Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides.

The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc). Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions. The results show that none of the 5 residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.     

Assignment of the histidine axial ligands to the cytochrome bH and cytochrome bL components of the bc1 complex from Rhodobacter sphaeroides by site-directed mutagenesis.

The cytochrome b subunit of the bc1 complex contains two cytochrome components, cytochrome bH and cytochrome bL. Sequence comparisons of this polypeptide from a number of organisms have revealed four invariant histidines which have been postulated to be the heme ligands for the two protoheme IX prosthetic groups. In Rhodobacter sphaeroides, these correspond to His97, His111, His198, and His212. In this paper, the results of amino acid substitutions at each of these positions are reported. Replacement of His97 by either Asp or Asn and of His198 by Asn or Tyr resulted in loss of both cytochrome components. However, His111Asn, His111Asp, and His212Asp all resulted in the selective loss of cytochrome bH and the retention of cytochrome bL. Furthermore, flash kinetics studies show that the myxothiazol-sensitive quinol oxidase (Qz) site associated with cytochrome bL is still functional. These data support the assignment of the axial ligands to cytochrome bH (His111 and His212) and cytochrome bL (His97 and His198). This pairing is consistent with current models of the cytochrome b subunit with eight transmembrane alpha-helices.     

Structure and reaction mechanisms of multifunctional mitochondrial cytochrome bc1 complex.

The cytochrome bc1 complex from bovine heart mitochondria is a multi-functional enzyme complex. In addition to electron and proton transfer activity, the complex also processes an activatable peptidase activity and a superoxide generating activity. The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, eight of which belong to cytochrome b, and five of which belong to cytochrome c1, Rieske iron-sulfur protein (ISP), subunits 7, 10 and 11, one each. The distances of 21 A between bL heme and bH heme and of 27 A between bL heme and the iron-sulfur cluster (FeS), accommodate well the observed fast electron transfers between the involved redox centers. However, the distance of 31 A between heme c1 and FeS, makes it difficult to explain the high electron transfer rate between them. 3D structural analyses of the bc1 complexes co-crystallized with the Qu site inhibitors suggest that the extramembrane domain of the ISP may undergo substantial movement during the catalytic cycle of the complex. This suggestion is further supported by the decreased in the cytochrome bc1 complex activity and the increased in activation energy for mutants with increased rigidity in the neck region of ISP.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.     

Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.     

On the mechanism of quinol oxidation at the QP site in the cytochrome bc1 complex: studied using mutants lacking cytochrome bL or bH

To elucidate the mechanism of bifurcated oxidation of quinol in the cytochrome bc1 complex, Rhodobacter sphaeroides mutants, H198N and H111N, lacking heme bL and heme bH, respectively, were constructed and characterized. Purified mutant complexes have the same subunit composition as that of the wild-type complex, but have only 9-11% of the electron transfer activity, which is sensitive to stigmatellin or myxothiazol. The Em values for hemes bL and bH in the H111N and H198N complexes are -95 and -35 mV, respectively. The pseudo first-order reduction rate constants for hemes bL and bH in H111N and H198N, by ubiquiniol, are 16.3 and 12.4 s(-1), respectively. These indicate that the Qp site in the H111N mutant complex is similar to that in the wild-type complex. Pre-steady state reduction rates of heme c1 by these two mutant complexes decrease to a similar extent of their activity, suggesting that the decrease in electron transfer activity is due to impairment of movement of the head domain of reduced iron-sulfur protein, caused by disruption of electron transfer from heme bL to heme bH. Both mutant complexes produce as much superoxide as does antimycin A-treated wild-type complex. Ascorbate eliminates all superoxide generating activity in the intact or antimycin inhibited wild-type or mutant complexes.     

Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. I. Characterization of the cytochrome bc1-type complex of the acidophilic ferrous ion-oxidizing bacterium Thiobacillus ferrooxidans.

