Chemical hypothesis for energy conservation in the mitochondrial respiratory chain

J. theor. Biol. (1970) 28, 233-259

Chemical Hypothesis for Energy Conservation in the Mitochondrial Respiratory Chain BAYARD T.

STOREY

Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pa. 19104, U.S.A. (Received 22 October 1969, and in revised,form

2 February

1970)

A chemical hypothesis for energy conservation in the mitochondrial respiratory chain is presented with particular emphasis on coupling sites II and III located between cytochrome b and oxygen. The basic premise underlying this hypothesis is that electron transport from substrate to oxygen and the reaction which conserves free energy are coupled at three coupling sites, as required by the ADP/O stoichiometry observed with intact, phosphorylating mitochondria. The coupling sites themselves participate in the redox reactions of electron transport. While the stoichiomerry of oxidative phosphorylation requires three coupling sites, the thermodynamics requires that the free energy available from the reaction of reduced cytochrome oxidase with oxygen be also conserved for this process. This hypothesis postulates that there is a fourth site of energy conservation at cytochrome oxidase which is thermodynamically coupled specifically to sites II and III. The coupling sites are postulated to be lipoprotein enzyme complexes, each specific to its region of the respiratory chain, which contain two tightly bound proteins, one which participates in the redox reaction, the other which acts as a transducer protein to store the free energy of electron transport. The redox protein has three thiol groups capable of forming two different disuhides when oxidized. One disulfide is a low energy form Es. Reduction of this primary disulfrde to the trithiol form ESH releases one thiol to form a thiol ester with a neighboring carboxyl group with no net free energy change, but producing one molecule of water. The other two thiol groups are then oxidized to the second disulfide, an energy-rich protein whose highly strained confirmation is held in place by the disulfide crosslink. The free energy of electron transport is conserved as -TAS in this high energy form Ed. The thiol ester group of Et contributes nothing to energy conservation,but actsasa functional group to facilitate energy transfer. Energy transfer takesplace by transesterification of the thiol estergroup of E, with a thiol and carboxyl group of the transducerprotein ET to regeneratethe primary disulfide ESand produce the thiolester crosslinkedprotein E-. It is at this stage that the water moleculeis removed. Energy distribution can then take place by further transesterificationsto produceenzymeswhoseenergizedstate is characterized by an active ester crosslink. Mechanismsby which these mediate 233

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phosphorylation of ADP, reverse electron transport, and energy-linked transhydrogenation are presented. Conservation of the free energy available from the reaction of oxygen and reduced cytochrome oxidase is postulated to involve a conformational change of this protein to a state which binds with high affinity at specific sites the water molecules produced at coupling sites II and III by electron transport through the redox protein Es. The dehydrated form of the oxidized cytochrome oxidase is the high energy form, while the hydrated form is the low energy form. The reduced oxidase is assumed to have a low affinity for water, and the water molecules are released to the medium from the reduced form of this enzyme.

1. Introduction One very fruitful hypothesis for the mechanism of oxidative phosphorylation by the mitochondrial respiratory chain has been the “chemical”, or “- ” (squiggle) hypothesis first formulated by Slater (1953), using both the concept of the energy-rich bond and the symbol “-” for this bond to a group of high transfer potential, which were introduced by Lipmann (1941). Slater stated specifically that his primary energy-rich intermediate, A - C, did not involve phosphate and could itself mediate other intramitochondrial energylinked processes. It was this postulate which differentiated his scheme from those proposed by Lipmann for other energy conserving processes. In the ensuing ten years, a large body of evidence supporting this postulate accumulated, which was reported in the first Johnson Foundation Colloquium (Chance, 1963). The recent elegant studies of Lee & Ernster (1966a,b, 1967, 1968a,b) on the energy-linked reactions of submitochondrial particles have virtually confirmed it. The work of Ter Wille & Slater (1967) indicates the existence of at least two successive intermediates of this type. The nature of A - C, or X N I as designated by Chance & Williams (1956). has remained an enigma. Four theories, those of Viklas & Lederer (1962), Grabe (1964), Wang (1967) and Griffiths (1965), which all postulate a chemical mechanism for oxidative phosphorylation in terms of functional groups, have avoided the enigma and reverted, despite the large body of evidence to the contrary, to the original Lipmann scheme which requires an oxidation to produce the energy-rich phosphorylated compound directly. Boyer (1965) has championed the acyl moiety of an activated carboxyl group as the most likely candidate for the group of high transfer potential attached to “-“. He has proposed that this potential comes directly from unspecified conformational changes of the respiratory carriers, and has extended this concept to other processes involving transduction of free energy. Falcone (1966) has proposed that the energy-rich intermediate formed by electron transport is a thiol ester, and has used his theory to interpret his results on mitochondrial ion transport

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(Faicone & Hadler, 1968). Chance, Lee & Mela (1967) have recently compared 13 contenders for the title of X - I, and conclude that six of these make good chemical sense, but may prove too unstable to identify. An excellent critical survey of these and similar hypotheses, and of pertinent model reactions, is available in the recent review by Lardy & Ferguson (1969). Inability to pin down X - I, to isolate it, or to find it in situ by various physical methods, has recently brought disenchantment with the whole concept, with the result that two major “non-chemical” hypotheses have appeared. Mitchell (1961, 1966a,b) has put forward a hypothesis which replaces X N I as a chemical entity with the free energy available from a potential and proton gradient across the mitochondrial membrane; he has named it the chemiosmotic hypothesis. This hypothesis requires that the primary business of the electron transport through the respiratory chain be formation of a proton gradient and membrane potential across the mitochondrial membrane, whose free energy is utilized to make ATP or carry out other energy-linked functions. The chemiosmotic hypothesis does provide a new and valuable viewpoint from which to examine the complexities of mitochondrial ion transport. But replacement of a chemical species by a gradient does not seem to have any conceptual advantage in the discussion of oxidative phosphorylation, except the urge to isolate a gradient is lacking. Some discrepancies between the predictions of the chemiosmotic hypothesis and experiment have been put forth by Chance, Lee 8t Mela (1967), and it has been critically reviewed by Slater (1967), Pullman & Schatz (1967) and Lardy & Ferguson (1969). Another viewpoint has been put forward by Penniston, Harris, Asai & Green (1968) and Harris, Penniston, Asai & Green (1968). They postulate that the free energy of electron transport is conserved directly by conformational changes of the mitochondrial membrane which can exist in an energized or non-energized state. These states are macroscopic as opposed to molecular phenomena, since the evidence for their existence comes from electron microscopy. A similar hypothesis has been put forward by Hackenbrock (1968), also on the basis of electron microscopy. Despite the ingenuity of the “non-chemical” hypotheses, the fact remains that phosphorylation of ADP to ATP coupled to electron transport is a chemical reaction with fixed stoichiometry towards electron transport. This can be expressed as the ratio ADP/O where 1 g atom 0 is used to symbolize transport of two electrons. The ADP/O stoichiometry of three for the oxidation of NAD-linked? substrates and of two for oxidation of succinate in the presence of rotenone is well documented (Lehninger, 1964; Racker, 1965). This stoichiometry holds with coupled mitochondria even under conditions t Abbreviations: NAD: oxidized form: NAD+). T.B.

