Proc. NatL Acad. Sci. USAVol. 79, pp. 1064-1068, February 1982Biochemistry

On the enzymic mechanism of oxidative phosphorylation(coupling unit/coupling sequences/transmembrane. orientation/phosphoryl transferase)

DAVID E. GREEN AND H. VANDE ZANDEInstitute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706

Contributed by David E. Green, October 28, 1981

ABSTRACT Oxidative phosphorylation, like substrate-levelphosphorylation, involves oxidative conversion of inorganic phos-phate to a reactive species followed by interaction of this specieswith enzyme-bound ADP to form enzyme-bound ATP. The re-active species is a phosphoryl ester in substrate-level phosphory-lation and a phosphonium ion of orthophosphate in oxidative phos-phorylation. The coupled synthesis is mediated by a combinationoftwo classical enzymes in substrate-level phosphorylation and bya set ofenergy-coupled enzymes in oxidative phosphorylation. Thefull range of experimental evidence supporting this proposed en-zymic mechanism ofoxidative phosphorylation is presented as wellas the rationalization of phenomena that hitherto have eludedexplanation.

Oxidative phosphorylation was discovered in the late 1930s (1,2), but no explicit mechanism based on experimental evidencehas been proposed for this coupled synthesis. The main thrustof the present communication is that oxidative phosphorylationis a variation of the enzymic mechanism that has been shownto apply to the oxidative synthesis of ATP from Pi and ADP,coupled either to the oxidation of 3-phosphoglyceraldehyde (3)(Eqs. 1 and 2) or to the oxidation of a-ketoglutarate (4) (Eqs.3 and 4).

3-phosphoglyceraldehyde + Pi + NAD+

1,3-diphosphoglycerate + NADH + H+ + H20 [1]

1,3-diphosphoglycerate + ADP = 3-phosphoglycerate + ATP [2]

a-ketoglutarate + CoASH + NAD+ = succinyl-SCoA+ NADH + C02 + H+ [3]

succinyl-SCoA + Pi + GDP = succinate + CoASH + GTP [4]

The obvious mechanistic analogy between oxidative and sub-strate-level phosphorylations was missed primarily because theconcept of energy-coupled enzymes had yet to be developed(5): the enzymic basis of oxidative phosphorylation was not ap-preciated until 40 years after the discovery ofthe phenomenon.

The development of the argument is divided into five parts:the description and underlying assumptions of the proposedmechanism of oxidative phosphorylation; the evidence for themechanism; the stoichiometry and control of oxidative phos-phorylation; energy coupling enzymes; and predictions of themechanism.Description and underlying assumptions of the proposedmechanism of oxidative phosphorylationThe unit ofoxidative phosphorylation, whether in mitochondriaor in bacterial cells, is a combination ofan electron transfer com-plex (I, III, or IV) and complex V which we shall also refer to

as F0-F1 or the synthetase complex. This same combination isoperative also in photosynthetic phosphorylation which in prin-ciple may be considered to be indistinguishable from oxidativephosphorylation. The paired complexes are oriented in the in-ner mitochondrial membrane so that one end faces the cytosolicor intercristal side of the membrane (I side) and the other endfaces the matrix side of the membrane (M side). The electrontransfer complex executes the driving sequence; the synthetaseexecutes the driven sequence. The structured domains withinwhich each sequence takes place are composites of three sec-tors-the sector in which a bond is ruptured and charges areseparated, the sector in which a bond is formed and charges arerecombined, and a sector in which the separated charges aretransferred to the site where bond formation takes place. Thethree sectors of the driving sequence are shown in Fig. 1A andthe three sectors of the driven sequence are shown in Fig. 1B.The driving sequence at each step is always coupled to thedriven sequence; neither sequence can proceed independentlyof the other. The separated charges in the driving sequence arean electron and a proton; the separated charges in the driven

0ll /O1 -M+

sequence are OH- and the phosphonium ion, +P\ ;M+O-M+

represents a monovalent cation. We shall represent the phos-phonium ion by the symbol Pi+. The stoichiometry of the reac-tants and charged species is always 1:1-i.e., H+/e = 1, Pi+/OH- = 1, e/Pi+ = 1, e/A = 1, Pi+/bound ADP (RADP) =1, etc. We could think of energy coupling as the pairing of twohalf-enzymes, one mediating the driving sequence and theother mediating the driven sequence.

