scheme: plastoquinol ~ Rieske Fe-S ~ cytochrome f--, plastocyanin ---,P-700. Study of the rates of the individual steps [34,233,234] strongly suggests that this is basically correct, with the exception that rates involving the Rieske iron-sulphur centre have not been measured directly. The disagreements at the experimental level are of two main kinds. First, widely different values for the characteristic potential of P-700 have been reported and these lead to different predictions for the behaviour of the linear model. Secondly, significantly different kinetic behaviour of cytochrome f or plastocyanin has been reported in different experiments. Published values for the mid-point potential of P-700 range from +375 to +520 mV [235]. The former would indicate an equilibrium constant between P-700 and cytochrome f of close to unity so that reduction of cytochrome f would be expected to proceed at almost the same rate as that of P-700 and without any significant lag phase. The latter would be equivalent to an equilibrium constant of 380 and a marked lag in cytochrome f reduction would be expected. Equilibrium should be nearly maintained throughout the reaction because electron transfer from f to P-700 is rapid compared with the initial donation of an electron by plastoquinol. Recent measurements on P-700 have revealed some significant factors influencing the apparent mid-point potential [235]. Low values are obtained with PS I preparations that have been extensively purified by detergent treatment. Artificially low values are also obtained when a preparation is equilibrated with the redox buffer at room temperature and then frozen in liquid nitrogen;

for this reason the estimate of + 375 mV obtained by Evans et al. [236] is probably too low. On the other hand, titrations can be distorted by chemical oxidation of bulk chlorophyll, an effect which tends to give erroneously high values. Serif and Mathis [235] have reported a value of +490 mV from measurements on the 820 nm band of P-700 + made at room temperature; it is not certain that this determination was entirely free of an effect on bulk chlorophyll, but the magnitude of the total AA was in reasonable agreement with that expected for P-700. This value is equivalent to an equilibrium constant of 120 with cytochrome f. The kinetic discrepancies refer mainly to the time courses of reduction of P-700, plastocyanin and cytochrome f following illumination. Most observations have been made on chloroplasts at room temperature, but Cox and colleagues [237241] have favoured the use of low temperatures, maintaining fluidity at sub-zero temperatures with a mixture of ethanediol and water, to slow the reaction and make it easier to measure. At - 3 2 ° C , with PS II active, the reduction of both cytochrome f and P-700 appeared to be strictly first order with tt/2 a t least 3-times greater for the former than the latter. This experiment would be difficult to reconcile with equilibrium between the two components with any chosen value for the equilibrium constant [237-239]. At 2°C in the presence of DCMU and of N A D P H plus ferredoxin as electron donor system, a slight lag in cytochrome f reduction could be seen which would be consistent with an equilibrium constant of 15 between the two components [241], but there is too little information about the effects of temperature on the characteristic potentials to be able to predict this value. In experiments at room temperature rapid reduction of cytochrome f, indicating a lack of equilibrium with P-700, has been observed by Haehnel [139,140] with chloroplasts and Bouges-Bocquet [130] with Chlorella suspensions. On the other hand, a small lag in cytochrome f reduction has been detected in chloroplasts by Whitmarsh and Cramer [242,243]. Earlier, Marsho and Kok [244] had reported apparent equilibrium constants between cytocbrome f and P-700 that vary according to experimental conditions, low values being obtained when the steady-state redox levels of the