nicotinamide

adenine dinucleotide

(reduced form: NADH; 16

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of respiration stimulated by valinomycin in the presence of potassium (Hijfer & Pressman, 1966). The oxidation of N,N-tetramethyl-p-phenylene diamine kept reduced by ascorbate gives an ADPjO stoichiometry of one in the presence of antimycin A which inhibits electron transport from reduced cytochrome b (Tyler, Estabrook & Sanadi, 1966). Higher and nonintegral ADPjO stoichiometries have been reported; the ephemeral nature of these values has been reviewed by Pullman & Schatz (1967). The ADP/O stoichiometry is in one-to-one correspondence with three sets of respirator> carriers which act as sites of interaction of ADP with the respiratory chain: these coupling sites were located by Chance & Williams (1956). Assays for each coupling site have been worked out: for site I between NADH and cytochrome b by Schatz (1967), for site IL between succinic dehydrogenasecytochrome b and cytochrome c by Lee, Sottocasa & Ernster (1967). and for site III between cytochrome c and oxygen by Sanadi & Jacobs (1967). In each case, the ADP/O or ADP/2e stoichiometry for the site approaches one. These stoichiometries are readily explained by the generation of one chemical species by transfer of two electrons between two carriers at a given coupling site. They are most difficult to explain by gradients or morphological changes whose functional parameters can assume a continuous range of values. The “non-chemical” hypotheses must ultimately be translatable into chemical terms if they are to account for oxidative phosphorylation of ADP in a satisfactory manner. It seems reasonable, therefore, to start from chemistry in formulating such a hypothesis, and this paper presents one based on known reactions of organic chemistry for the mechanism by which free energy from electron transport through the mitochondrial respiratory chain may be conserved. 2. Thermodynamic (A)

Considerations

NOMENCLATURE

Thermodynamic coupling in open systems (Prigogine, 1961) may be considered a process by which the affinity or negative free energy of one chemical reaction may be conserved or stored as positive free energy in a chemical species which can undergo another reaction to release that free energy. Consider the reversible reaction T+ T*. Conversion of T to T* is endergonic;? reversion of T* to T is exergonic-1 and this process can be thought of as releasing the free energy stored in T *. In this case, T* is an energy-rich molecule. If the reaction T $ TX is coupled to an exergonic reaction, the free ./_Coryell (1940) introduced

the terms “endergonic”

and “exergonic”

specifically Ibt

reactionsoccurringwith AG” 3, 0 and AC’ _ 0, respectively, in contrast to the terms endothermicand exothermicreferringto heatchange.These terms were adopted by Lipmann (1941). By extension,the term “isergonic”refersto reactionswith AG’ : 0.

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energy released by the latter reaction can be considered as stored in the high energy species T*. In this paper, most of the reactions which interconvert low and high energy chemical species are of the type AG’ > 0. T+T*+H,O; (1) The species T* is an anhydride of T; chemical free energy stored in T* is designated the anhydride storage potential of T*, and is defined by AG” or AC’ for the reverse of (1): T*+H20=T (2) This is closely analogous to the method for defining a scale for the group transfer potential of a group X (Lipmann, 1941), proposed by Klotz (1967), as AC” or AG ’ for the reaction : XNI+H~O~XOH+IH (3) where X is the group transferred to the oxygen atom of water. The same convention is used in this paper. In describing redox potential values, the symbol E,,,, for midpoint potential (referred to the Normal Hydrogen Electrode) at pH = x is used, as recommended by Clark (1960). The approximation is made that all activity coefficients are unity in this system, even in the lipoprotein milieu of the mitochondrial membrane, so that potentials can be related directly to concentrations. (The approximation is extreme, but it is perhaps the only reasonable one to make in the absence of any information concerning these coefficients, and it has the virtue of simplicity.) The difference in midpoint potentials of a pair of chemicals capable of undergoing redox reactions is called a potential span and is designated AE,,,, at pH = 7. (B)

FREE ENERGY

REQUIREMENTS

OF OXIDATIVE

PHOSPHORYLATION

The anhydride storage potential of X w I required to maintain the phosphate potential observed in the controlled or state 4 respiration of mitochondria (Chance & Williams, 1956) in the presence of EDTA to complex all external Mg *’ , has been calculated to be 15.6 kcal/g atom 0 by Cockrell, Harris & Pressman (1966). They used a value of AGbb, = -9.6 kcal/mol for the hydrolysis of ATP to ADP and Pi in the absence of Mg*+ at pH = 7.8, where AGb,,, is the standard free energy at the given pH, and they measured the concentrations of ADP, ATP and Pi present in the medium in state 4 at this pH. A comparable result was obtained by Klingenberg, Heldt 8z Pfaff (1969). Slater (1969) has reported that the external phosphate potential in state 4 is constant over the pH range 6.85 to 8.45 with an average of 15.5 kcal/ mol by the same method, using the same values of AGb,,. A recent recalculation of the values of AG’,, as a function of pH and Mg*+ concentration by