The redox components of the cytochrome bc1 complex from the acidophilic chemolithotrophic organism Thiobacillus ferrooxidans were investigated by potentiometric and spectroscopic techniques. Optical redox titrations demonstrated the presence of two b-type hemes with differing redox midpoint potentials at pH 7.4 (-169 and + 20 mV for bL and bH, respectively). At pH 3.5, by contrast, both hemes appeared to titrate at about +20 mV. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide, and stigmatellin induced distinguishable shifts of the b hemes' alpha-bands, providing evidence for the binding of antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide near heme bH (located on the cytosolic side of the membrane) and of stigmatellin near heme bL (located on the periplasmic side of the membrane). The inhibitors stigmatellin, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, and 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone affected the EPR spectrum of the Rieske iron-sulfur center in a way that differs from what has been observed for cytochrome bc1 or b6f complexes. The results obtained demonstrate that the T. ferrooxidans complex, although showing most of the features characteristic for bc1 complexes, contains unique properties that are most probably related to the chemolithotrophicity and/or acidophilicity of its parent organism. A speculative model for reverse electron transfer through the T. ferrooxidans complex is proposed.     

Ubiquinol:cytochrome c oxidoreductase. Effects of inhibitors on reverse electron transfer from the iron-sulfur protein to cytochrome b

The effects of inhibitors on the reduction of the bis-heme cytochrome b of ubiquinol: cytochrome c oxidoreductase (complex III, bc1 complex) has been studied in bovine heart submitochondrial particles (SMP) when cytochrome b was reduced by NADH and succinate via the ubiquinone (Q) pool or by ascorbate plus N,N,N', N'-tetramethyl-p-phenylenediamine via cytochrome c1 and the iron-sulfur protein of complex III (ISP). The inhibitors used were antimycin (an N-side inhibitor), beta-methoxyacrylate derivatives, stigmatellin (P-side inhibitors), and ethoxyformic anhydride, which modifies essential histidyl residues in ISP. In agreement with our previous findings, the following results were obtained: (i) When ISP/cytochrome c1 were prereduced or SMP were treated with a P-side inhibitor, the high potential heme bH was fully and rapidly reduced by NADH or succinate, whereas the low potential heme bL was only partially reduced. (ii) Reverse electron transfer from ISP/c1 to cytochrome b was inhibited more by antimycin than by the P-side inhibitors. This reverse electron transfer was unaffected when, instead of normal SMP, Q-extracted SMP containing 200-fold less Q (0. 06 mol Q/mol cytochrome b or c1) were used. (iii) The cytochrome b reduced by reverse electron transfer through the leak of a P-side inhibitor was rapidly oxidized upon subsequent addition of antimycin. This antimycin-induced reoxidation did not happen when Q-extracted SMP were used. The implications of these results on the path of electrons in complex III, on oxidant-induced extra cytochrome b reduction, and on the inhibition of forward electron transfer to cytochrome b by a P-side plus an N-side inhibitor have been discussed.     

Evidence for electron equilibrium between the two hemes bL in the dimeric cytochrome bc1 complex

Structural analysis of the dimeric mitochondrial cytochrome bc1 complex suggests that electron transfer between inter-monomer hemes bL-bL may occur during bc1 catalysis. Such electron transfer may be facilitated by the aromatic pairs present between the two bL hemes in the two symmetry-related monomers. To test this hypothesis, R. sphaeroides mutants expressing His6-tagged bc1 complexes with mutations at three aromatic residues (Phe-195, Tyr-199, and Phe-203), located between two bL hemes, were generated and characterized. All three mutants grew photosynthetically at a rate comparable to that of wild-type cells. The bc1 complexes prepared from mutants F195A, Y199A, and F203A have, respectively, 78%, 100%, and 100% of ubiquinol-cytochrome c reductase activity found in the wild-type complex. Replacing the Phe-195 of cytochrome b with Tyr, His, or Trp results in mutant complexes (F195Y, F195H, or F195W) having the same ubiquinol-cytochrome c reductase activity as the wild-type. These results indicate that the aromatic group at position195 of cytochrome b is involved in electron transfer reactions of the bc1 complex. The rate of superoxide anion (O2*) generation, measured by the chemiluminescence of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one hydrochloride-O2* adduct during oxidation of ubiquinol, is 3 times higher in the F195A complex than in the wild-type or mutant complexes Y199A or F203A. This supports the idea that the interruption of electron transfer between the two bL hemes enhances electron leakage to oxygen and thus decreases the ubiquinol-cytochrome c reductase activity.     