142

two components under weak illumination were compared, and high values during reduction by ascorbate after oxidation in far-red light. In contrast with these variable observations on the relative redox states of cytochrome f and P-700, plastocyanin and P-700 seem to react together with an equilibrium constant of about 20 between them (AE m --~80 mV) [130,140,244,245]. This would suggest an E m for P-700 nearer to +450 mV than to + 4 9 0 mV. Published values of E m for plastocyanin range from +340 to + 390 mV, and the value increases below pH 7 (pK 5.6) [246,247]. The original report that P-700 + is reduced in two phases (t~/z-~20 and 200 /~s) following a very short flash [248] has been confirmed several times and has led to suggestions either of a second donor to P-700 or of an additional intermediate between plastocyanin and P-700 [249]. Cytochrome f has been ruled out as an alternative donor and evidence presented that the 20 /~s phase represents reduction by plastocyanin bound to P-700 [250,250a]. The kinetics of P-700 reduction can also be explained as a bimolecular reaction with plastocyanin [240], but plastocyanin does not always behave as if it is reacting in solution because a change in internal volume affected the amplitudes but not the half-times of the two phases [250a]. In the latter experiments all the active plastocyanin seems to have been adsorbed to the membrane surface, but it is not clear whether the two phases represent electron transfer between plastocyanin molecules in two states or exchange of the two kinds of plastocyanin molecules. Anomalous kinetics of cytochrome f tend to arise when plastoquinol, rather than an artificial system, acts as the electron donor. Perhaps this is not surprising in view of the poorly understood complexities of the system, in particular the electron pathways within the cytochrome b-f compex and the lateral heterogeneity of the membrane. It may be that variations in rate constants, as a result of pH changes, electrostatic effects or conformational changes, will have to be taken into account. The redox p K of 5.6 on the reduced form of plastocyanin [246,247] may be significant when the internal p H falls under energized conditions, as Freeman [251] has emphasized. Whitmarsh and Cramer [242,243] have developed a theory that involves an effect of the redox state, i.e., the

charge state, of the system on rate constants governing the loss of successive electrons from plastoquinol and they show how this could introduce a lag into the reduction kinetics of cytochrome f. The most clear-cut case has appeared to be that of Bouges-Bocquet [130] who found conditions under which plastocyanin oxidation could be observed at the same time as cytochrome f reduction following flash illumination of Chlorella cells, but unequivocal detection of plastocyanin, with its weak, diffuse spectrum, must be especially difficult with whole cells. Nevertheless, it is not difficult to imagine that a small movement of cytochrome f relative to the membrane surface might have a large effect on its rate of oxidation.

VE. Possible functions for cytochromes b-559 Apart from the cytochrome b-f complex, chloroplasts contain cytochrome b-559cp which is loosely associated with the complex in an undefined manner, cytochrome b-559Hp being clearly associated with PS II, and cytochrome b-560. To discuss possible functions for the last of these would be premature, except to enter a plea to remember it when interpreting difference spectra. In this section we will discuss possible functions of the two cytochromes b-559. Cytochrome b-559cp has been neglected in the past and there is little firm information to guide speculations as to its possible functions. No lightinduced redox changes have been reported that could not be ascribed to the high-potential cytochrome or a modified form of it. Its pH-independent E m of + 2 0 mV [21] means that it is not readily reduced in equilibrium with the plastoquinone pool (Era,7 = + 118 mV [150]) except at rather alkaline pH. The ease of reduction by dithionite [25] or by N A D P H and ferredoxin (Bendall, D.S., unpublished results) suggests that the haem may be accessible from the exterior of the thylakoid membrane, but this is no more than a tentative conclusion. The failure to detect any ascorbate-reducible cytochrome b-559 in heterocysts of Nostoc muscorum [69] suggests that cytochrome b-559cp may be associated with Photosystern II, but this result is equivocal because ascorbate gives slow and incomplete reduction of the cytochrome, depending on conditions. Cytochrome b-559 H, has been detected in a Chlamy-