Whether the oxidation proceeds by a pair of one-electronsteps (NADH -) NAD -- NAD+, QH2 -) QH -- Q) or in asingle one-electron step (ferrocytochrome c -* ferricytochromec), the coupled reaction is completed with the transmembranemovement of one electron. In other words, the source of theelectron-complex I, III, or IV, or QH2 or QH, or ferrocyto-chrome c-is immaterial in respect to the ATP/electron ratio.

Whereas Pi moves across the membrane as a charged speciesafter ionization to Pi+ and OH-, ADP moves across the mem-brane as a neutral species via a carrier system that exchangesADP moving inward for ATP moving outward. The ionizationof Pi to produce Pi stripped of OH- (Pi+) takes place in the F0(membrane) subdivision ofthe synthetase; the interaction of Pi+with bound ADP takes place in F1 (the subdivision of the syn-thetase projecting from the membrane). The chain for transportof Pi+ (Pi+TC) is in FO; the enzyme catalyzing the formation ofthe POP bond is in F1.

Abbreviations: A, terminal electron acceptor; ETC, electron transfer.chain; Pi+, Pi stripped ofOH- (phosphonium ion); Pi+TC, transfer chainfor Pi'; RADP, bound form of ADP; RATP, bound form of ATP; DH,primary hydrogen donor.

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Proc. Natl. Acad. Sci. USA 79 (1982) 1065

FIG. 1. (A) Electron transfer sequence for complexes I, Im, and IV. The primary hydrogen donor (DH) is the reduced flavoprotein for complexI, QH2 for complex Im, and ferrocytochrome c for complex IV. The terminal electron acceptor (A) is Q for complex I, ferricytochrome c for complexm, and °2 for complex IV. The electron transfer chain (ETC) includes all oxidation-reduction components between DH and A. AH, reduced formof A. (B) Sequence for coupled ATP synthesis. (C) Sequence for cyclical cation transport. ITC, cation transfer chain. The cardiolipin channel is notcation specific but, for simplicity, we have indicated K+ as the cation.

Pi' reacts with RADP to form bound ATP (RATP),Pi + RADP - RATP + H+, [5]

and, in turn, RATP reacts with freeADP to form ATP and RADPin a kinase-mediated reaction. The kinase is associated with F1but is not one of the subunits of Fl. The coupled reaction ter-minates with the formation ofRATP; the transfer ofa phosphorylgroup to ADP in the matrix space is mediated by a classicalenzyme and hence there is no coupling involved in this phos-phoryl transfer.One final point is critical for the mechanism. Each electron

transfer complex is a complete coupling unit without any sup-plementation. The coupled sequence is cyclical cation transport(Fig. IC). We shall refer to this sequence as "intrinsic coupling"to distinguish it from oxidative phosphorylation that requirestwo complexes. We shall refer to coupling requiring two com-plexes as "extrinsic coupling." Both intrinsic and extrinsic cou-pling can take place under any set of experimental conditionssuitable for coupling. This optionality with respect to the cou-pling mode has important implications for the mechanism ofoxidative phosphorylation; these will be considered in the sec-tion on stoichiometry and control.

Evidence for the mechanismUnit of Coupling. Racker et al. (6-8) have demonstrated in

reconstitution experiments with liposomes that the combinationof an electron transfer complex and the F0-F1 complex is re-quired to mediate oxidative phosphorylation. The intrinsic cou-pling capability of each electron transfer complex was demon-strated by studies in our laboratory (9-11).