Abundance, subunit composition, redox properties, and catalytic activity of the cytochrome bc1 complex from alkaliphilic and halophilic, photosynthetic members of the family Ectothiorhodospiraceae.

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complexes were demonstrated to be present in the membranes of the alkaliphilic and halophilic purple sulfur bacteria Ectothiorhodospira halophila, Ectothiorhodospira mobilis, and Ectothiorhodospira shaposhnikovii by protoheme extraction, immunoblotting, and electron paramagnetic resonance spectroscopy. The gy values of the Rieske [2Fe-2S] clusters observed in membranes of E. mobilis and E. halophila were 1.895 and 1.910, respectively. In E. mobilis membranes, the cytochrome bc1 complex was present in a stoichiometry of approximately 0.2 per reaction center. This complex was isolated and characterized. It contained four prosthetic groups: low-potential cytochrome b (cytochrome bL; Em = -142 mV), high-potential cytochrome b (cytochrome bH; Em = 116 mV), cytochrome c1 (Em = 341 mV), and a Rieske iron-sulfur cluster. The absorbance spectrum of cytochrome bL displayed an asymmetric alpha-band with a maximum at 564 nm and a shoulder at 559 nm. The alpha bands of cytochrome bH and cytochrome c1 peaked at 559.5 and 553 nm, respectively. These prosthetic groups were associated with three different polypeptides: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, with apparent molecular masses of 43, 30, and 21 kDa, respectively. No evidence for the presence of a fourth subunit was obtained. Maximal ubiquinol-cytochrome c oxidoreductase activity of the purified complex was observed at pH 8; the turnover rate was 57 mol of cytochrome c reduced.(mol of cytochrome c1)-1.s-1. The complex showed a strikingly low sensitivity towards typical inhibitors of cytochrome bc1 complexes.     

Proton pumping in the bc1 complex: a new gating mechanism that prevents short circuits

The Q-cycle mechanism of the bc1 complex explains how the electron transfer from ubihydroquinone (quinol, QH2) to cytochrome (cyt) c (or c2 in bacteria) is coupled to the pumping of protons across the membrane. The efficiency of proton pumping depends on the effectiveness of the bifurcated reaction at the Q(o)-site of the complex. This directs the two electrons from QH2 down two different pathways, one to the high potential chain for delivery to an electron acceptor, and the other across the membrane through a chain containing heme bL and bH to the Qi-site, to provide the vectorial charge transfer contributing to the proton gradient. In this review, we discuss problems associated with the turnover of the bc1 complex that center around rates calculated for the normal forward and reverse reactions, and for bypass (or short-circuit) reactions. Based on rate constants given by distances between redox centers in known structures, these appeared to preclude conventional electron transfer mechanisms involving an intermediate semiquinone (SQ) in the Q(o)-site reaction. However, previous research has strongly suggested that SQ is the reductant for O2 in generation of superoxide at the Q(o)-site, introducing an apparent paradox. A simple gating mechanism, in which an intermediate SQ mobile in the volume of the Q(o)-site is a necessary component, can readily account for the observed data through a coulombic interaction that prevents SQ anion from close approach to heme bL when the latter is reduced. This allows rapid and reversible QH2 oxidation, but prevents rapid bypass reactions. The mechanism is quite natural, and is well supported by experiments in which the role of a key residue, Glu-295, which facilitates proton transfer from the site through a rotational displacement, has been tested by mutation.     

The oxidation of ubiquinol by the isolated Rieske iron-sulfur protein in solution

The pre-steady-state redox reactions of the Rieske iron-sulfur protein isolated from beef heart mitochondria have been characterized. The rates of oxidation by c-type cytochromes is much faster than the rate of reduction by ubiquinols. This enables the monitoring of the oxidation of ubiquinols by the Rieske protein through the steady-state electron transfer to cytochrome c in solution. The pH and ionic strength dependence of this reaction indicate that the ubiquinol anion is the direct reductant of the oxidized cluster of the iron-sulfur protein. The second electron from ubiquinol is diverted to oxygen by the isolated Rieske protein, and forms oxygen radicals that contribute to the steady-state reduction of cytochrome c. Under anaerobic conditions, however, the reduction of cytochrome c catalyzed by the protein becomes mechanicistically identical to the chemical reduction by ubiquinols. The present kinetic work outlines that: (i) the electron transfer between the ubiquinol anion and the Rieske cluster has a comparable rate when the protein is isolated or inserted into the parent cytochrome c reductase enzyme; (ii) the Rieske protein may be a relevant generator of oxygen radicals during mitochondrial respiration.     