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domonas mutant (BF 25) that lacks the high-potential component and O2-evolving activity but retains the reaction centre of PS II, and also in another mutant (F 34) in which PS II is completely missing (Sanguansermsri, M. and Bendall, D.S., unpublished results); these observations argue against any close connection between cytochrome b-559Lp and PS II. The evidence more strongly favours a connection with the cytochrome b-f complex. For example, recent studies of the lateral distribution of cytochromes in thylakoid membranes [35,35a] have shown that cytochromes b559ep, b-563 and f are all rather evenly distributed, whereas P S I and PS II show a marked segregation into non-appressed and appressed membranes, respectively, and cytochrome b-559Hp follows PS II. The same three cytochromes are also known to be extracted together by digitonin [24,49]. Nevertheless, the association appears to be loose because cytochrome b-559Lp is not extracted by a cholate/octylglucoside mixture which solubilises an active form of the cytochrome b-f complex [36] and it is present at the normal concentration in mutants of Chlamydomonas that completely lack cytochromesf and b-563 (Sanguansermsri, M. and Bendall, D.S., unpublished results). Mitchell [115,252] suggested, by analogy with a similar proposal for mitochondria, that cytochrome b-559Lp might be involved in a proton-motive Q-cycle. In Mitchell's scheme the electrogenic arm of the cycle consisted of electron transfer from cytochrome b-563 situated near the inner surface of the thylakoid to b-559Lp near the outside. Direct evidence for such an arrangement is lacking and might be difficult to obtain, although the scheme would be consistent with the oxidantinduced reduction of cytochrome b-563 (subsections VB and VD) and the tentative conclusion that cytochrome b-559Lp is external. It seems more likely that cytochromes b-566 (by) and b-562 (b K ) of mitochondria should be related to the two haems of cytochrome b-563, rather than to cytochromes b-563 and b-559Lp. Nevertheless, even if Mitchell's formulation of the Q-cycle is not strictly correct, cytochrome b-559Lp should be considered as a candidate for the external electron acceptor of the electrogenic arm, i.e., C in Bouges-Bocquet's scheme [135,222], although the spectrum obtained for C is not that of a cytochrome. A closely related

possiblity is that cytochrome b-559Lp might behave as an electron acceptor for ferredoxin in the PS I-dependent cyclic pathway as part of the ferredoxin-plastoquinone reductase, V, of Crowther and Hind [134,162]. Little or no experimental evidence can be brought forward to support such speculations, but on the other hand appropriate spectral changes do not seem to have been specifically looked for, and they would in any case be difficult to distinguish in the presence of redox changes of cytochrome b-563 unless they can be resolved in time. It is worth noting that the width at half the peak height in a difference spectrum representing cytochrome b-563 alone (taken from the line joining the two absorption minima) is about 10½ nm. Any broadening or shift of position to below 563 nm might be taken as evidence for a contribution from b-559Lp. The difference spectrum reported for cytochrome b-563 under cyclic conditions by Crowther and Hind [162] is very close to what would be expected for pure cytochrome b-563. By contrast, although cytochrome b-559Hp has been extensively studied no definite function has emerged. The problem has been dealt with in previous reviews [19,26,27,253] and only a few comments are appropriate here. Whitmarsh and Cramer [254] showed that photoreduction of the cytochrome is too slow for it to be an essential component in electron transfer between the two light reactions. The cytochrome can be fairly rapidly oxidized by PS II, but only when 02 evolution is impaired. These observations make it difficult to support a simple role in electron transport. There is also good evidence that in its high-potential form it is not essential for O 2 evolution per se; the most clear-cut is provided by trypsin-treated chloroplasts [255] but flash-greened leaves may also show a high rate of 02 evolution with only a low concentration of cytochrome b559Hp [256,257]. It might be argued, however, that low-potential forms of this component are still functional in 02 evolution, and all known mutants which lack the cytochrome are also incapable of O2-evolving activity. A close parallelism has been demonstrated between the manganese content of chloroplasts and the content of cytochrome b-559,p, both in extraction experiments [258] and, conversely, in a tem-