Orientation of Coupling Complexes in the Mitochondrion.The directionality of active transport of cations is an absoluteindicator of the orientation ofenergy-coupling complexes in theinner mitochondrial membrane. Because the three electrontransfer complexes as well as complex V can mediate the activetransport of cations into the matrix space (9-12), it necessarilyfollows that these complexes have the same orientation and thatcoupling begins on the I side of the complex and terminates onthe M side. Because electron transfer in the inner membranecan drive either transport of cations or synthesis of ATP, theconclusion can logically be drawn that the sequence for coupledATP synthesis also begins on the I side and terminates on the

M side. Consistent with this conclusion is the fact that the ADP/ATP carrier is required to move ADP into the matrix space (13);it is on the matrix side that coupled ATP synthesis should ter-minate with the interaction of Pi' with RADP.

Diagnostics of an Energy Coupling System. The basic as-sumptions implicit in the concept of an energy coupling en-zyme, assumptions carried over to the proposed mechanism ofoxidative phosphorylation, are: (a) energy coupling begins withbond rupture and charge separation on the I side of the mem-brane and terminates with bond formation and charge recom-bination on the M side; (b) the combination of a chain (or chan-nel) and a membrane-water interface is required for both chargeseparation and charge recombination; (c) in oxidative phos-phorylation the separated charges are e- and H' in the electrontransfer sequence and Pi' and OH- in the sequence for coupledATP synthesis; and (d) the pairing (coupling) principle appliesto all steps in the coupling sequence. Proton release duringenergy coupling has been shown to be correlated with chargeseparation in complexes I, III, IV, and V (9, 11, 12). Similarly,proton uptake in these same complexes has been correlated withcharge recombination during energy coupling (9, 11, 12).The reality ofcharge separation is attested to by the fact that,

during coupled electron transfer, the electron moves into themembrane whereas the proton is extruded into the aqueousphase. Similarly, during coupled cation transport, the cationmoves across the membrane whereas the anion with which itwas originally associated is left behind in the aqueous phase.In coupled ATP pyrophosphorolysis mediated by complex V,

M+O- 0\ 11O- moves across the membrane whereas ADP+

(ADP stripped ofa hydroxyl group) is extruded into the aqueousphase (12). Whenever such long-range charge separation takesplace, a molecular device has to be present in the membraneto capture the moving charge. Such a device is the electrontransfer chain (ETC) for capture of the electron (14-16) and thecardiolipin channel for capture of the cation (17). Both of thesedevices have been shown to be present in each of the threeelectron transfer complexes. In complex V, a specific ionophore(calciphorin) isolated by Jeng and Shamoo (18, 19) has been im-

Biochemistry: Green and Vande Zande

1066 Biochemistry: Green and Vande Zande

plicated in the active transport of Ca2" driven by pyrophos-phorolysis of ATP. An ionophore may be considered to be thefunctional equivalent of an ion transport channel. We thus havethree documented examples ofchain- or channel- or ionophore-mediated charge separations during energy coupling. This is theexperimental foundation for assuming a chain for Pi' in coupledATP synthesis. The evidence for the tactic ofa membrane-waterinterface for separating charge is the extrusion of protons intothe aqueous phase on the I side of the inner membrane and theuptake ofprotons from the aqueous phase on the M side (9, 11).

That the separated charges in electron transfer are the elec-tron and the proton is attested to by the fact of an ETC andproton extrusion. Similarly, the evidence that the separatedcharges in cation transport are the cation and anion is that thecardiolipin chain (20), as well as calciphorin (19), is cation-spe-cific. Hence, the anion would be extruded into the aqueousphase at the initiation of coupled transport. That the movingspecies in coupled ATP synthesis is Pi' has been established bythe 180 studies in the laboratories of Boyer (21) and Cohn (22).During oxidative phosphorylation the bridge oxygen of the ter-minal POP bond of ATP was found to be derived exclusivelyfrom ADP. Furthermore, the oxygen of orthophosphate wasfound to equilibrate rapidly with the oxygen of water, and thisequilibration was shown to be sensitive to uncoupler (23). Tri-phenylmethylphosphonium ion, which is analogous to Pi', isactively transported during energy coupling driven by oxidationof D-lactate in Escherichia coli (24). This observation demon-strates the feasibility of Pi+ transport in energy coupling sys-tems. If Pi+ is the species that interacts with ADP to form ATP,then OH- must be the other charged species of the pair orig-inating from Pi.The evidence for pairing during energy coupling would be

