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On looking back at a lifetime of research, it is interesting to see, in the light of current progress, how things came to be, and to speculate on how things might be. I am delighted in the context of the Mitchell prize to have that excuse to present this necessarily personal view of developments in areas of my interests. I have focused on the Q-cycle and a few examples showing wider ramifications, since that had been the main interest of the lab in the 20 years since structures became available, - a watershed event in determining our molecular perspective. I have reviewed the evidence for our model for the mechanism of the first electron transfer of the bifurcated reaction at the Q o -site, which I think is compelling. In reviewing progress in understanding the second electron transfer, I have revisited some controversies to justify important conclusions which appear, from the literature, not to have been taken seriously. I hope this does not come over as nitpicking. The conclusions are important to the final section in which I develop an internally consistent mechanism for turnovers of the complex leading to a state similar to that observed in recent rapid-mix/freeze-quench experiments, reported three years ago. The final model is necessarily speculative but is open to test., (Copyright © 2021. Published by Elsevier B.V.)

A key feature of the modified Q-cycle of the cytochrome bc 1 and related complexes is a bifurcation of QH 2 oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H + are released on complete turnover of the Q-cycle (2H + /ubiquinol (QH 2 ) oxidized). We identified different partial processes of the Q o -site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron‑sulfur protein. In the absence of Q o -site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q o -site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH 2 on both turnovers. However, a minor lag in proton release found at pH 7 but not at pH 8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range., (Copyright © 2018. Published by Elsevier B.V.)

The Q-cycle mechanism of the bc 1 complex is now well enough understood to allow application of advanced computational approaches to the study of atomistic processes. In addition to the main features of the mechanism, these include control and gating of the bifurcated reaction at the Q o -site, through which generation of damaging reactive oxygen species is minimized. We report a new molecular dynamics model of the Rhodobacter sphaeroides bc 1 complex implemented in a native membrane, and constructed so as to eliminate blemishes apparent in earlier Rhodobacter models. Unconstrained MD simulations after equilibration with ubiquinol and ubiquinone respectively at Q o - and Q i -sites show that substrate binding configurations at both sites are different in important details from earlier models. We also demonstrate a new Q o -site intermediate, formed in the sub-ms time range, in which semiquinone remains complexed with the reduced iron sulfur protein. We discuss this, and a spring-loaded mechanism for modulating interactions of the iron sulfur protein with occupants of the Q o -site, in the context of control and gating roles. Such atomistic features of the mechanism can usefully be explored through simulation, but we stress the importance of constraints from physical chemistry and biology, both in setting up a simulation and in interpreting results.

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.

Enzymes of the bc1 complex family power the biosphere through their central role in respiration and photosynthesis. These enzymes couple the oxidation of quinol molecules by cytochrome c to the transfer of protons across the membrane, to generate a proton-motive force that drives ATP synthesis. Key for the function of the bc1 complex is the initial redox process that involves a bifurcated electron transfer in which the two electrons from a quinol substrate are passed to different electron acceptors in the bc1 complex. The electron transfer is coupled to proton transfer. The overall mechanism of quinol oxidation by the bc1 complex is well enough characterized to allow exploration at the atomistic level, but details are still highly controversial. The controversy stems from the uncertain binding motifs of quinol at the so-called Qo active site of the bc1 complex. Here we employ a combination of classical all atom molecular dynamics and quantum chemical calculations to reveal the binding modes of quinol at the Qo-site of the bc1 complex from Rhodobacter capsulatus. The calculations suggest a novel configuration of amino acid residues responsible for quinol binding and support a mechanism for proton-coupled electron transfer from quinol to iron-sulfur cluster through a bridging hydrogen bond from histidine that stabilizes the reaction complex.

