Structure and Mechanism of Respiratory III-IV Supercomplexes in Bioenergetic Membranes
In the final steps of energy conservation in aerobic organisms, free energy from electron transfer through the respiratory chain is transduced into a proton electrochemical gradient across a membrane. In mitochondria and many bacteria, reduction of the dioxygen electron acceptor is catalyzed by cytochrome c oxidase (complex IV), which receives electrons from cytochrome bc1 (complex III), via membrane-bound or water-soluble cytochrome c. These complexes function independently, but in many organisms they associate to form supercomplexes. Here, we review the structural features and the functional significance of the nonobligate III2IV1/2Saccharomyces cerevisiae mitochondrial supercomplex as well as the obligate III2IV2 supercomplex from actinobacteria. The analysis is centered around the Q-cycle of complex III, proton uptake by CytcO, as well as mechanistic and structural solutions to the electronic link between complexes III and IV.
Aerobic organisms extract energy by linking oxidation of environmental compounds to production of ATP. In eukaryotes, these compounds are initially degraded to yield NADH, which is used to reduce molecular oxygen to water. Electrons from NADH are transferred through a number of enzymes that reside in the inner mitochondrial membrane. These enzymes are collectively referred to as the respiratory chain because they are wired to transfer electrons consecutively from low-potential electron donors, via a number of intermediate electron carriers, to the final, high-potential electron acceptor, O2. The electron current through the respiratory chain drives proton translocation across the membrane, from the inside mitochondrial matrix (negative side, n) to the outside intermembrane space (positive side, p) (Figure 1A). As a result of this process, a difference in voltage and proton concentration is maintained across the membrane, referred to as an electrochemical proton gradient or protonmotive force (PMF).1 The free energy that is stored in this electrochemical gradient is typically in the order ∼0.2 eV,2,3 and it is used for production of ATP from ADP by the ATP synthase (also known as F1Fo-ATP-synthase and sometimes referred to as complex V) or for transport of molecules or ions across the membrane.4
In mitochondria, the energy-conversion machinery is found in protrusions of the inner membrane which define subcompartments called cristae. Here, the respiratory chain is located in the flat regions, while the ATP synthases are restricted mainly to the bent end regions5,6 (Figure 1B). In aerobic bacteria the respiratory chain is found in the cytoplasmic membrane where protons are translocated from the cytoplasm to the periplasm (for review, see refs (2,7−9)).
In mammalian mitochondria, the first component of the respiratory chain is an integral membrane protein called NADH:ubiquinone oxidoreductase (also named complex I), which catalyzes oxidation of NADH and reduction of quinone (Q) to quinol (QH2) (Figure 1A). This electron-transfer reaction is linked to pumping of protons across the membrane. Many yeast species such as Saccharomyces (S.) cerevisiae do not harbor a complex I, but in these mitochondria, oxidation of NADH and reduction of Q is catalyzed by other, membrane peripheral NADH dehydrogenases located both on the inner (Ndi1) and outer (Nde1 and Nde2) surfaces of the inner mitochondrial membrane10−12 (Figure 1A). Electron transfer to Q is also performed by succinate dehydrogenase (also named complex II). Reduced QH2 diffuses within the membrane to donate electrons to ubiquinol-cytochrome c reductase (also named cytochrome (cyt.) bc1 or complex III), which transfers electrons to water-soluble cyt. c that resides in the intermembrane space. Reduced cyt. c is an electron donor to cytochrome c oxidase (CytcO, also named complex IV), which catalyzes oxidation of cyt. c and reduction of molecular oxygen to water. Aerobic bacteria utilize a wide range of electron donors, and a specific organism may harbor many different respiratory chains that are expressed depending on environmental conditions and are often branched. General reviews of these pathways are found in refs (2,7,13−15).
Because the mobile electron carriers of the mitochondrial electron-transport chain, i.e., QH2 and cyt. c, can diffuse freely in the membrane and water phases, respectively, a functional link between the components of the respiratory chain does not require a physical linkage between these complexes. Experimental data and theoretical analyses supported a model where all respiratory complexes diffuse independently in the membrane, as do the electron carriers Q and cyt. c.16 This perception changed gradually with the invention of blue native polyacrylamide gel electrophoresis (BN-PAGE), which made it possible to identify larger complexes, referred to as respiratory supercomplexes, composed of different combinations of the respiratory enzymes with variable stoichiometry.17 Functionally active respiratory supercomplexes were found in a wide range of organisms.17−28 Recent structural studies of the inner mitochondrial membrane using electron cryo-tomography in situ demonstrated that the electron-transport chain components are organized in supercomplexes in mammals, yeast and plants,29 i.e., the observation of supercomplexes is not a consequence of the isolation procedures used. A wide range of these supercomplexes with different composition and stoichiometry of the components have been isolated using “weak” detergents, and in recent years a number of high-resolution supercomplex structures have been obtained using electron cryomicroscopy (cryo-EM) (reviewed in refs (30,31) and listed in Table 1).