the value of unity for the e/cation ratio during active transport(9, 11), the value of unity for the cotransport of Pi and Ca2+during ATP-driven transport of Ca2+ (12), and the identical di-rectionality of electron and cation movements in active trans-port (9-11) and of electron and Pi' movements in coupled ATPsynthesis (see second paragraph of this section).

Origin of the Protons Extruded During Energy Coupling.Implicit in the proposed mechanism is that the protons extrudedduring coupled oxidation in the three electron transfer com-plexes arise exclusively from the respective substrates of thesecomplexes (NADH, QH2, and ferrocytochrome c). We haveshown that, for each electron removed by oxidation fromNADH* or ferrocytochrome c, one proton is released (26).There can be no question that the oxidation of QH2 to Q alsoinvolves the two-step release of two electrons and two protons.Hence, we can rule out the possibility that the H+/e ratio canexceed unity because all the protons extruded during energycoupling arise exclusively from the substrates undergoingoxidation. tThe evidence is equally strong that the protons taken up dur-

* Although NADH is the ultimate reductant for complex I, there is goodreason to assume that the protons extruded during oxidation ofNADHin complex I arise from the reduced flavin prosthetic group and notfrom NADH. That means that energy coupling actually begins withthe oxidation of the reduced flavoprotein and not with the oxidationof NADH. The transfer of a hydride ion and a proton to the flavinprosthetic group catalyzed by NADH dehydrogenase (25) is catalyzedby a classical enzyme (NADH dehydrogenase).

t The 1:1 relationship between proton release and electron release dur-ing coupled oxidation applies only when electron flow is coupled tothe transport of a univalent cation. When a divalent cation is trans-ported, an additional proton is released arising from the ionizable res-idues in the ion transport channel. Thus, Ca2+ is transported as[Ca2+A-]+', A- representing a dissociated residue of the channel orof an ionophore (calciphorin). The proton thereby dissociated is re-

ing energy coupling are taken up in the reduction ofthe terminaloxidants. We have shown this to be the case for the reductionofNAD' in complex I and for the reduction of ferricytochromec in complex III (26). Because the observed ratio of protonstaken up per electron is 1 for cytochrome oxidase, it follows thatthe theoretical value of 4 protons per 02 reduced is implicit inthat ratio.

Stoichiometry of Coupled Reactions. Because oxidativephosphorylation is carried out by a combination of two com-plexes, it is impossible to separate intrinsic coupling from ex-trinsic coupling. For that reason, determinations of stoichi-ometry would be ambiguous in the oxidative phosphorylationsystem. But that ambiguity would not apply to determinationsof the stoichiometry for intrinsic coupling. Studies of coupledcation transport in complexes I, III, and IV have clearly shownthe predicted 1:1 stoichiometry (9-11). For each electron trav-ersing the inner membrane, one proton was released on the Iside, one cation was transported, and one proton was taken upon the M side (9-11). There are many reports in the literaturethat are consistent with this 1:1 stoichiometry for energy cou-pling (27-31). But in an extended series of papers Lehningerand his group reported values of2 for H+/e and K+/e in studiesof valinomycin-mediated active transport of K+ in liver mito-chondria (32-34). We have been unable to reproduce their re-sults under the experimental conditions they specified.ADP/ATP Carrier Protein. In the laboratories of Klingen-

berg and Vignais the protein that mediates ADP/ATP exchangeduring oxidative phosphorylation has been isolated (13, 35). Theexchange is a consequence ofa spontaneous nonenergized equi-librium process. During the exchange, protons are released intothe aqueous phase on the I side (36) and presumably are takenup from the aqueous phase on the M side. The exchange re-action establishes two critical points. Because ATP arises in thematrix space, it necessarily follows that Pi in its reactive formhad to traverse the inner membrane. Furthermore, becauseADP enters the matrix space by the exchange reaction, we mayconclude that ATP is formed by the enzymic interaction of freeADP with an activated form of Pi on the matrix side.ATP/ADP Kinase-Mediated Phosphoryl Transfer. Beyer