1. Recent results suggest that the major flux is carried by a monomeric function, not by an intermonomer electron flow. 2. The bifurcated reaction at the Qo-site involves sequential partial processes, - a rate limiting first electron transfer generating a semiquinone (SQ) intermediate, and a rapid second electron transfer in which the SQ is oxidized by the low potential chain. 3. The rate constant for the first step in a strongly endergonic, proton-first-then-electron mechanism, is given by a Marcus-Brønsted treatment in which a rapid electron transfer is convoluted with a weak occupancy of the proton configuration needed for electron transfer. 4. A rapid second electron transfer pulls the overall reaction over. Mutation of Glu-295 of cyt b shows it to be a key player. 5. In more crippled mutants, electron transfer is severely inhibited and the bell-shaped pH dependence of wildtype is replaced by a dependence on a single pK at ~8.5 favoring electron transfer. Loss of a pK ~6.5 is explained by a change in the rate limiting step from the first to the second electron transfer; the pK ~8.5 may reflect dissociation of QH. 6. A rate constant (<10(3)s(-1)) for oxidation of SQ in the distal domain by heme bL has been determined, which precludes mechanisms for normal flux in which SQ is constrained there. 7. Glu-295 catalyzes proton exit through H(+) transfer from QH, and rotational displacement to deliver the H(+) to exit channel(s). This opens a volume into which Q(-) can move closer to the heme to speed electron transfer. 8. A kinetic model accounts well for the observations, but leaves open the question of gating mechanisms. For the first step we suggest a molecular "escapement"; for the second a molecular ballet choreographed through coulombic interactions. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We re-examine the pH dependence of partial processes of ubihydroquinone (QH(2)) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer. Loss of the pK at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6-8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to the wildtype. Occupancy of the Q(o)-site by semiquinone (SQ) was similar in the wildtype and the Glu→Trp mutant. Since heme b(L) is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant <10(3)s(-1) for reduction of heme b(L) by SQ from the domain distal from heme b(L), a value 1000-fold smaller than that expected from distance. If the pK ~8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q(•-) as electron donor to heme b(L), then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H(+) transfer from QH•, and delivery of the H(+) to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme b(L) by allowing Q(•-) to move closer to the heme, accounts well for the observations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The homodimeric bc(1) complexes are membrane proteins essential in respiration and photosynthesis. The ~11Å distance between the two b(L)-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc(1) complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The rate-determining step in the overall turnover of the bc(1) complex is electron transfer from ubiquinol to the Rieske iron-sulfur protein (ISP) at the Q(o)-site. Structures of the ISP from Rhodobacter sphaeroides show that serine 154 and tyrosine 156 form H-bonds to S-1 of the [2Fe-2S] cluster and to the sulfur atom of the cysteine liganding Fe-1 of the cluster, respectively. These are responsible in part for the high potential (E(m)(,7) approximately 300 mV) and low pK(a) (7.6) of the ISP, which determine the overall reaction rate of the bc(1) complex. We have made site-directed mutations at these residues, measured thermodynamic properties using protein film voltammetry to evaluate the E(m) and pK(a) values of ISPs, explored the local proton environment through two-dimensional electron spin echo envelope modulation, and characterized function in strains S154T, S154C, S154A, Y156F, and Y156W. Alterations in reaction rate were investigated under conditions in which concentration of one substrate (ubiquinol or ISP(ox)) was saturating and the other was varied, allowing calculation of kinetic terms and relative affinities. These studies confirm that H-bonds to the cluster or its ligands are important determinants of the electrochemical characteristics of the ISP, likely through electron affinity of the interacting atom and the geometry of the H-bonding neighborhood. The calculated parameters were used in a detailed Marcus-Brønsted analysis of the dependence of rate on driving force and pH. The proton-first-then-electron model proposed accounts naturally for the effects of mutation on the overall reaction.

The proton environment of the reduced [2Fe-2S] cluster in the water-soluble head domain of the Rieske iron-sulfur protein (ISF) from the cytochrome bc(1) complex of Rhodobacter sphaeroides has been studied by orientation-selected X-band 2D ESEEM. The 2D spectra show multiple cross-peaks from protons, with considerable overlap. Samples in which (1)H(2)O water was replaced by (2)H(2)O were used to determine which of the observed peaks belong to exchangeable protons, likely involved in hydrogen bonds in the neighborhood of the cluster. By correlating the cross-peaks from 2D spectra recorded at different parts of the EPR spectrum, lines from nine distinct proton signals were identified. Assignment of the proton signals was based on a point-dipole model for interaction with electrons of Fe(III) and Fe(II) ions, using the high-resolution structure of ISF from Rb. sphaeroides. Analysis of experimental and calculated tensors has led us to conclude that even 2D spectra do not completely resolve all contributions from nearby protons. Particularly, the seven resolved signals from nonexchangeable protons could be produced by at least 13 protons. The contributions from exchangeable protons were resolved by difference spectra ((1)H(2)O minus (2)H(2)O), and assigned to two groups of protons with distinct anisotropic hyperfine values. The largest measured coupling exceeded any calculated value. This discrepancy could result from limitations of the point dipole approximation in dealing with the distribution of spin density over the sulfur atoms of the cluster and the cysteine ligands, or from differences between the structure in solution and the crystallographic structure. The approach demonstrated here provides a paradigm for a wide range of studies in which hydrogen-bonding interactions with metallic centers has a crucial role in understanding the function.

Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.

The Rieske [2Fe-2S] iron-sulfur protein of cytochrome bc(1) functions as the initial electron acceptor in the rate-limiting step of the catalytic reaction. Prior studies have established roles for a number of conserved residues that hydrogen bond to ligands of the [2Fe-2S] cluster. We have constructed site-specific variants at two of these residues, measured their thermodynamic and functional properties, and determined atomic resolution X-ray crystal structures for the native protein at 1.2 A resolution and for five variants (Ser-154-->Ala, Ser-154-->Thr, Ser-154-->Cys, Tyr-156-->Phe, and Tyr-156-->Trp) to resolutions between 1.5 A and 1.1 A. These structures and complementary biophysical data provide a molecular framework for understanding the role hydrogen bonds to the cluster play in tuning thermodynamic properties, and hence the rate of this bioenergetic reaction. These studies provide a detailed structure-function dissection of the role of hydrogen bonds in tuning the redox potentials of [2Fe-2S] clusters.

The interaction of the reduced[2Fe-2S] cluster of isolated Rieske fragment from the bc1 complex of Rhodobacter sphaeroides with nitrogens (14N and 15N) from the local protein environment has been studied by X- and S-band pulsed EPR spectroscopy. The two-dimensional electron spin echo envelope modulation spectra of uniformly 15N-labeled protein show two well resolved cross-peaks with weak couplings of approximately 0.3-0.4 and 1.1 MHz in addition to couplings in the range of 6-8 MHz from two coordinating Ndelta of histidine ligands. The quadrupole coupling constants for weakly coupled nitrogens determined from S-band electron spin echo envelope modulation spectra identify them as Nepsilon of histidine ligands and peptide nitrogen (Np), respectively. Analysis of the line intensities in orientation-selected S-band spectra indicated that Np is the backbone N-atom of Leu-132 residue. The hyperfine couplings from Nepsilon and Np demonstrate the predominantly isotropic character resulting from the transfer of unpaired spin density onto the 2s orbitals of the nitrogens. Spectra also show that other peptide nitrogens in the protein environment must carry a 5-10 times smaller amount of spin density than the Np of Leu-132 residue. The appearance of the excess unpaired spin density on the Np of Leu-132 residue indicates its involvement in hydrogen bond formation with the bridging sulfur of the Rieske cluster. The configuration of the hydrogen bond therefore provides a preferred path for spin density transfer. Observation of similar splittings in the 15N spectra of other Rieske-type proteins and [2Fe-2S] ferredoxins suggests that a hydrogen bond between the bridging sulfur and peptide nitrogen is a common structural feature of [2Fe-2S] clusters.

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.

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 kinetics of the cytochrome (cyt) components of the bc(1) complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The "traditional" set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c(tot) (cyt c(1)+cyt c(2)), cyt b(L), cyt b(H), and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c(1) and c(2) is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c(1), c(2), b(L), and b(H)) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc(1) complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc(1) complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.

The cytochrome (cyt) bc(1) complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc(1) complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc(1) complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc(1) turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b(L). From LS analysis of the chromophoric components (RC, c(tot), b(H) and b(L)), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc(1) complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.

The pH-dependent resonance Raman (RR) spectral changes of the cytochrome bc1-associated, high-potential Rieske proteins have frequently been invoked to explain the redox-linked ionization behavior. We report herein RR spectral data of archaeal and bacterial Rieske proteins that directly demonstrate the pH-dependent changes near and above pKa,ox2, but not around pKa,ox1, of the visible circular dichroism (CD) transitions. The RR spectral changes are attributed to modification of the immediate [2Fe-2S] cluster environment due to deprotonation of some exchangeable amide groups in the polypeptide backbone, rather than previously assumed simple changes of the Fe-Nimid stretching vibrations.