From the above discussion, it becomes apparent that the term “respiratory supercomplex” is used to describe a phenomenon, i.e., formation of membrane-bound clusters of respiratory complexes rather than entities with a well-defined composition (see Table 1). This variation in the constituents and their stoichiometry has contributed to the difficulty in uncovering a functional role of respiratory supercomplexes, which is reflected in ongoing discussions (e.g., refs (53−55)).
Many Gram-negative prokaryotes also harbor respiratory supercomplexes, but much less is known about their composition or structure (reviewed in ref (56)). For example, in Paracoccus (P.) denitrificans, which under aerobic conditions harbors a respiratory chain similar to that of mitochondria, supercomplexes composed of complexes III and IV were isolated already in 1985,57 and a larger supercomplex that included also complex I was identified later.58 In a recent study, a complex III–IV supercomplex from Rhodobacter (R.) sphaeroides that contains a membrane-anchored cyt. cy was isolated and functionally characterized.59 In another recent study, the cryo-EM structure of a Rhodobacter capsulatus supercomplex composed of complex III, a cbb3-type complex IV and a membrane-anchored cyt. cy was presented.46 Furthermore, in Escherichia (E.) coli cytoplasmic cell membranes a segregation of respiratory complexes into subdomains was observed in vivo, although these bacteria do not harbor supercomplexes.60,61 Gram-positive bacteria, which belong to the phylum Actinobacteria, e.g., Mycobacterium (M.) smegmatis, Mycobacterium tuberculosis, and Corynebacterium (C.) glutamicum, lack small c-cytochromes and harbor an obligate supercomplex composed of a complex III dimer flanked by two monomers of complex IV (denoted III2IV2), which are electronically linked by the diheme cyt. cc domain of complex III.62−67 A supercomplex composed of complexes III and IV was also isolated from the Gram-positive bacterium Bacillus PS3.68
The S. cerevisiae respiratory supercomplex is composed of a cyt. bc1 dimer, flanked by either one or two CytcOs on each side of the central dimer.17,18,69−77 Recently determined cryo-EM structures of this supercomplex33−35,37 revealed its molecular architecture (Figure 2A) but also showed that the association of cyt. bc1 and CytcO does not lead to any significant structural changes of the components. This observation suggests that the functionality of the S. cerevisiae supercomplex is simply that of the sum of the components, except that the components reside at a fixed intercomplex distance. In contrast, structural and functional studies of the M. smegmatis(43,44) (Figure 2B) and C. glutamicum(45,62,65) supercomplexes revealed intercomplex connections that presumably modulate the functionality of the components, consistent with the obligate nature of these supercomplexes.
Recent progress in development of methods to isolate pure respiratory supercomplexes has allowed functional studies using biochemical and biophysical techniques, previously employed in studies of the individual respiratory complexes. Major advancement in the field was contributed by the use of cryo-EM to determine the overall architecture of supercomplexes, high-resolution structures of their components as well as positions and distances between all cofactors (shown for the S. cerevisiae and M. smegmatis supercomplexes in Figure 3). These studies are still in an early phase, but the data available to date allows a discussion of possible links between the molecular architecture and function of respiratory supercomplexes. This review is centered around the S. cerevisiae supercomplex, but we also discuss the M. smegmatis and C. glutamicum obligate III2IV2 supercomplexes while focusing on functional similarities and differences to the mitochondrial counterpart. The emphasis is put on the biological processes at the molecular level in terms of physical mechanisms.
Complex III (cyt. bc1) is an obligate homodimer. Each monomer is composed of three main, functionally important catalytic subunits (Figure 4A): (i) cyt. b (QcrB in actinobacteria), which harbors two hemes B and two quinone-binding sites; (ii) cyt. c1, which harbors a heme C (QcrC, which harbors two hemes C in actinobacteria); (iii) the Rieske iron–sulfur protein (ISP, called QcrA in actinobacteria or Rip1 in S. cerevisiae), which harbors a 2Fe-2S center (FeS) that is bound in an ectodomain on the p side of the membrane (reviewed, e.g., in ref (78−85)). In addition to these three catalytic subunits, in S. cerevisiae, each cyt. bc1 monomer is composed of an additional 7 subunits (Figure 2A), collectively shown in gray in the inset to Figure 4A (lower left).