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Phillips, George & Rutman (1969) and Alberty (1969) showsthat thesevalues are low, and that the phosphate potentials as measuredare nearly - 17.0 kcal! mol. The measurementswere made with succinate as substrate in the presence of rotenonc, conditions under which complications invol\ ing substrate phosphorylation and energy-linked reduction of cndogenous pyridine nucleotide are absent, and under which the ADP/O stoichiometry is two. In order to maintain an external phosphate potential of 17.0 kcal/mol in state 4, which with slowly respiring, tightly coupled mitochondria lies near equilibrium (Slater, 1969), the oxidation of succinate must provide 2 x 17.0 kcal or 34.0 kcal/mol. The midpoint potential Em7 of the succinate-fumarate couple is 430 mv (Borsook, 1935; Burton & Krebs, 1953). The value of AEm between succinate and oxygen is +780 mv, using Em, = +810 mv for the O,--H,O couple (Clark, 1960). This potential span corresponds to AC’ = 36.0 kcal/mol succinate oxidized or g atom 0 consumed, and thus the free energy is available without need for contribution from extreme concentration ratio terms. The efficiency of sitesII and III which are involved in energy conservation during succinate oxidation is evidently close to 100:‘,‘,. That the efficiency of converting exergonic substrate oxidation to endergonic ATP formation from ADP and Pi is close to 1000,: has been demonstrated by the enthalpy measurementsof Poe & Estabrook (1968) with rat liver mitochondria treated with rotenone, using succinate as substrate. The quantities AEn,, and AC ’ are accessiblefor the carriers of the respiratory chain, and they are used directly to balance the free energy accounts of mitochondrial energy conservation. While this is an approximation, it is a fairly good one for the carriers of the respiratory chain in the aerobic steady state. The ratio of oxidized to reduced form of a given carrier in isolated mitochondria in this state is rarely outside the limits of 0.1 to 10. and AC z AC’ to within 2 kcal. Chance & Williams (1956) located site II for ADP interaction with the respiratory chain between cytochromes b and c; site I is located between NADH and cytochrome b. The potential span between NADH and cytochrome b (Schatz, 1967) should give an estimate of the free energy available to site I. The value of E,n7 for the NAD+/NADH couple is -320 mv (Burton & Krebs, 1953; Burton & Wilson, 1953; Rodkey, 1955, 1959). The Em7value for cytochrome b in submitochondrial particles is about + 70 mv (Holton & Colpa-Boonstra, 1960; Urban & Klingenberg, 1969), and in intact mitochondria appears to be about +30 mv (Caswell, 1968). A compromise value of + 50 mv gives a potential span AE,,, = f370 mv, corresponding to 17.0 kcal/2e. The potential span between NADH and cytochrome b, is therefore adequate to maintain the observed phosphate potential. The potential span between cytochrome b and oxygen, which encompassessites

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II and III, is + 760 mv, corresponding to AG ’ = - 35.0 kcal/2e, which is still sufficient to provide an average of 17.0 kcal/2e at each coupling site. However, the potential span of 180 to 200 mv between cytochrome b and c, corresponding to site II, is too small to maintain this phosphate potential, as is the potential span between cytochromes c and cytochrome oxidase (Slater, 1966); the latter corresponds to site III for interaction of ADP with the respiratory chain (Chance & Williams, 1956). The free energy obtained from oxidation of reduced cytochrome oxidase and oxygen-a potential span of at least +400 mv-must be made available to sites II and III if the high, observed efficiency of phosphorylation is to be maintained. A fourth site of energy conservation must therefore be postulated between cytochrome oxidase and oxygen. The ADP/O stoichiometry of the respiratory chain requires three coupling sites, each of which produces one chemical species per two electrons transported which in turn results in the phosphorylation of ADP. The free energy requirements of the respiratory chain demand utilization of the additional source of free energy provided by oxidation of reduced cytochrome oxidase for coupling sites II and III, a point emphasized by Slater (1969). It is evident from the thermodynamic requirements of the respiratory chain that the model compounds usually proposed as high-energy intermediates in oxidative phosphorylation do not have a large enough anhydride storage potential. Simple thiol esters are known to phosphorylate ADP to ATP in the presence of phosphate (Racker, 1965). The group transfer potential of the acyl moiety of a thiol ester is close to that of the phosphoryl moiety of ATP (Klotz, 1967; Jencks, 1968), so the reaction is isergonic. This group transfer potential is 8 to 10 kcal/mol, which falls far short of the required free energy. Another means of storing free energy in a chemical in addition to that of the group transfer potential of an active ester is required to provide a mechanism for oxidative phosphorylation which is in accord with experiment. The hypothesis presented in the following section specifies an anhydride storage potential for X - I of 17.0 kcal corresponding to nearly perfect energy conservation; the hypothesis can evidently accommodate lower values of this potential. (C)

MIDPOINT

POTENTIAL

REQUIREMENT

FOR

THE COUPLING

REACTION

The midpoint potentials of the carriers of the respiratory chain range from Em7 = -320 mv for pyridine nucleotide to Em7 = > + 330 mv for cytochrome uag (Caswell, 1968). The midpoint potentials of the redox reactions which couple electron transport to energy conservation should match approximately those of the carriers with which they react in order that both the oxidized and reduced forms of the reactants be available in kinetically

240

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useful concentrations. One reaction exists which is biochemically reasonable. and whose Em7 depends on reaction conditions and covers the range. It is the thiol-disulfide reaction: H2 -i- RSSR +2RSH. In aqueous solution, Em7 for this reaction is - 320 to - 340 mv, as measured for cysteine, glutathione, thiolglycolic and thiolactic acids (Krebs & Kornberg, 1957; Clark, 1960). The reaction in the vapor phase has a much more positive midpoint potential. The AGJ in the vapor phase of the symmetrical dialkyl disulfides and corresponding thiols from methyl through ir-butyl and AC” for the redox reaction are shown in Table 1. The values of AC” are TABLE

1

AG,” values for RSH and RSSR at 298 K; vapor phase, ideal gas R CH,GH,nC, H7 &Ho-

AGj, kcal/molt RSH -11.91 - 10.66 -9.02 -6.90

RSSR

AC”, kcal/mol H, i- RSSR + 2RSH

-15.56 -13.76 - 10.24 -6.22

--8.2 -- 7.5 --7.8 -- 7.6

t Values are those of Scott & McCollough

(1961).

essentially constant for ethyl through n-butyl with an average value of -7.8 kcal/mol, corresponding to Em, = + 170 mv= Em,. It is postulated that the vapor phase is a far better approximation to the hydrophobic interior of a protein than is aqueous solution. The difference in values between the vapor phase and aqueous solution arises from free energies of solvation associated with the latter. The standard potential of + 170 mv is about the most positive which could be anticipated for the disulfide-thiol reaction in a protein. More negative values would arise from interaction of other groups on the protein with the polar thiol groups in question to provide a form of solvation. The thiol-disulfide reaction is peculiarly well fitted to electron transport through the coupling sites of the respiratory chain since its midpoint potential, depending on environment, can vary from that of pyridine nucleotide at - 320 mv to nearly that of cytochrome c at around + 250 mv (Rodkey & Ball, 1950). Experimental support for the involvement of this reaction in the energy conservation has been provided by Sanadi, Lam & Kurup (1968) who find that thiol groups are essential to energy conservation in submitochondrial particles.