Kinetics of Electron Transfer within Cytochrome bc 1 and Between Cytochrome bc 1 and Cytochrome c

In this minireview an overview is presented of the kinetics of electron transfer within the cytochrome bc (1) complex, as well as from cytochrome bc (1) to cytochrome c. The cytochrome bc (1) complex (ubiquinone:cytochrome c oxidoreductase) is an integral membrane protein found in the mitochondrial respiratory chain as well as the electron transfer chains of many respiratory and photosynthetic bacteria. Experiments on both mitochondrial and bacterial cyatochrome bc (1) have provided detailed kinetic information supporting a Q-cycle mechanism for electron transfer within the complex. On the basis of X-ray crystallographic studies of cytochrome bc (1), it has been proposed that the Rieske iron-sulfur protein undergoes large conformational changes as it transports electrons from ubiquinol to cytochrome c (1). A new method was developed to study electron transfer within cytochrome bc (1) using a binuclear ruthenium complex to rapidly photooxidize cytochrome c (1). The rate constant for electron transfer from the iron-sulfur center to cytochrome c (1) was found to be 80,000 s(-1), and is controlled by the dynamics of conformational changes in the iron-sulfur protein. Moreover, a linkage between the conformation of the ubiquinol binding site and the conformational dynamics of the iron-sulfur protein has been discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method has also been developed to measure electron transfer from cytochrome c (1) to cytochrome c. The kinetics of electron transfer are interpreted in light of a new X-ray crystal structure for the complex between cytochrome bc (1) and cytochrome c.     

Regulatory interactions between ubiquinol oxidation and ubiquinone reduction sites in the dimeric cytochrome bc1 complex

We have obtained evidence for conformational communication between ubiquinol oxidation (center P) and ubiquinone reduction (center N) sites of the yeast bc1 complex dimer by analyzing antimycin binding and heme bH reduction at center N in the presence of different center P inhibitors. When stigmatellin was occupying center P, concentration-dependent binding of antimycin occurred only to half of the center N sites. The remaining half of the bc1 complex bound antimycin with a slower rate that was independent of inhibitor concentration, indicating that a slow conformational change needed to occur before half of the enzyme could bind antimycin. In contrast, under conditions where the Rieske protein was not fixed proximal to heme bL at center P, all center N sites bound antimycin with fast and concentration-dependent kinetics. Additionally, the extent of fast cytochrome b reduction by menaquinol through center N in the presence of stigmatellin was approximately half of that observed when myxothiazol was bound at center P. The reduction kinetics of the bH heme by decylubiquinol in the presence of stigmatellin or myxothiazol were also consistent with a model in which fixation of the Rieske protein close to heme bL in both monomers allows rapid binding of ligands only to one center N. Decylubiquinol at high concentrations was able to abolish the biphasic binding of antimycin in the presence of stigmatellin but did not slow down antimycin binding rates. These results are discussed in terms of half-of-the-sites activity of the dimeric bc1 complex.     

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c(6)), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c(6) through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.     

Anti-cooperative oxidation of ubiquinol by the yeast cytochrome bc1 complex

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc(1) complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c(1) reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is approximately 200 mV and in a bc(1) complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c(1) reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc(1) complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc(1) complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b(H) hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc(1) complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc(1) dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc(1) complex dimer.     