Complex III catalyzes net oxidation of QH2 and reduction of cyt. c in a reaction sequence that is referred to as the proton-motive Q-cycle, which contributes to maintaining the proton electrochemical potential across the inner mitochondrial membrane.86 The QH2 electron donor binds in a Q-binding site referred to as QP, which is located near the p side of the membrane (also called Qo) (Figure 4A). In the mitochondrial cyt. bc1, this site is characterized by a conserved PEWY (Pro-Glu-Trp-Tyr) motif87 (residues 270–273 in Figure 4B). The equivalent in M. smegmatis is PDFY (PDVY in C. glutamicum) residues 301–304 in Figure 4C. The first electron from QH2 is transferred to the FeS center and then to cyt. c1 along a branch that is referred to as the “C branch” (Figure 4A). This electron transfer is accompanied by release of two protons to the aqueous solution on the membrane p side. The second electron is transferred along the “B branch”, consecutively to the low-potential heme bL, the high-potential heme bH and a Q in the QN site (also called Qi), which forms a semiquinone, SQ•–. After oxidation of QH2 in the QP site, the product Q is replaced by another QH2, and the sequence of electron and proton-transfer reactions is repeated. As a result, a doubly reduced QH2 is formed at the QN site after proton uptake from the n side. The QH2 is released from the QN site by equilibration with the Q/QH2 pool in the membrane. The overall reaction catalyzed by cyt. bc1 is (see also Figure 4A):
Overall reaction:1cwhere subscripts n and p refer to the two sides of the membrane, respectively, and N and P refer to the two Q-binding sites, respectively.
Crystal structures of cyt. bc1 complexes have revealed a single bound Q in the QN site for each monomer, but the QP site is typically empty. The putative position of the QP site was instead revealed by the location of inhibitors such as stigmatellin or myxothiazol (reviewed in refs (78,81,84)). In the cryo-EM structures of the S. cerevisiae cyt. bc1 complexes33−35,37 a Q could not be modeled convincingly in the QP site, but a ubiquinone (UQ) was found to be bound in the QN site, in line with the earlier structural studies using X-ray crystallography. A recent cryo-EM study of the mammalian I1III2 supercomplex88 revealed a UQ in the QP site, but only in one monomer of the cyt. bc1 dimer (the other QP site was empty). In another recent cryo-EM structure of complex III2 from C. albicans, density for a UQ was found in both QP sites of the dimer (as well as in the QN sites), although at low occupancy.89
On the basis of the observation of an empty QP site and a UQ bound in the QN site in the S. cerevisiae complex III, it was recently suggested that a higher affinity for UQ at the QN site would prevent release of a semiquinone that would give rise to superoxide upon reaction with O2.35 However, we note that (i) the difference in affinity for UQ at the two binding sites is not directly related to the affinity of the negatively charged semiquinone radical, SQ•–, at these sites,90 (ii) SQ•– is not released to the membrane, i.e., the reaction of O2 with SQ•– is more likely to occur in situ, but (iii) it occurs at the QP rather than at the QN site.81,91,92 We instead suggest that observation of a bound UQ in the QN site reflects a higher affinity for the substrate UQ in that site, compared to the product UQ in the QP site (all structures were obtained with the oxidized state of complex III).