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Falcone (1966) proposed that the structure of X N I be a thiol ester formed by reduction of a carboxyl group to an aldehyde, followed by oxidative coupling of the aldehyde with a thiol group. The value of AG” for the reduction of acetic acid to acetaldehyde in the vapor phase corresponds to E = - 76 mv = Em7 as the most positive value applicable to this reaction (I?izer & Weltner, 1949; Rossini, Wagman, Evans, Levine & Jaffe, 1952; Weltner, 1961; Child & Hay, 1964). In the aqueous phase, E,,,, = - 598 mv (Krebs & Kornberg, 1957). The range of values covered by this model redox reaction are too negative to meet the midpoint potential requirements of the respiratory chain, and, despite its attractive features, this reaction was discarded in favor of the thiol-disulfide reaction in formulating this hypothesis. 3. Outline of the Hypothesis One basic premise underlying this hypothesis is that exergonic electron transport from substrate to oxygen and the endergonic reaction, which produces the primary intermediate conserving free energy as the anhydride storage potential of X N I, are chemically coupled at three coupling sites. This requires that the equivalents pass through the coupling sites which themselves act as respiratory chain carriers and are present at concentrations comparable to those of the other carriers. It is also postulated that the same chemical reactions occur at the three different coupling sites of the respiratory chain, designated site I, II and III (Chance & Williams, 1956) and that the free energy conserved at these sites is then transferred by similar chemical reactions to its points of utilization in the mitochondrial membrane. It is also a basic premise of this hypothesis that, in addition to the three chemical coupling sites required by the observed ADP/O stoichiometry, there is a fourth site of energy conservation involving the reaction of oxygen with the terminal oxidase, thermodynamically coupled to site II and III and used directly in the production of X N I at these two sites. (A)

CHEMICAL

REACTIONS

OF THE COUPLING

SITES

Each coupling site is postulated to be a lipoprotein-enzyme complex in the mitochondrial membrane, consisting of an electron accepting and electron donating site and two tightly bound proteins in a hydrophobic region which function in energy conservation. One of these proteins, &, takes part in electron transport by means of three active thiol groups and exists in its energized form E,* as a disulfide-crosslinked thiol ester. The other protein is a transducer enzyme, designated E,, which reacts rapidly with E,* to acquire a thiol ester crosslink, yielding E; and regenerating Es. Since both proteins are essential to the function of the coupling site, the overall reaction is written:

242

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E,.E,-,E,*.E,+Es* ET. Each coupling site has its specific enzyme complex Es . E,. The detailed mechanism for this sequence of reactions is shown in Figs 1, 2 and 3, in which electron transport occurs as hydrogen atom transfer in one-electron stages from one hypothetical respiratory carrier M to another carrier N of more positive E,,,,. In Fig. 1, the coupling site protein

i

-

FIG. I, Reduction of the primary disulfide Es of the de-energized coupling site enzyme complex Es . E, by sequential electron transport (shown as H atom transport) to give the reduced form Hz . Es . ET, followed by intramolecular transfer to reduce Es to the trithiol form ESH . E,. The trithiol with free carboxyl is isergonic with the form dithiolthiol ester pius water.

starts with one free carboxyl, one free thiol, and one disulfide bridge; it is referred to as the primary disulfide, Es. The transducer protein has one free carboxyl and one free thiol group. In this state, the coupling site is deenergized and is designated Es .E,. Reduction of the E, moiety cleaves the disulfide bridge and yields the trithiol Es,, *E,. The free carboxyl group of the trithiol is in a hydrophobic environment so that it remains unionized,

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243

and it is situated so that it can readily interact with one thiol to form thiol ester and water, as shown in Fig. 1. This reaction occurs with AG ’ = 0; the two isergonic forms of the trithiol are considered the same species GH -ET for free energy calculations. The reaction of the thiol group in proximity to the unionized carboxyl group to form thiol ester and water is analogous to the reaction of the serine group of chymotrypsin to form acyl enzyme directly from substrate with an unionized carboxyl group, which has been demonstrated spectrophotometrically by Miller & Bender (1968). Formation of thiol ester and water changes the spatial relation of the other two thiol groups to the oxidizing site, and oxidation of one thiol to thiyl radical ensues as shown in Fig. 2. Abstraction of the hydrogen atom from the remaining thiol by the thiyl radical is followed by concerted oxidation and coupling of the two thiyl radicals to form the disulfide crosslink. This

LllzJIL+ SH

HOOC

-

2

H.N, E:E, SH

3 HOOC

FIG. 2. Oxidation of the reduced form of the coupling site enzyme ESH . E, in the dithiol-thiol ester form to the energized disulfide-crosslinked thiol ester E* . ET by sequential hydrogen atom transport to the oxidized carrier N.

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crosslink freezes the coupling site protein into a highly strained conformation which also removes the thiol ester from proximity with the bound water molecule. The negative entropy change of the crosslinking reaction provides an anyhydride storage potential equivalent to the potential span between Es, and N,,, all of which is conserved as - TAS. The thiol ester of E,* contributes nothing per se to the anhydride storage potential since its formation from carboxyl and thiol is isergonic, but it does provide a functional group by which the anhydride storage potential of Et is transferred to E, by transesterification. This transesterification is shown in Fig. 3. Interaction of the three thiol groups is essential to this step. The thiol group liberated by reaction of E,* with E-r to give the mixed anhydride reacts rapidly with the strained disulfide bridge to regenerate the original, unstrained disulfide bridge and thiol group

- H,O

!i

tHzO

Es E; --1

,1 HSsy;ooH

o;z -

;-

FIG. 3. Energy transfer by transesterification from the disulfide-crosslinked thiol ester Ei to the transducer enzyme E, * to produce the energized form of the coupling site enzyme complex ES . I?,“.

of the primary disulfide Es. Formation of the thiol ester EF also regenerates the carboxyl group of Es, during which processthe water molecule is removed from the binding site. This dissociation step is a crucial one to the energy conservation process. It is postulated that removal of the water molecule from coupling sites II and III is the reaction which thermodynamically couples the fourth energy conservation site involving the reaction of oxygen