(37, 38) has shown that a kinase is required for the phosphor-ylation of external ADP during oxidative phosphorylation. TheP/O ratio is depressed when this enzyme is extracted from testparticles and is restored to the original level of activity by ad-dition of the purified kinase. The kinase catalyzes the transferof a phosphoryl group from ATP to ADP and undergoes phos-phorylation during that transfer. It is probable that the kinasecan also catalyze the transfer of a phosphoryl group from boundATP to external ADP. On the basis of the 180 data of Boyerreferred to above, it is reasonable to assume that the phosphorylgroup transferred by the kinase is a phosphonium ion ratherthan a phosphoanion of orthophosphate.

Essentiality of Bound Nucleotides for Oxidative Phosphor-ylation. Leimgruber and Senior (39, 40) have shown that thecomplement oftightly boundADP in F1 is essential for oxidativephosphorylation. Removal ofbound nucleotides leads to the lossofthe coupling function of F1. This lost capacity can be restoredby reincorporating ADP into F1 so depleted. In our proposedmechanism we postulate that RADP is first phosphorylated to

leased into the aqueous space on the I side of the membrane. In cou-pled ATP synthesis, the proton released by separation from the elec-tron is neutralized by the OH- released by ionization of Pi. Net protonrelease therefore is zero. In the energized phase of oxidative phos-phorylation, the H'/e ratio is indeed unity. But as discussed, in thesection on stoichiometry and control, nonenergized cation cyclingleads to the balancing of proton release and uptake. Hence the netprotonic change is zero.

Proc. Nad Acad. Sci. USA 79 (1982)

Proc. Natl. Acad. Sci. USA 79 (1982) 1067

RATP and then a phosphoryl group is transferred to externalADP, mediated by the Beyer kinase. Smith and Boyer (41)found that RADP is indeed phosphorylated under the condi-tions of oxidative phosphorylation, but they suggested thatphosphorylation takes place only after dissociation of the nu-

cleotide from Fl. Considering the very tight binding of ADPto F1 shown by Leimgruber and Senior (39, 40), such dissocia-tion of RADP is unlikely. More likely is the kinase-mediatedtransfer of a phosphoryl group from RATP to external ADP.

Stoichiometry and control of oxidative phosphorylationIn the system of two complexes that executes oxidative phos-phorylation, electron flow can be coupled either to cyclical ca-

tion transport (intrinsic coupling) or to ATP synthesis (extrinsiccoupling). This optionality for coupling may not be a happen-stance. A strong case can be made for the thesis that the car-

diolipin channel, the molecular instrument for cyclical cationtransport, plays an essential role in the regulation of oxidativephosphorylation. The mechanism of coupled ATP synthesis re-

quires entry of Pi and ADP into the mitochondrion and effluxofATP from it. In round numbers, Pi and ADP would enter inassociation with four monovalent cations as counterions, andATP would efflux with three monovalent cations. For each ca-

tion thus accumulated in the matrix space, one proton wouldhave to be taken up to maintain charge neutrality. The inevi-table accumulation of cations as well as the progressive alka-linization of the matrix fluid would rapidly lead to the suppres-sion of oxidative phosphorylation. The cation/proton exchangereaction mediated by the cardiolipin channel in each electrontransfer complex (20, 42) would correct both of these imbal-ances. Intrinsic coupling as such merely provides competitionfor oxidative phosphorylation, but the cardiolipin channel im-poses homeostasis on an otherwise unbalanced coupling system.The proposed mechanism for oxidative phosphorylation re-