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perature-sensitive mutant of Scenedesmus (LF-2) with which incubation of a cell suspension with Mn 2+ leads to restoration of PS II activity and the high-potential form of the cytochrome [259]. However, the existence of fully active preparations with the cytochrome in a low-potential form suggests that Mn 2~ in the membrane is influencing the potential of the cytochrome not by a direct electrostatic effect, but indirectly through a conformational change of the whole PS II complex. Under conditions where photo-oxidation of cytochrome b-559Hp occurs the reaction is rapid. At low temperature (77 K) a half-time of 4.6 ms has been reported [260] and at room temperature in the presence of tetraphenylboron the reaction is biphasic, with half-times of about 30/xs and 4 ms [261]. Under normal functional conditions, such fast reactions evidently do not occur because they would lead to a serious loss of efficiency in PS II. The presence of the cytochrome may nevertheless be essential as a safety mechanism to keep P-680 in the reduced form in the event of loss of activity by an O2-evolving centre. P-680 ÷ is such a strong oxidant that if it could no longer be reduced by the normal pathway in a single centre, unspecific photo-oxidations would tend to spread rapidly through the pigments and lipids of the whole chloroplast. The reductant for the cytochrome would probably be ascorbate, which occurs in high concentration in the stroma of chloroplasts [262]. Cytochrome b-559Hp provides a useful probe into the redox reactions on the oxidizing side of PS I1, which have recently been reviewed [169,263]. It probably donates electrons to P-680 either directly or via the component Zj (in the terminology adopted by Bouges-Bocquet [169]), which is the immediate acceptor from states SO and S l (as defined by Kok et al. [264]) of the photosynthetic water dehydrogenase. Direct donation to S2 and S3 does not occur, as is shown by the very slow deactivation of these states in isolated chloroplasts [265,266], even though the cytochrome is present in the reduced form. The reason for this is presumably that Z l (or P-680) provides a kinetic barrier which depends on its relatively high redox potential [169]. The so-called A D R Y reagents, however, which catalyse the dark reduction of S2 and S) by endogenous donors [267,268], also induce the photo-oxidation of cytochrome b-559 m,

[269,270]. At the same time an inhibition of water oxidation becomes apparent at low light intensities so that the cytochrome can now compete successfully with the water dehydrogenase as an electron donor to P-680. The common feature of all A D R Y reagents is that they are lipid-soluble anions at neutral pH [268,270,271]. Velthuys [261,272] has found tetraphenylboron to be particularly effective in promoting cytochrome oxidation. Although the effects of this reagent are complicated by its own ability to act as an electron donor to PS II, he has suggested that its primary effect is to stabilise species such as Z ] occurring in a hydrophobic environment and thus to lower the potential of a couple like Z~/Z. If there is no corresponding effect on the S-states (either because their immediate environment is less hydrophobic or because the positive charge is released as a proton) they would cease to be effective reducing agents for Zi t and cytochrome oxidation would be promoted. A further effect of tetraphenylboron was found to be the promotion of a DCMU-insensitive photoreduction of cytochrome b-559Hp in response to a flash in the presence of ferricyanide to pre-oxidize the cytochrome [261,272]. This was described as an oxidant-induced reduction such as can be observed with other b-type cytochromes, and in this case the reaction would depend upon the ability of tetraphenylboron to act as a two-equivalent donor. Velthuys suggested that the behaviour of cytochrome b-559Hp could be rationalized by postulating the existence of a two-equivalent carrier, labelled q, through which oxidation and reduction of the cytochrome always takes place, oxidation, for example, depending upon the generation of the half-reduced species q - . Such a scheme remains speculative as direct evidence is lacking, but nevertheless there is evidence for a role of a quinone species (presumably plastoquinone) on the oxidizing side of PS II [273,274]. Observation of dibromothymoquinone- and DCMU-sensitive photo-oxidation of cytochrome b-559Hp by far-red light under certain conditions has suggested that P S I is involved, acting through the plastoquinone pool [275,276]. Such experiments should be interpreted cautiously because dibromothymoquinone is easily photoreduced, even when standing in solution on the bench, and

145 so may effectively act as a reducing agent. Heber et al. [269] demonstrated that in preparations of intact chloroplasts treated with FCCP, far-red light oxidizes cytochrome b-559Hp almost entirely through PS II. Any suggestion for a role of the cytochrome in electron flow between the two light reactions must also take account of Whitmarsh and Cramer's observation that the maximum rate of reduction by PS II (tl/2 = 100 ms) is slower than the maximum uncoupled rate of electron flow by a factor of about 10 [254]. Butler [27] has proposed an ingenious, but speculative, scheme in which only one in four of the electrons needs to pass through cytochrome b-559Hp. The suggestion is made that the energy that is potentially available as the cytochrome passes from a low-potential to a high-potential form could be used to extract one proton from water as it is oxidized by the water dehydrogenase of PS Ii, possibly in the transition S l ~ S2 which does not release a proton to the internal phase [277,279]. The cytochrome would cycle between a high-potential protonated form and a low-potential deprotonated form; in the reduced state the pK would be relatively high and in the oxidized state relatively low. This arrangement demands that the measured E m should vary with pH between the two pK values and this is difficult to fit with observation because E m has been found constant at + 370 mV, at least between pH 6 and 8 [21,280]. A proton-linked function for cytochrome b559Hp has also been suggested on the basis of an unusual fall in mid-point potential at low pH with a pK around 5.5 [20]. There is conflicting evidence, however, as the potential has also been reported to rise at low pH [280]. Despite the wealth of experimental information about the behaviour of cytochrome b-559Hp in situ, we still do not understand what its function is. VI. Conclusions