Ubiquinol oxidation in the cytochrome bc1 complex: reaction mechanism and prevention of short-circuiting

This review is focused on the mechanism of ubiquinol oxidation by the cytochrome bc1 complex (bc1). This integral membrane complex serves as a "hub" in the vast majority of electron transfer chains. The bc1 oxidizes a ubiquinol molecule to ubiquinone by a unique "bifurcated" reaction where the two released electrons go to different acceptors: one is accepted by the mobile redox active domain of the [2Fe-2S] iron-sulfur Rieske protein (FeS protein) and the other goes to cytochrome b. The nature of intermediates in this reaction remains unclear. It is also debatable how the enzyme prevents short-circuiting that could happen if both electrons escape to the FeS protein. Here, I consider a reaction mechanism that (i) agrees with the available experimental data, (ii) entails three traits preventing the short-circuiting in bc1, and (iii) exploits the evident structural similarity of the ubiquinone binding sites in the bc1 and the bacterial photosynthetic reaction center (RC). Based on the latter congruence, it is suggested that the reaction route of ubiquinol oxidation by bc1 is a reversal of that leading to the ubiquinol formation in the RC. The rate-limiting step of ubiquinol oxidation is then the re-location of a ubiquinol molecule from its stand-by site within cytochrome b into a catalytic site, which is formed only transiently, after docking of the mobile redox domain of the FeS protein to cytochrome b. In the catalytic site, the quinone ring is stabilized by Glu-272 of cytochrome b and His-161 of the FeS protein. The short circuiting is prevented as long as: (i) the formed semiquinone anion remains bound to the reduced FeS domain and impedes its undocking, so that the second electron is forced to go to cytochrome b; (ii) even after ubiquinol is fully oxidized, the reduced FeS domain remains docked to cytochrome b until electron(s) pass through cytochrome b; (iii) if cytochrome b becomes (over)reduced, the binding and oxidation of further ubiquinol molecules is hampered; the reason is that the Glu-272 residue is turned towards the reduced hemes of cytochrome b and is protonated to stabilize the surplus negative charge; in this state, this residue cannot participate in the binding/stabilization of a ubiquinol molecule.     

Evidence for a concerted mechanism of ubiquinol oxidation by the cytochrome bc1 complex

To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and cytochrome b(L) within the cytochrome bc(1) complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bc(1) complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of cytochrome c(1) reduction by approximately 10-fold but had no effect upon the rate of cytochrome c(1) reduction in bc(1) complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone cytochrome c(1), reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, cytochrome b(H) and b(L), exert negative control on the rate of reduction of cytochrome c(1) and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from cytochrome b(H) and b(L) to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.     

Insights from the structure of the yeast cytochrome bc1 complex: crystallization of membrane proteins with antibody fragments

The ubiquinol:cytochrome c oxidoreductase (EC 1.20.2.2, QCR or cytochrome bc1 complex) is a component of respiratory and photosynthetic electron transfer chains in mitochondria and bacteria. The complex transfers electrons from quinol to cytochrome c. Electron transfer is coupled to proton translocation across the lipid bilayer, thereby generating an electrochemical proton gradient, which conserves the free energy of the redox reaction. The yeast complex was crystallized with antibody Fv fragments, a promising technique to obtain well-ordered crystals from membrane proteins. The high-resolution structure of the yeast protein reveals details of the catalytic sites of the complex, which are important for electron and proton transfer.     

The diheme cytochrome b subunit (Narl) of Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols

Significant recent advances have been made in studies of the major dissimilatory nitrate reductase (NarGHI) of Escherichia coli. This enzyme is a complex iron-sulfur ([Fe-S]) molybdoenzyme that oxidizes menaquinol or ubiquinol at a periplasmically oriented Q-site (Qp site), and reduces nitrate at a cytoplasmically-oriented molybdo-(bismolybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor. The Qp site, as well as two hemes, termed bL and bH, are localized in a hydrophobic diheme cytochrome b(Narl) that: (i) provides a conduit for electron-transfer from the periplasmically-oriented Qp-site; (ii) provides a membrane anchoring functionality for the membrane-extrinsic subunits (NarGH) that coordinate the Mo-bisMGD (NarG) and four [Fe-S] clusters (NarH); and (iii) helps ensure the separation of sites of H+-yielding and H+-consuming reactions such that enzyme turnover leads to the generation of a proton-electrochemical potential across the cytoplasmic membrane. This minireview focuses on recent advances and future prospects for the diheme cytochrome b subunit (Narl) of NarGHI.