Exchangeable protons in the immediate neighborhood of the semiquinone (SQ) at the Qi-site of the bc1 complex (ubihydroquinone:cytochrome c oxidoreductase (EC 1.10.2.2)) from Rhodobacter sphaeroides have been characterized using electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation spectroscopy (HYSCORE) and visualized by substitution of H2O by 2H2O. Three exchangeable protons interact with the electron spin of the SQ. They possess different isotropic and anisotropic hyperfine couplings that allow a clear distinction between them. The strength of interactions indicates that the protons are involved in hydrogen bonds with SQ. The hyperfine couplings differ from values typical for in-plane hydrogen bonds previously observed in model experiments. It is suggested that the two stronger couplings involve formation of hydrogen bonds with carbonyl oxygens, which have a significant out-of-plane character due to the combined influence of bulky substituents and the protein environment. These two hydrogen bonds are most probably to side chains suggested from crystallographic structures (His-217 and Asp-252 in R. sphaeroides). Assignment of the third hydrogen bond is more ambiguous but may involve either a bond between Asn-221 and a methoxy O-atom or a bond to water. The structural and catalytic roles of the exchangeable protons are discussed in the context of three high resolution crystallographic structures for mitochondrial bc1 complexes. Potential H-bonds, including those to water molecules, form a network connecting the quinone (ubiquinone) occupant and its ligands to the propionates of heme bH and the external aqueous phase. They provide pathways for exchange of protons within the site and with the exteriors, needed to accommodate the different hydrogen bonding requirements of different quinone species during catalysis.

The rate-limiting reaction of the bc(1) complex from Rhodobacter sphaeroides is transfer of the first electron from ubihydroquinone (quinol, QH(2)) to the [2Fe-2S] cluster of the Rieske iron-sulfur protein (ISP) at the Q(o)-site. Formation of the ES-complex requires participation of two substrates (S), QH(2) and ISP(ox). From the variation of rate with [S], the binding constants for both substrates involved in formation of the complex can be estimated. The configuration of the ES-complex likely involves the dissociated form of the oxidized ISP (ISP(ox)) docked at the b-interface on cyt b, in a complex in which N(epsilon) of His-161 (bovine sequence) forms a H-bond with the quinol -OH. A coupled proton and electron transfer occurs along this H-bond. This brief review discusses the information available on the nature of this reaction from kinetic, structural and mutagenesis studies. The rate is much slower than expected from the distance involved, likely because it is controlled by the low probability of finding the proton in the configuration required for electron transfer. A simplified treatment of the activation barrier is developed in terms of a probability function determined by the Brønsted relationship, and a Marcus treatment of the electron transfer step. Incorporation of this relationship into a computer model allows exploration of the energy landscape. A set of parameters including reasonable values for activation energy, reorganization energy, distances between reactants, and driving forces, all consistent with experimental data, explains why the rate is slow, and accounts for the altered kinetics in mutant strains in which the driving force and energy profile are modified by changes in E(m) and/or pK of ISP or heme b(L).

The bc1 complexes are intrinsic membrane proteins that catalyze the oxidation of ubihydroquinone and the reduction of cytochrome c in mitochondrial respiratory chains and bacterial photosynthetic and respiratory chains. The bc1 complex operates through a Q-cycle mechanism that couples electron transfer to generation of the proton gradient that drives ATP synthesis. Genetic defects leading to mutations in proteins of the respiratory chain, including the subunits of the bc1 complex, result in mitochondrial myopathies, many of which are a direct result of dysfunction at catalytic sites. Some myopathies, especially those in the cytochrome b subunit, exacerbate free-radical damage by enhancing superoxide production at the ubihydroquinone oxidation site. This bypass reaction appears to be an unavoidable feature of the reaction mechanism. Cellular aging is largely attributable to damage to DNA and proteins from the reactive oxygen species arising from superoxide and is a major contributing factor in many diseases of old age. An understanding of the mechanism of the bc1 complex is therefore central to our understanding of the aging process. In addition, a wide range of inhibitors that mimic the quinone substrates are finding important applications in clinical therapy and agronomy. Recent structural studies have shown how many of these inhibitors bind, and have provided important clues to the mechanism of action and the basis of resistance through mutation. This paper reviews recent advances in our understanding of the mechanism of the bc1 complex and their relation to these physiologically important issues in the context of the structural information available.