quires only one electron to drive the formation of one POPbond. Although it is not possible to determine directly the stoi-chiometry of coupled ATP synthesis because two coupled pro-cesses are proceeding simultaneously, nonetheless this stoichi-ometry can be deduced by indirect means. In the transport ofCa2" driven by pyrophosphorolysis of ATP, one Ca2' is trans-ported for each POP bond ruptured (12). Similarly, in the trans-port of Ca2' driven by electron flow, one Ca2+ is transportedper electron released by oxidation (43). Therefore, the ruptureof one POP bond generates the equivalent driving force of a

single electron. Ifcoupled ATP synthesis and coupled ATP pyr-ophosphorolysis follow the same mechanistic pattern, then itwould follow that the 1:1 equivalence ofan electron and a POPbond should apply both to the rupture and to the formation ofthis bond.

Calculations are often made of the maximum number ofmol-ecules ofATP that can be formed per site per pair of electrons.Such calculations are made on the basis of the oxidation-reduc-tion potentials of the free forms of the reductants and oxidantsfor each site. All the reductants and oxidants of the three-elec-tron transfer complexes are bound species, and these calcula-tions are unreliable because the potentials of free and boundspecies can be vastly different [for example, compare the po-tentials of flavin adenine dinucleotide and flavoproteins con-

taining the dinucleotide (44, 45)].Energy coupling enzymes

The characteristic reaction pattern of an energy coupling en-

zyme system subtends paired charge separation on one side ofa membrane, paired charge recombination on the other side ofthe membrane, and paired charge transfer within the mem-

brane. This pattern has been shown to apply to coupled ATPsynthesis (this communication), coupled cyclical cation trans-

port (9-11), and ATP-driven Ca2+ transport (12). Althoughthere still are gaps in the body of evidence for this pattern inthe three systems listed above, the pattern in all cases is un-mistakable. Classical enzymes are known which catalyze, re-spectively, the four-electron reduction of 02 to water (46-49)(the cytochrome oxidase reaction), the oxidative synthesis ofATP from Pi and ADP (3, 4), and the pyrophosphorolysis ofATPto Pi and ADP (49) (the oligomycin-insensitive reaction cata-lyzed by F1). The fact that these three reaction sequences cat-alyzed by energy coupling systems can be duplicated by classicalenzymes reemphasizes the enzymic nature of energy couplingand points up that enzymic catalysis can be expressed either ina form that leads to the formation ofnew bonds (classical enzymecatalysis) or in a form that leads to energy coupling. The powerofthe enzymic approach to energy coupling is that precise limitsare set on what is possible mechanistically and what is not. Theprinciples underlying enzymic catalysis require direct couplingand unit stoichiometry and forbid energy storage as in the formof a membrane potential (25, 50). For 40 years, biochemistsattempted to find a substitute for enzyme catalysis to rationalizeenergy coupling. It now appears that this substitution is notnecessary.Predictions of the proposed mechanism

a. Reagents that uncouple oxidative phosphorylation (uncou-plers and the valinomycin/nigericin combination) should undergothe same sequence ofchange as does Pi-i. e., charge separationat the I side of the membrane, charge transfer across the mem-brane, and charge neutralization on theM side ofthe membrane(51). These reagents should displace Pi from the charge sepa-ration site and, when ionized, should be transferred preferen-tially in the transfer chain for Pi'. The positively charged speciesthat accomplishes this displacement would be a cation complexof either uncoupler or valinomycin.

b. The reagents that specifically suppress coupled ATP syn-thesis [DCCD (52), oligomycin (53), and tripropyltin (54)]should combine with residues in the Pi' transfer chain that areessential for the transfer of Pi' from the site ofcharge separationto the site of phosphorylation of bound ADP.

We are grateful to Dr. Alan E. Senior for his valuable advice.

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Proc. Natl. Acad. Sci. USA 79 (1982)'