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with the terminal oxidase to these two sites. This postulate is developed in the following section. The anhydride storage potential of E; consists of two parts: the anhydride storage potential of EB derived from oxidation of &n and the negative free energy accompanying removal of water and rebinding of the carboxyl of Es to the coupling site complex. These parts sum, on the average, to 17.0 kcal/ 2e. The additional free energy in ET” over that provided by the group transfer potential of the thiol ester is conserved as -TAS of the strained conformation of this protein held in place by the thiol ester crosslink. The strain arises from

the loss of vibrational and rotational degrees of freedom of the protein caused by the crosslink (Flory, 1953). This mechanism provides a specific molecular model for the conservation of free energy by conformational change as proposed by Boyer (1965). The series of reactions in Figs 1, 2 and 3 provides a redox cycle involving thiols and disulfides whose end product is the crosslinked, energy-rich thiol ester E; with the required anhydride storage potential. In this hypothesis, ET” is the source from whence “N ” is distributed to its various destinations in the mitochondrial membrane. Thiol groups are peculiarly fitted to the sequence of reactions in Fig. 2 since they are uniquely reactive in hydrogen atom transfer and radical coupling reactions (Walling, 1957; Pryor, 1962). The inter- or intra-molecular coupling of polymeric radicals to form a covalent bond proceeds very rapidly with large negative entropy change (Walling, 1957) so the coupling of two thiyl radicals to form disulfide (Pryor, 1962) is also well fitted to store free energy in the manner postulated. The oxidation reaction requires two sequential one-electron transfers and is thus compatible with a respiratory chain containing one-electron carriers. All the reactions shown in Figs 1, 2 and 3 are taken to be reversible. Hydrogen atom transfer to a sulfur atom of disulfide, required to reverse the coupling reaction of Fig. 2, is one among a number of known, rapid radical displacement reactions on sulfur in disulfides (Pryor, 1962). The other reverse reactions are fully consistent with the known chemistry of the various species postulated. Reverse electron transport from carrier N to carrier M requires the disulfide-crosslinked thiol ester E: as an intermediate, in accord with the observed energy requirement for this process. (B)

ENERGY

CONSERVATION

AT

THE

TERMINAL

OXIDASE

This hypothesis postulates that the terminal oxidase in the mitochondrial membrane interacts with coupling sites II and III to utilize the free energy of reaction of this enzyme complex with oxygen. The specific interaction proposed is removal of the water molecule which is generated at each site by transport of two electrons through the site. The two coupling sites are assumed

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to be so arranged in the mitochondrial membrane that the appropriate water binding sites of the cytochrome oxidase complex are readily accessible to the water molecules. These binding sites have low affinity for water and readily release it to the medium when the oxidase is reduced. Upon reaction of lhc reduced cytochrome oxidase with oxygen, the enzyme complex undergoes a conformational change to a form in which the water binding site is empty but has a high affinity for water. In the intact mitochondrial membrane, the only water molecules available to the site are those from the two coupling sites; entry of water from the medium is prohibited by the membrane structure. The oxidized, dehydrated form of the oxidase is thus an energy-rich form, in which the free energy of oxidation is largely conserved. The free energy relationships are qualitatively the following: AG; r0 XR.H20=XR+H20 (4) x R- -x*

ox

AG;rO

(3

AC; < 0. X,*, + H,O + X,, * H,O (6) In this scheme, X stands for a water binding site on the cytochrome oxidase complex; the subscripts R and ox stand for reduced and oxidized forms of the enzyme. The values of AC ’ for the first two reactions are approximately zero. The value of AGk is negative but large, since it corresponds to the potential span between cytochrome oxidase and oxygen. The energy rich form X,*, has, therefore, an anhydride storage potential corresponding to AGb. The potential spans for coupling sites II and III and the cytochrome au,-oxygen reaction are listed in Table 2, on a two electron basis. Four water molecules are bound per active unit of cytochrome oxidase of four equivalents capacity-presumably two hemes corresponding to cytochromes a and a3 and two copper atoms (van Gelder, 1966)-capable of reducing one molecule of 02, or two water molecules/g atom 0. The midpoint potential of cytochrome oxidase, here represented as aa3, is chosen to be + 370 mv to give AEm = +200 mv between the coupling site trithiol Es, at site III. This, in turn, is assigned a midpoint potential at + 170 mv, the most positive one applicable to the thiol-disulfide equilibrium (Table 1). The value of + 370 mv is close to that of E,,,7,4= + 330 mv obtained by Caswell(1968) for cytochrome aa in cyanide-inhibited, uncoupled rat liver mitochondria with a combined potentiometric and spectrophotometric method. He points out that, under the conditions of his measurement, this value is a minimum and that the actual value is probably more positive. Caswell’s value of + 330 mv is some 50 mv more positive than the values +278 mv reported by Minnaert (1965) and + 285 mv reported by Tzagaloff & Wharton (1965) for isolated cytochrome oxidase. (The complexity of the isolated cytochrome oxidase system and the difficulties inherent in making such a potential measurement have been

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2

Values of Em,, AE,, and AC’ pertinent to energy conservation at sites ZZ and ZZZ Site II Carriers

E&v)

Cyt. b

t50

Em.

$50

t

AEdmv)

0 II

Cyt. c

$200

+250

AC’ (kcal/I!e)

0

-9.2 -

Site III Cyt. c

$250

E 8H. III

+170

Cyt. au3

$370

Cyt. aa3

+370

02

+810

-80

+3.7

$200

-9.2

+440

-20.2

t See text for source of Em7 values.

reviewed by Slater, van Gelder & Minnaert (1965)). This complexity is underlined in a recent paper of Horio & Ohkawa (1968), who reported three values of E,,,, for cytochrome oxidase: + 360, + 300 and + 208 mv. Some very recent data of Dutton & Wilson (1970), which are the most reliable obtained so far with intact mitochondria, give E,,,, values of + 190 and -f-395 mv for cytochromes a and a3, respectively, which suggests that coupling site III actually lies between these two cytochromes. These values are satisfyingly close to the Em, values of + 170 and + 370 mv proposed for site III. It makes no difference to this formulation whether the coupling site enzyme lies on the path of electron transport between a and a3, or whether it lies between c and aa as in the generally accepted placement of site III, as long as the low potential carrier for this site has a midpoint potential near + 170 mv. To accommodate the Dutton-Wilson scheme, it suffices to rewrite the site III sequence as c *aE,,, Ill -a3 rather than as c * E,, ,,, *aas. The latter, generally