In the M. smegmatis and C. glutamicum III2IV2 supercomplexes, menaquinone (MQ) was observed in the QP and QN sites but also at additional sites on the p side of complex III.43−45 The MQ in the QP site of the M. smegmatis complex III overlaps in space with that of UQ in the mammalian complex III. In C. glutamicum, the QP cavity is larger than in M. smegmatis, and the data suggest that MQ could also occupy a position just outside of the QP site, suggesting two possible binding modes, one inside and one just outside of the QP site.45 Furthermore, in both M. smegmatis(44) and C. glutamicum(45) supercomplexes, clear density corresponding to an additional MQ on the p side was observed. In the M. smegmatis supercomplex, this MQ is positioned near the Tyr of the PDFY motif, at the vertex of a triangle formed the FeS center (at a distance of ∼20 Å) and heme bL (at a distance of ∼20 Å). In the C. glutamicum supercomplex structure, the second MQ is located at a distance of ∼14 Å from heme bL and ∼35 Å from the FeS center. The role of an additional MQ binding site on the p side is unknown, but identification of these Q-binding sites in both C. glutamicum and M. smegmatis suggests a functional role, for example, to bypass energy conservation in complex III at low O2 concentrations.45
A bifurcated electron transfer from QH2 at the QP site is required by the Q-cycle mechanism. As outlined above, in this process, one electron from QH2 is transferred to FeS and one to heme bL along the C and B branches, respectively (Figure 4A), which is schematically outlined in the following equation, assuming a putative semiquinone intermediate:2
The detailed mechanism of this bifurcation at the QP site remains enigmatic.78,81,82 Transfer from QH2 to FeS with a midpoint potential Em7 ≥ 300 mV is thermodynamically more favorable than transfer to heme bL with Em7 ≅ 0 mV (when heme bH is oxidized). Thus, oxidation of QH2 results first in reduction of FeS along the C branch. The second electron could in principle also be transferred along the same C branch to FeS after reoxidation of FeS– by cyt. c1, i.e., without energy conservation.82 Instead, the electron is transferred along the B branch in a reaction that is strictly controlled yielding almost complete reduction of heme bL. This phenomenon was clearly illustrated in an experiment where transfer to the QN site, along the B branch, was inhibited by binding of the QN-site inhibitor antimycin. Even though, in principle, the enzyme could turnover by electron transfer via the C branch only, this block of the B branch resulted in reduction of both hemes bL and bH and almost full inhibition of the cyt. bc1 turnover complex.93
Crystal structures of canonical cyt. bc1 complexes revealed that the FeS ectodomain could adopt different positions where in the two extreme orientations the FeS cluster is found in proximity to either cyt. c1 (C position) or heme bL (B position).94−96 These two FeS ectodomain positions are indicated schematically in the right-hand side inset to Figure 4A (see also inset to Figure 3A). The distance spanned by the FeS cluster while moving between the B and C positions is almost 20 Å, and the structural data suggested that the FeS cluster could accept electrons from QH2 (in site QP) only in the B position, while electron transfer to cyt. c1 would occur only in the C position. However, the link between Q/QH2 binding in the QP site, the redox state of FeS and the equilibrium constant for the two FeS-domain positions remains enigmatic.78,81,97
Structural studies with different types of inhibitors bound in the QP site indicate that the position of the FeS ectodomain depends on its interactions with the inhibitor as well as minor structural changes caused by the inhibitor binding.78,89,95,97−103 There are two classes of QP-site inhibitors referred to as Pf (f for fix) and Pm (m for mobile), respectively. The Pf class of inhibitors, such as the UQ analogue stigmatellin, fix the FeS ectodomain in the B position, presumably due to formation of a hydrogen bond between the inhibitor and the FeS ectodomain. The Pm class of inhibitors, such as, e.g., myxothiazol or azoxystrobin, displace the FeS ectodomain from the B position yielding a mobile domain that adopts different positions, including the C position. A recent cryo-EM study with the Pm-type fungal complex III2 inhibitor Inz-5 revealed the distribution of these positions.89
Crystal structures of complex III2 revealed also intermediate positions of the ectodomain, in between the B and C positions.104 This variability in the ectodomain position was explained by differences in crystal packing (summarized in ref (84)). However, in the cryo-EM structures of the S. cerevisiae cyt. bc1(34,35) the FeS ectodomain also adopts an intermediate position (shown in Figure 4A), i.e., the intermediate ectodomain position is not a consequence of protein crystallization. Interestingly, in a recent cryo-EM structure of the C. albicans cyt. bc1 several classes of particles were observed in which the FeS head domain is either in the B position, C position, or in between these positions,89 suggesting a statistical distribution of these states, which is consistent with spectroscopic data.292 Similarly, in the cryo-EM structure of the R. capsulatus cyt. bc1, subpopulations were identified with the FeS ectodomain either in the B or C position with an empty QP site.46 In the cryo-EM structure of the mammalian cyt. bc1, only one QP site of complex III2 dimer is occupied,88 but the FeS domain adopts the C position in both monomers. Furthermore, in the recently determined structure of the plant supercomplex from Vigna (V.) radiata, both FeS domain positions were observed in the absence of bound Q in the QP site.32 Hence, all these data suggest that the position of the FeS ectodomain is stochastic when the QP site is empty or occupied by an oxidized Q.78,81,88,97 On the other hand, binding of a reduced hydroquinone in the QP site when the FeS cluster is oxidized may shift the equilibrium of the FeS domain toward the B position, similarly to binding of stigmatellin.89,105−110
Because movement of the FeS domain is involved in transfer of the first electron from QH2 to cyt. c1, the equilibrium constant and/or time constant for the FeS domain transition between the B and C positions determines the kinetics of this electron transfer.82,84 A stochastic FeS domain movement after oxidation of QH2 in the QP site implies that the B–C transition is not required to accomplish the electron bifurcation from the QP site,107 i.e., electron branching in the Q-cycle is possible without movement of the FeS domain. Indeed, the FeS domain is permanently fixed near the B position in the M. smegmatis and C. glutamicum III2IV2 supercomplexes.43−45 Rich and colleagues107 discussed the thermodynamics and kinetics of electron bifurcation in the framework of eq 2 above and concluded that the mechanism could be explained by a concerted two-electron oxidation of QH2.