Rieske [2Fe-2S] clusters can be classified into two groups, depending on their reduction potentials. Typical high-potential Rieske proteins have pH-dependent reduction potentials between +350 and +150 mV at pH 7, and low-potential Rieske proteins have pH-independent potentials of around -150 mV at pH 7. The pH dependence of the former group is attributed to coupled deprotonation of the two histidine ligands. Protein-film voltammetry has been used to compare three Rieske proteins: the high-potential Rieske proteins from Rhodobacter sphaeroides (RsRp) and Thermus thermophilus (TtRp) and the low-potential Rieske ferredoxin from Burkholderia sp. strain LB400 (BphF). RsRp and TtRp differ because there is a cluster to serine hydrogen bond in RsRp, which raises its potential by 140 mV. BphF lacks five hydrogen bonds to the cluster and an adjacent disulfide bond. Voltammetry measurements between pH 3 and 14 reveal that all the proteins, including BphF, have pH-dependent reduction potentials with remarkably similar overall profiles. Relative to RsRp and TtRp, the potential versus pH curve of BphF is shifted to lower potential and higher pH, and the pK(a) values of the histidine ligands of the oxidized and reduced cluster are closer together. Therefore, in addition to simple electrostatic effects on E and pK(a), the reduction potentials of Rieske clusters are determined by the degree of coupling between cluster oxidation state and histidine protonation state. Implications for the mechanism of quinol oxidation at the Q(O) site of the cytochrome bc(1) and b(6)f complexes are discussed.

Pulsed EPR spectroscopy was used to explore the structural neighborhood of the semiquinone (SQ) stabilized at the Qi site of the bc1 complex of Rhodobacter sphaeroides (EC 1.10.2.2) and to demonstrate that the nitrogen atom of a histidine imidazole group donates an H-bond to the SQ. Crystallographic structures show two different configurations for the binding of ubiquinone at the Qi site of mitochondrial bc1 complexes in which histidine (His-201 in bovine sequence) is either a direct H-bond donor or separated by a bridging water. The paramagnetic properties of the SQ formed at the site provide an independent method for studying the liganding of this intermediate species. The antimycin-sensitive SQ formed at the Qi site by either equilibrium redox titration, reduction of the oxidized complex by ascorbate, or addition of decylubihydroquinone to the oxidized complex in the presence of myxothiazol all showed similar properties. The electron spin echo envelope modulation spectra in the 14N region were dominated by lines with frequencies at 1.7 and 3.1 MHz. Hyperfine sublevel correlation spectroscopy spectra showed that these were contributed by a single nitrogen. Further analysis showed that the 14N nucleus was characterized by an isotropic hyperfine coupling of approximately 0.8 MHz and a quadrupole coupling constant of approximately 0.35 MHz. The nitrogen was identified as the N-epsilon or N-delta imidazole nitrogen of a histidine (it is likely to be His-217, or His-201 in bovine sequence). A distance of 2.5-3.1 A for the O-N distance between the carbonyl of SQ and the nitrogen was estimated. The mechanistic significance is discussed in the context of a dynamic role for the movement of His-217 in proton transfer to the site.

Crystallographic structures of the bc1 complex from different sources have provided evidence that a movement of the Rieske iron-sulfur protein (ISP) extrinsic domain is essential for catalysis. This dynamic feature has opened up the question of what limits electron transfer, and several authors have suggested that movement of the ISP head, or gating of such movement, is rate-limiting. Measurements of the kinetics of cytochromes and of the electrochromic shift of carotenoids, following flash activation through the reaction center in chromatophore membranes from Rhodobacter sphaeroides, have allowed us to demonstrate that: (i) ubiquinol oxidation at the Qo-site of the bc1 complex has the same rate in the absence or presence of antimycin bound at the Qi-site, and is the reaction limiting turnover. (ii) Activation energies for transient processes to which movement of the ISP must contribute are much lower than that of the rate-limiting step. (iii) Comparison of experimental data with a simple mathematical model demonstrates that the kinetics of reduction of cytochromes c1 and bH are fully explained by the modified Q-cycle. (iv) All rates for processes associated with movement of the ISP are more rapid by at least an order of magnitude than the rate of ubiquinol oxidation. (v) Movement of the ISP head does not introduce a significant delay in reduction of the high potential chain by quinol, and it is not necessary to invoke such a delay to explain the kinetic disparity between the kinetics of reduction of cytochromes c1 and bH.