248

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accepted placement of site III is used in this paper, pending further elaboration of the work of Dutton & Wilson (1970). The midpoint potential for cytochromc c, here taken to include cytochrome cl, is taken as +250 mv. This value is the one estimated by Caswell (1968) in rat liver mitochondria, and is also the one measured recently in submitochondrial particles (Drs P. L. Dutton and C. P. Lee, personal communication). In addition, Rodkey SCBall (1950) report Em7 = +250 mv for isolated cytochrome c from an extensive potentiometric study. Green, Jarnefelt & T&dale (1960) found Em7 for their cytochrome c1 preparation to be + 220 mv. Cytochrome b is assigned E,,,, = + 50 mv, as discussed previously, and the coupling site trithiol E,,, at site II is given the same E,,,7. [Ubiquinone has nearly the same midpoint potential as cytochrome b (Urban & Klingenberg, 1969) and so contributes nothing to this analysis. This carrier will be discussed in the next section in connection with site I.] The potential span AE,,,, between cytochrome b and oxygen is + 760 mv, corresponding to 35.0 kcal/g atom 0. Conservation of 17.0 kcal/2e at each coupling site consumes 34.0 kcal. It is assumed that the loss of 1.O kcal to irreversibility occurs in the reaction of cytochrome oxidase with oxygen. leaving 19.2 kcal/g atom 0 available to sites II and 111. There is, a priori, no reason why the sites of water binding should have different anhydride storage potentials; in this hypothesis they are each assigned an anhydride storage potential Ac;L = -9.6 kcali2e equivalent to -9.6 kcal/H,O. Two binding sites, each binding one water molecule, or one site binding two water molecules, are considered equivalent in this formulation. The free energy available at coupling site II sums to 18.8 kcal/%e, while that of coupling site III sums to 15.1 kcal/2e; together the two sites sum to 33.9 kcal/2e for an average of 17.0 kcal/2e at each site. In considering the operation of the mitochondrion in vivo, sites II and 111 must be considered as a unit. The mitochondrion in vivo does not subsist on ascorbate plus N,N-tetramethyl-p-phenylene diamine in the presence of antimycin A, nor does it subsist on succinate plus ferricyanide. All physiological substrates which bypass site I provide reducing equivalents for both sites 11 and III. Further, these two coupling sites utilize the cytochrome chain for electron transport, rather than the flavoprotein chain (Chance, Bonner & Storey, 1968). Both in terms of architecture within the mitochondrial membrane and in terms of architecture within the electron transport carriers themselves, sites II and III appear to be a single functional unit in vivo for energy conservation. Experimental support for the proposed energy conservation reaction at the cytochrome oxidase site comes from the observation of Bonner & Plesnicar (1967) that, in mitochondria isolated from etiolated bean seedlings, cytochrome a3 is but partially reduced in the transition to anaerobiosis from

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state 4. All other respiratory chain carriers are reduced. A similar observation was made with rat liver mitochondria by Wilson (1967). Complete reduction was attained on adding uncoupler (Bonner & Plesnicar, 1967) or on adding ADP (Wilson, 1967). These observations imply that there exists an energized form of the oxidase in which cytochrome +---the hemoprotein which reacts directly with oxygen (Keilin & Hartree, 1939)-tan no longer accept electrons from cytochrome a. Hydration of this form by addition of uncoupler or ADP releases the inhibition. The existence of such an inhibited form of cytochrome a3 explains the observation that evolution of oxygen by mitochondria from water has never been observed, even though such evolution would be predicted from a free energy analysis of the overall system. The inhibited form of a, may have a midpoint potential equal to or lower than that of a, thus making electron transport between a and (I~ energetically neutral or unfavorable and making oxidation of water energetically most unfavorable. This situation is compatible with the formulation of equations (4), (5) and (6). Experimental support for it has been given by Dutton & Wilson (1970) who find that addition of ATP to the mitochondrial suspension reduces the Em7 of a3 from +395 to +3OO mv. Additional support for a site of energy conservation at the terminal oxidase is provided by Muraoko & Slater (1969). They studied the transitions in the redox state of cytochromes (c+ cl) and (a+~) between state 3 and state 4 in rat liver mitochondria utilizing ascorbate plus TMPD as substrate, and concluded that a site of energy coupling occurred between cytochrome a3 and oxygen by application of the crossover theorem (Chance, Holmes, Higgins & Connelly, 1958). (C)

DISTRIBUTION

AND

UTILIZATION

OF CONSERVED

FREE ENERGY

The distribution of chemical free energy through the mitochondrial membrane is postulated to occur by isergonic active ester transfer from E; to a similar enzyme E,, yielding E,” with the same anhydride storage potential as Ep ; E; acts as the general “N ” carrier for the mitochondrion. The process is shown in Fig. 4(a). It is postulated that E,” has access to all coupling sites through their respective transducer enzymes E,. It is further postulated that the total content of E, in the mitochondrion is enough greater than the content of coupling sites that E; can function as a central pool for all the energylinked functions occurring in the mitochondrial membrane. The group XH in Fig. 4(a) is not specified; two likely candidates are thiol or imidazole. The enzyme E,- may perform its function in two ways. It may move through the membrane as a single protein to its point of utilization. Or there may be actuai “w” transport resulting from active ester transfer between membrane-bound proteins according to the process E,“, + E,, Z$ E, r + E,“. In the latter case, one

250

E,+E,.

E” P

y E,+E,

E;P

EP (b)

FIG. 4. (a) Isergonic energy transfer from the transducer enzyme ET of the coupling site complex to the carrier enzyme E, by active ester transfer. The reaction is freely reversible. (b) Scheme for oxidative phosphorylation of ADP with P, involving energy transfer from ED- to E, to give the crosslinked active ester E,“, followed by reaction with phosphate to give E,“P which phosphorylates ADP. All reactions arc reversible.

might expect a number of speciesof E,, and one or a combination of these may correspond to coupling factors F1 and F, (Pullman, Penefsky, Datta & Racker, 1960; Kagawa & Racker, 1966; Fessenden& Racker, 1966). A reaction sequenceby which the free energy stored in E, can be used to synthesize ATP from ADP and Pi is shown in Fig. 4(b). The reactions are all reversible so that the free energy of ATP hydrolysis can be utilized to produce E” resulting in reversed electron transport (Chance, 1961; Chance & H%unger, 1961) according to the reaction scheme shown in Fig. 3. The phosphorylation schemeinvolving E, in its three forms could account for the various features of the Pi-ATP exchange and ADP-ATP exchange reviewed by Racker (1965), and the oxygen bridge to the terminal phosphate of ATP originating from ADP (Hill & Boyer, 1967), all observed during oxidative phosphorylation. The rapid exchange of ‘*O from Hz”0 into inorganic phosphate (Cohn, 1953; Cohn & Drysdale, 1958) and loss of ‘*O label from phosphate into water could be accommodated by competition of water with ADP for E,“P. The site of oligomycin inhibition is tentatively placed between EC and EP P and that of aurovertin between E,” P and ATP, as suggestedby Ernster, Lee & Janda (1967) to account for the differences between these two inhibitors when used with submitochondrial particles. Inhibition ot‘ E, hydrolysis by oligomycin would account for the restoration of respiratory control in submitochondrial particles by this inhibitor first reported by Lee & Ernster (1966~). Note that oligomycin has no effect on the reactions of Er .