In the M. smegmatis supercomplex, the cyt. cc domain of complex III displayed two conformations in the two halves of the supercomplex, a closed conformation in which it is located near the electron acceptor at complex IV, and an open conformation where the electronic connection between the two complexes is interrupted44 (Figure 3B). We hypothesized that movement of the cyt. cc domain, instead of movement of the FeS ectodomain, could mediate electron transfer from MQH2 within the supercomplex.44 However, at this point, it is unknown whether or not the cyt. cc domain movement is stochastic or linked to other reactions. In the C. glutamicum supercomplex45 as well as in another structure of the M. smegmatis supercomplex,43 all elements of the electron-transfer chain appear to be fixed, which suggests that the Q-cycle can be realized without any domain movements. Collectively, these data suggest a variability in the structural solution to a mechanistic realization of the Q cycle, which is discussed in the next subsection.
The electron bifurcation from QH2 along the C and B branches, respectively, is functionally linked to proton release to the membrane p side.82,87,97,110−114 In the canonical cyt. bc1, binding of QH2 at the QP site has been suggested to shift the equilibrium of the FeS head domain toward the B position where one of the QH2 protons would form a hydrogen bond with the FeS ligand His161 (mammalian complex III numbering, His181 in S. cerevisiae). It is well established that upon transfer of the first electron from QH2 to FeS, the first proton is transferred to this His161.82,87,97,111−114 The second proton has been suggested to be transferred to Glu271 (Glu272 in S. cerevisiae) of the PEWY motif (Figure 4B), followed by rotation of the protonated Glu271 toward the heme bL propionate upon electron transfer to heme bL (Figure 4B). After transfer of the second electron along the B branch, the FeS head domain would transiently adopt the C position (see discussion in the previous section), from where the first electron is transferred to cyt. c1, linked to proton release from His161 to the p side of the membrane. In other words, this mechanism implies that part of the proton-transfer route for the first proton would involve the rotation of the FeS head domain.
It is likely that a spatial distribution of the two proton-transfer paths and the link between proton and electron transfer yields the bifurcated proton transfer. While the transfer route of the first proton from QH2 is relatively well characterized, the route of the second proton remains to be explored. The proton from Glu271 has been suggested to be transferred consecutively to Arg79 (not shown in Figure 4B) and the p side aqueous phase.111 However, functional studies of structural variants at position Glu271 indicate that this residue is not a unique proton acceptor from QH2,115,116 and there are presumably alternative proton-release pathways.81 In the structure of S. cerevisiae complex III, residues Glu272 and Tyr274 (equivalent of Asp302 and Tyr304, respectively, in M. smegmatis, Figure 4BC), together with other residues, coordinate a network of water molecules between heme bL and the QP site, which may be involved in proton transfer, and determines the dielectric environment of the site.
The mechanism described above outlines that deprotonation of His161 to the p side occurs only when the FeS head domain had moved to transiently adopt the electron donating C position. Because in M. smegmatis and C. glutamicum the FeS domain is fixed in the B position, a different proton-release route is presumably utilized in these complexes. In complex III from M. smegmatis and C. glutamicum, a Q was found to be bound in a site equivalent to the canonical QP site.43−45 His368, the equivalent of His161, is presumably the acceptor of the first proton from QH2 also in these complexes III (Figure 4C). In the M. smegmatis complex III, the equivalent of Glu271 is a shorter side chain Asp302, which cannot approach the QP site sufficiently closely to act as an acceptor of the second proton. Instead, Asp309 (M. smegmatis numbering) is found in proximity to the second proton of QH2 (Figure 4C). Furthermore, Asp309 is found at ∼4 Å from His368, suggesting a possible common proton-release route of the two QH2 protons.45 Many actinobacteria harbor a Glu residue instead of Asp309, which could also serve as a proton acceptor.