Following addition of myxothiazol to antimycin-treated chromatophores from Rhodobacter sphaeroides poised at an ambient redox potential (E(h)) of approximately 300 mV, the amplitude of the flash-induced cytochrome c(1) oxidation in the ms range increased, indicating a decrease in the availability of electrons from the immediate donor to c(1), the Rieske iron-sulfur protein (ISP). Because the effect was seen only over the limited E(h) range, we conclude that it is due to a decrease in the apparent midpoint redox potential (E(m)) of the ISP by about 40 mV on addition of myxothiazol. This is in line with the change in E(m) previously seen in direct redox titrations. Our results show that the reduced ISP binds with quinone at the Q(o) site with a higher affinity than does the oxidized ISP. The displacement of ubiquinone by myxothiazol leads to elimination of this preferential binding of the ISP reduced form and results in a shift in the midpoint potential of ISP to a more negative value. A simple hypothesis to explain this effect is that myxothiazol prevents formation of hydrogen bond of ubiquinone with the reduced ISP. We conclude that all Q(o) site occupants (ubiquinone, UHDBT, stigmatellin) that form hydrogen bonds with the reduced ISP shift the apparent E(m) of the ISP in the same direction to more positive values. Inhibitors that bind in the domain of the Q(o) site proximal to heme b(L) (myxothiazol, MOA-stilbene) and displace ubiquinone from the site cause a decrease in E(m) of ISP. We present a new formalism for treatment of the relation between E(m) change and the binding constants involved, which simplifies analysis. Using this formalism, we estimated that binding free energies for hydrogen bond formation with the Q(o) site occupant, range from the largest value of approximately 23 kJ mol(-1) in the presence of stigmatellin (appropriate for the buried hydrogen bond shown by structures), to a value of approximately 3.5 kJ mol(-1) in the native complex. We discuss this range of values in the context of a model in which the native structure constrains the interaction of ISP with the Q(o) site occupant so as to favor dissociation and the faster kinetics of unbinding necessary for rapid turnover.

Since available structures of native bc(1) complexes show a vacant Q(o)-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme-substrate complex at the Q(o)-site. This is formed by binding of both ubiquinol (QH(2)) and the dissociated oxidized iron-sulfur protein (ISP(ox)). A binding constant of approximately 14 can be estimated from the displacement of E(m) or pK for quinone or ISP(ox), respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme-product complex is also formed at the Q(o)-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E(m) of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E(m) reflects a binding constant of approximately 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q(o)-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.

The cytochrome (cyt) bc(1) complex is central to energy transduction in many species. Most investigators now accept a modified Q-cycle as the catalytic mechanism of this enzyme. Several thermodynamically favorable side reactions must be minimized for efficient functioning of the Q-cycle. Among these, reduction of oxygen by the Q(o) site semiquinone to produce superoxide is of special pathobiological interest. These superoxide-producing bypass reactions are most notably observed as the antimycin A- or myxothiazol-resistant reduction of cyt c. In this work, we demonstrate that these inhibitor-resistant cyt c reductase activities are largely unaffected by removal of O(2) in the isolated yeast cyt bc(1) complex. Further, increasing O(2) tension 5-fold stimulated the antimycin A-resistant reduction by a small amount ( approximately 25%), while leaving the myxothiazol-resistant reduction unchanged. This most likely indicates that the rate-limiting step in superoxide production is the formation of a reactive species (probably a semiquinone), capable of rapid O(2) reduction, and that in the absence of O(2) this species can reduce cyt c by some other pathway. We suggest as one possibility that a semiquinone escapes from the Q(o) site and reduces either O(2) or cyt c directly. The small increase in antimycin A-resistant cyt c reduction rate at high O(2) can be explained by the accumulation of a low concentration of a semiquinone inside the Q(o) site. Under aerobic conditions, addition of saturating levels of superoxide dismutase (SOD) inhibited 50% of cyt c reduction in the presence of myxothiazol, implying that essentially all bypass reactions occur with the production of superoxide. However, SOD inhibited only 35% of antimycin A-resistant cyt c reduction, suggesting the presence of a second, slower bypass reaction that does not reduce O(2). Given that myxothiazol blocks cyt b reduction whereas antimycin A promotes it, we propose that this second bypass occurs by reduction of the Q(o) site semiquinone by prereduced cyt b(L).

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.