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Accessibility of E,” to all coupling sites allows energy conserved at one site to be utilized at another one. The first recognized example of this phenomenon was the energy-linked reduction of pyridine nucleotide by succinate reported by Chance (1961) and Chance & Hollunger (1961). A variety of other examples are reported in the first Johnson Foundation Colloquium (Chance, 1963). Energy-linked reduction of pyridine nucleotide by succinate can occur by reaction of E,” formed at sites II and III to form ET at site I which then supports reversed electron transport. The scheme for electron transport in reverse through coupling site I is shown in Fig. 5. Q -‘=b,

\

Es, ,/PC,,

-

NAD

FIG. 5. Reversed electron transfer formulation of site I. Reduced ubiquinone is the high potential reservoir of reducing equivalents transported to NAD which, as NADH, acts as the low potential reservoir. Free energy for the reverse electron transport is supplied by active ester transfer from E,” to Es * & Reverse electron transport occurs as shown in Figs 1, 2 and 3 from H - Nn to M,,.

Ubiquinone is taken to be the high potential reservoir for reducing equivalents which feeds to pyridine nucleotide, the low potential reservoir. Two flavoprotems, Fp,, and FpDl are taken to be the respiratory chain carriers directly associated with coupling site I according to the formulation of Chance, Ernster, Garland, Lee, Light, Ohnishi, Ragan 8c Wong (1967). Site I connects to site II at cytochrome b either through ubiquinone (Krbger & Klingenberg, 1967) as recent experiments of Ernster, Lee, Norling 8z Persson (1969) would strongly imply, or through one or more of the high potential flavoproteins which can react with both ubiquinone and cytochrome b, as suggested for plant mitochondria by Storey & Bahr (1969). Either formulation is perfectly compatible with this hypothesis. A mechanism by which E,” might interact with the energy-linked transhydrogenase to give NADPH from NADH is depicted in Fig. 6. Reduction of ET;i by NADH to the aldehyde intermediate is exactly analogous to the reaction of NADH with the thiol ester intermediate of glyceraldehyde phosphate dehydrogenase (Harting & Chance, 1953; Boyer & Segal, 1954; Park, 1966; Racker, 1965). The group -YH of E, in Fig. 6 could be thiol by analogy with the dehydrogenase, or carboxyl. Oxidation of the aldehyde to the carboxyl group by NADP+ gives NADPH and ETH in an exergonic 17 l-3.

252

R.

T.

STOREY v

c=o c

+

CH +NADH

.NAD’ -

tNAD Y-

EM

H20cNADP’

_

+

a

e CH

+NADPH

z= Y‘ ETH

FIG. 6. Proposed mechanism for the energy-linked transhydrogenation of NADP+ by NADH. The group -YH is not specified but the two most probable candidates are -SH or -COOH. The hydride ion from NADH is transferred directlv to NADP+ via the aldehyde group and cannot exchange with the hydrogens of water or the enzyme.

reaction. It is postulated that NADP+ binds to the enzyme on the opposite side of the aldehyde group from NADH, so that labeled hydride transferred from NADH via the aldehyde to NADP+ gives NADPH with stereochemistry opposite to NADH, as required by the findings of Ernster (1965) and Lee, Simard-Duquesne, Ernster & Hoberman (1965). Hydride transferred via the aldehyde intermediate cannot exchange with water and thus is equivalent to direct transfer between pyridine nucleotides, in accord with the results of Ernster (1965) and Lee et al. (1965). The transhydrogenation equilibrium is shifted by -85 mv or +4 kcal/mol from its normal equilibrium value (Ernster, 1967). The loss of some 13 kcal/mol EC probably arises from re-equilibration of the two species through the non-energy-linked transhydrogenase also present in this system (Ernster, 1967) and possibly through direct hydrolysis of E& itself. In the reaction schemes of Figs 4, 5 and 6, phosphorylation, reverse electron transport, and energy-linked transhydrogenation all compete for the same intermediate E,’ ; this competition has been well documented by Lee & Ernster (1967). In this hypothesis, uncoupling is postulated to occur primarily by hydrolytic cleavage of the active ester group of the energy transducing proteins, in particular EC” and E;, to dissipate directly the anhydride storage potential as heat and regenerate the primary disulfide Es. E, at the coupling sites. The uncoupling agent need not be a weak acid as postulated by Van Dam & Slater (1967). Any compound which promotes hydration of the membrane such that water molecules have ready access to the coupling sites will act as uncoupler. In this class fall the group of uncouplers which increase the permeability ofartificial membranes to hydrogen ions (Liberman, Topaly, Tsofina, Jasaitis & Skulachev, 1969). These experiments, which show a correlation between increased hydrogen ion conductance of these membranes and potency

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253

as uncouplers, were interpreted as evidence for the chemiosmotic hypothesis (Mitchell, 1961) since such compounds should allow the collapse of a proton gradient. Since the hydrogen ion being conducted is actually a hydronium ion, these data are equally well interpreted as showing that such compounds give access in the membrane to protonated water molecules. Another type of uncoupler would be a compound which promoted direct electron transport between respiratory chain carriers and bypassed the coupling sites; no direct evidence for this type of uncoupler exists at present. Energy-linked cation transport may also be supported via E,“. Figure 7 t wembraneHA, R HA’

FIG. 7. Speculation for the mechanism of energy-linked ion-transport utilizing the anhydride storage potential of E;. The carrier with two carboxylate groups as ligands binds Ma+ in the membrane and transfers it to an interior binding site by ion exchange. Reaction of the carrier with E,” converts its carboxyl groups to an anhydride crosslink, trapping the ion inside. Hydrolysis of the carrier anhydride on the outer surface of the membrane in concert with binding a second Ma+ is not shown.

254

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T.