From the functional point of view cytochromes cannot be considered in isolation from the rest of the electron-transport system. Perhaps the most important developments of the last 5 years are those that help us to see in closer perspective the key role of the cytochrome b-f complex as a func-

tional unit. Improvements in its purification have given precise information about its composition and no doubt further refinements will both help to remove some of the ambiguities about its function by use of model and reconstitution experiments, and also provide material for structural studies such as have been carried out with cytochrome oxidase and the mitochondrial b-c 1 complex. The cytochrome b-f complex is more complicated than seems necessary for a simple catalyst of plastoquinol oxidation, which would have no obvious role for b-type cytochrome. The extra complexity is likely to be connected with the H ÷pumping properties. To understand these, comparison with the cytochrome b-c 1 complex is helpful: the similarities are obvious, but the facultative behaviour of the electrogenic H + pump in chloroplasts (and not in mitochondria) indicates the importance of the differences. The most significant difference may be that E m for cytochrome b-563 is pH independent, a fact which favours the Q-cycle rather than the b-cycle model. In addition, the relatively high potential of cytochrome f and low potential of cytochrome b-563 compared with their mitochondrial counterparts, and also the equality of potential of the two haems of cytochrome b-563, may all be elements of the mechanism that switches off the electrogenic H + pump as the proton-motive force rises. Energetically, this seems a sensible way for the system to be organized as it removes any energetic limitation to electron flow at high light intensities. The second distinctive feature of the cytochrome b-f complex is its ability to participate in cyclic phosphorylation. It remains uncertain whether the cytochrome b-f complex itself functions differently in the cyclic and non-cyclic modes, but the apparently specific effect of antimycin on cyclic phosphorylation suggests that it does, A major gap in our knowledge of the cyclic electron pathway is the mechanism by which electrons get back into the membrane and the cytochrome b-f complex from reduced ferredoxin. As discussed above, there is likely to be a specific catalyst for this reaction, possibly a ferredoxin-plastoquinone oxidoreductase. Electron transfer within the cytochrome b-f complex is a solid-state process which obeys different rules and requires different experimental

146

approaches from the more familiar liquid-state reactions. Such solid-state reactions probably have a large rate constant and require a high degree of structural organization; the mechanisms cannot be understood without detailed structural information which at present is almost entirely lacking. In this review it has seemed appropriate to stress the liquid-state behaviour of plastoquinone. Although the scheme shown in Fig. 5 seems to provide a workable model which resolves some of the apparent contradictions between the concepts of the pool and of bound quinones, much experimental work remains to be done to substantiate the model, and there are further interesting questions, recognizing that the model is an oversimplification. Some of the available evidence demonstrates mobility between components without clearly showing it to be due to the quinone pool. To what extent can relative mobility be ascribed to direct interaction between freely diffusing complexes without involving the quinone pool? Evidence for direct interaction between PS II and the cytochrome b-f complex has been discussed above. Does channelling have a part to play? How fast do different components such as the protein complexes, plastoquinone and plastocyanin diffuse laterally within the membrane? The new information about the fairly extreme lateral differentiation of thylakoid membranes in grana-containing chloroplasts demonstrates the need for quantitative information about rates of diffusion. Finally, there remain the problems of the extra components, the two cytochromes b-559 and now cytochrome b-560. Cytochrome b-559Hp is clearly intimately involved in PS II in a way which will ultimately be understood - a safety valve explanation may prove adequate. There is little information available about the other two components but an intriguing possibility is a role in the recently discovered respiratory function of chloroplasts [282].

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