On the basis of this analysis of the structure, we speculated that a possible Q-cycle mechanism in C. glutamicum and M. smegmatis complex III may involve the following sequence of events:45 (i) transfer of the first proton/electron to His368/FeS, (ii) transfer of the second proton/electron to Asp309/heme bL, (iii) electron transfer from heme bL to heme bH, linked to deprotonation of Asp309, and (iv) electron transfer from FeS to the nearest cyt. cI of the cyt. cc domain.
The electron transfer from FeS to cyt. cI in (iv) is assumed to occur only if it is linked to deprotonation of the FeS ligand His368, which is possible only after deprotonation of Asp309, i.e., after electron transfer from heme bL to heme bH. Indeed, the electron transfer in (iii), from FeS to cyt. cc along the C branch, was shown to be rate-limiting for turnover of the C. glutamicum supercomplex,65 i.e., it would occur after electron transfer along the B branch. In addition, on the basis of analysis of one of the M. smegmatis supercomplex structures, we hypothesize that the transition between the open and closed conformation of the cyt. cc domain (Figure 3B) may provide a mechanism to gate electron transfer from complex III to complex IV.44 However, as indicated above, it is presently unclear how this movement would be linked to the binding of QH2 at the QP site and the proton-transfer reactions. It should be stressed that the mechanism outlined above is based on analyses of structures and is presented only to serve as a guide in the design of experiments aimed at testing this hypothesis.
The mitochondrial complex IV is a member of the heme-copper oxidase family, which is characterized by a catalytic site that is composed of a heme group and a copper ion where dioxygen is reduced to water. Other oxidases, such as the UQH2-O2 oxidoreductases, cytochrome bd(118,119) and alternative oxidases120 also catalyze reduction of O2 to water in respiratory chains, but these oxidases harbor catalytic sites of different composition and do not belong to the heme-copper oxidase family. The heme-copper oxidase family is defined by homology in subunit I (Figure 5A), which harbors six conserved histidine residues that coordinate three redox-active metal sites: (i) a six-coordinated heme group with two axial His ligands (heme a in Figure 5A); (ii) a five-coordinated heme group with one axial His ligand (heme a3 in Figure 5A); and (iii) a copper ion called CuB, which is coordinated by three His ligands. The latter heme and CuB form a catalytic site where O2 binds and is reduced. In bacteria, the two heme groups may be of the same or different types: hemes a, b, or o. In mitochondria both hemes are of the same a type, hence these complexes are sometimes also referred to as cytochromes aa3.
The heme-copper oxidase family can be divided in two functional subgroups, based on the origin of the electron donor: quinol oxidases and CytcOs. The former receive electrons from membrane-soluble QH2, while the latter receive electrons from cyt. c. The quinol oxidase from, e.g., E. coli (cytochrome bo3) has an overall structure that is similar to those of bacterial CytcOs but lacks the electron acceptor metal site (CuA, see below) and instead harbors a Q-binding site at which QH2 donates electrons.
The primary electron acceptor of the mitochondrial CytcOs, including that of S. cerevisiae, is a dinuclear Cu-center called CuA, located near the p side in subunit II (Figure 5A). Because electrons from cyt. c are donated at the p side of the membrane, while protons are taken up from the opposite, n, side of the membrane, the reaction yields a charge separation across the membrane that is equivalent to moving one positive charge from the n to the p side. In addition, for each electron transferred to the catalytic site, one proton is pumped from the n to the p side, thereby increasing the total charge-separation stoichiometry. The proton-pumping stoichiometry varies between CytcOs from different organisms. Thus, the reaction catalyzed by the CytcOs is3where δ is the proton-pumping stoichiometry, i.e., number of H+ pumped per electron transferred to O2, typically 0.5 ≤ δ ≤ 1 (δ = 1 for mitochondrial CytcOs), subscripts n and p refer to the two sides of the membrane, and the subscript “pump” refers to pumped protons released on the p side (for more detailed reviews on the structure and function of CytcOs, see refs (121−130)).