STOREY

shows a speculation on how this process might function for a divalent cation. A cation from the outside medium is bound to a membrane carrier with high affinity, the necessary free energy being provided by hydrolysis 01‘;t dicnrboxylic anhydride crosslink of the carrier at a specific site HI the memhran~medium interface. lsergonic ion exchange of the cation with a polyanion in the mitochondrial interior frees the carboxyl groups of the carrier which are reconverted to the dicarboxylic anhydride by E;. The cation is then trapped inside the mitochondrion. Migration of the carrier in anhydride form to its hydrolysis site completes the cycle. The schemein Fig. 7 is drawn for the binding of one divalent cation per carrier, and thus implies a 1 : I stoichiometry for cation: EC. The actual stoichiometry of ion transport will be strongly dependent on the anions present outside and inside the rnitochondrion. If an outside anion A- binds strongly to the cation to form MA ‘. then two such specieswill be transported per E;. Such a stoichiometry has been observed with Ca’+ under the proper conditions (Chance, 1965; Lehninger, Carafoli & Rossi, 1967). If the anions inside the mitochondrion can bind the cation strongly, then the carrier shown in Fig. 7 can deliver cations ud libitum to this anion, the free energy of transport being provided by formation of the complex rather than by hydrolysis of E;. In this case, E, is needed only to start the process of transport, and many cations would be transported per E;. This scheme is therefore quite compatible with the “super-stoichiometry” observed for Ca’ + transport under certain conditions (Lehninger et al., 1967). Chance (1965) has postulated a scheme for Ca’ ’ transport in mitochondria with a carrier similar to this. Falcone (1966) has postulated a detailed scheme of this general type which also includes the action of various antibiotics on mitochondrial ion transport. While intact mitochondria take up Ca2+ ion readily (Chance, 1965). submitochondrial particles obtained by sonication do not (Chance & Mela, 1967). Submitochondrial particles obtained by osmotic shock appear to accumulate some Ca’+, but by means of a process which appears very different from that operating in intact mitochondria (Loyter, Christiansen, Steensland, Saltzgaber & Racker, 1969). The brief speculation presented here predicts that CaZf is not transported in submitochondrial particles becauseit has nowhere to go; theseparticles do not contain anions and polyanions to bind the C‘s’ ’ 4. Discussion The chemical hypothesis developed in this paper differs from the chemiosmotic hypothesis of Mitchell (1961,1966u,b) in the fundamental postulate that the free energy derived from electron transport through the respiratory chain of the mitochondrial membrane is conserved as active ester crosslinks of

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specific mitochondrial proteins in the membrane, rather than as a generalized proton gradient or membrane potential across the membrane. The reactions E + E” consume one potentially ionizable carboxyl group per active ester formed, which in turn reduces the cation exchange capacity of the membrane. This should be observable as an uptake of hydrogen ion from the medium in exchange for bound cations. This hydrogen ion uptake has been observed in submitochondrial particles by Chance & Mela (1967) at a rate more compatible with a relatively slow esterification reaction than with the rapid rates of electron transport which the respiratory carriers can mediate. If the proposed hypothesis is correct, the hydrogen ion uptake should be observable upon energization in all preparations of mitochondrial membrane fragments which are free of matrix protein and capable of energy conservation, whether the membrane vesicles are “inside out” with respect to the inner membrane orientation of the intact mitochondrion (Chance & Mela, 1967; Chance, Lee & Mela, 1967), or “right side out”. This hypothesis would also predict that the response of the fluorochrome, 8-anilino-1-naphthalene sulfonic acid, to the energized state, observed by Azzi, Chance, Radda & Lee (1969), should be the same in both types of particle preparation. Both responses must be observed free of complications from transport of other cations. This aspect of the chemical hypothesis should be accessible to experiment. This hypothesis also predicts a high energy form of cytochrome oxidase which should be observable at phosphate potentials high enough that, on the basis of overall free energy considerations, oxygen evolution from water by reverse electron transport might be expected. This form may be the partially reduced form of cytochrome a3 in anaerobiosis observed by Wilson (1967) and by Bonner dc Plesnicar (1967). Verification of such a species may well have been found by Dutton & Wilson (1970). The chemical hypothesis also stipulates that the transducer enzyme E, at each site be a thiol ester in the energized form ET”, and proposes that E,” be either a thiol ester or an acyl imidazole. These two species have characteristic absorption spectra in the ultraviolet. Thiol esters have an absorption maximum at 235 nm (Racker, 1950; Noda, Kuby & Lardy, 1953; Harting & Chance, 1953), while acyl imidazoles have a maximum at 245 nm (Stadtman, 1954). The extinction coefficients of these species are unfortunately rather low, being in the range 2 to 4 rnM-l cm-l. In the case of ET”, the problem of spectrophotometric identification is quite formidable, since one is attempting to identify a component present at about the concentration of cytochrome c with one-tenth the extinction coefficient in the ultraviolet region of the spectrum where technical problems abound, particularly with turbid particle suspensions (Chance & Redfearn, 1961; Chance, 1964; Storey, 1967). Since

256

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virtually no spectrophotometry has been carried out with mitochondrial preparations in the ultraviolet region 210 to 260 nm, it is not surprising that a speciesidentifiable directly asET or Ez has not yet been found. The difficulties are not insuperable, however; a firm technical basisfor proceeding with such direct spectrophotometric methods in this region of the spectrum has been laid down by Chance (1964). This hypothesis predicts that uncoupling is caused by direct hydrolysis of either ET or EC, and hence requires that this hydrolysis, on transition from the energized to the uncoupled state with addition of uncoupler, be kinetically compatible with the same transition observed for the respiratory chain carriers. Direct spectrophotometric observation of this hydrolysis would test the prediction. The hypothesis presented in this paper has been biased towards mitochondria from mammalian tissues and yeast. In particular, only one cytochrome b has been postulated. Mitochondria isolated from the tissues of‘ higher plants have three b cytochromes identifiable by ditrerence spectrophotometry at low temperature (Bonner, 1965) and by their differing kinetic behavior (Storey, 1969). For the hypothesis to be properly validated, its ability to correlate the observations concerning energy conservation in plant mitochondria must also be tested. Whether this hypothesis can be extended to energy conservation processesin bacterial and photosynthetic organisms has not yet been investigated. At present, it is applied only to mitochondrial respiratory chain. I thank ProfessorJ. Higgins for exhaustive discussionsconcerningkinetic and thermodynamictheory, and ProfessorD. Garfinkel for hisintroduction to computer simulationtechniquesand computer time at the University of Pennsylvania,School of Medicine Computer Facility supportedby N.I.H. Grant FR-15. Many helpful ideaswere provided by ProfessorsB. Chance,C. P. Lee, and D. F. Wilson, of this Department, by ProfessorA. Azzi of the University of Paduaand ProfessorE. C. Slater of the University of Amsterdam. This work was supported by USPHS GM-12202 and was carried out during the tenure of Career Development Award 5-K3-GM-731 l-02. REFERENCES

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