The Cytochrome bc 1 Complex and its Homologue the b 6 f Complex: Similarities and Differences

The ubihydroquinone:cytochrome c oxidoreductase (also called complex III, or bc (1) complex), is a multi subunit enzyme encountered in a very broad variety of organisms including uni- and multi-cellular eukaryotes, plants (in their mitochondria) and bacteria. Most bacteria and mitochondria harbor various forms of the bc (1) complex, while plant and algal chloroplasts as well as cyanobacteria contain a homologous protein complex called plastohydroquinone:plastocyanin oxidoreductase or b (6) f complex. Together, these enzyme complexes constitute the superfamily of the bc complexes. Depending on the physiology of the organisms, they often play critical roles in respiratory and photosynthetic electron transfer events, and always contribute to the generation of the proton motive force subsequently used by the ATP synthase. Primarily, this review is focused on comparing the 'mitochondrial-type' bc (1) complex and the 'chloroplast-type' b (6) f complex both in terms of structure and function. Specifically, subunit composition, cofactor content and assembly, inhibitor sensitivity, proton pumping, concerted electron transfer and Fe-S subunit large-scale domain movement of these complexes are discussed. This is a timely undertaking in light of the structural information that is emerging for the b (6) f complex.     

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

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.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

Photoinduced electron transfer between the Rieske iron-sulfur protein and cytochrome c(1) in the Rhodobacter sphaeroides cytochrome bc(1) complex. Effects of pH,temperature, and driving force

Electron transfer from the Rieske iron-sulfur protein to cytochrome c(1) (cyt c(1)) in the Rhodobacter sphaeroides cytochrome bc(1) complex was studied using a ruthenium dimer complex, Ru(2)D. Laser flash photolysis of a solution containing reduced cyt bc(1), Ru(2)D, and a sacrificial electron acceptor results in oxidation of cyt c(1) within 1 micros, followed by electron transfer from the iron-sulfur center (2Fe-2S) to cyt c(1) with a rate constant of 80,000 s(-1). Experiments were carried out to evaluate whether the reaction was rate-limited by true electron transfer, proton gating, or conformational gating. The temperature dependence of the reaction yielded an enthalpy of activation of +17.6 kJ/mol, which is consistent with either rate-limiting conformational gating or electron transfer. The rate constant was nearly independent of pH over the range pH 7 to 9.5 where the redox potential of 2Fe-2S decreases significantly due to deprotonation of His-161. The rate constant was also not greatly affected by the Rieske iron-sulfur protein mutations Y156W, S154A, or S154A/Y156F, which decrease the redox potential of 2Fe-2S by 62, 109, and 159 mV, respectively. It is concluded that the electron transfer reaction from 2Fe-2S to cyt c(1) is controlled by conformational gating.     

Function of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions of the mitochondrial respiratory chain

Resolution and reconstitution has been used to examine the involvement of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions in this region of the mitochondrial respiratory chain. The iron-sulfur protein is required for electron transfer from succinate and from ubiquinol to cytochrome c1. It is not required for reduction of cytochrome b under these conditions, but it is required for oxidation of cytochrome b by cytochrome c plus cytochrome c oxidase. Removal of the iron-sulfur protein from the b-c1 complex prevents reduction of both cytochromes b and c1 by succinate or ubiquinol if antimycin is added to the depleted complex. As increasing amounts of iron-sulfur protein are reconstituted to the depleted complex, the amounts of cytochromes b and c1 reduced by succinate in the presence of antimycin increase and closely parallel the amounts of ubiquinol-cytochrome c reductase activity restored to the reconstituted complex, measured before addition of antimycin. The function of the iron-sulfur protein in these oxidation-reduction reactions is consistent with a cyclic pathway of electron transfer through the cytochrome b-c1 complex, in which the iron-sulfur protein functions as a ubiquinol-cytochrome c1/ubisemiquinone-cytochrome b oxidoreductase.     

Cytochrome bc1 complexes of microorganisms.

The cytochrome bc1 complex is the most widely occurring electron transfer complex capable of energy transduction. Cytochrome bc1 complexes are found in the plasma membranes of phylogenetically diverse photosynthetic and respiring bacteria, and in the inner mitochondrial membrane of all eucaryotic cells. In all of these species the bc1 complex transfers electrons from a low-potential quinol to a higher-potential c-type cytochrome and links this electron transfer to proton translocation. Most bacteria also possess alternative pathways of quinol oxidation capable of circumventing the bc1 complex, but these pathways generally lack the energy-transducing, protontranslocating activity of the bc1 complex. All cytochrome bc1 complexes contain three electron transfer proteins which contain four redox prosthetic groups. These are cytochrome b, which contains two b heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a 2Fe-2S iron-sulfur protein. The mechanism which links proton translocation to electron transfer through these proteins is the proton motive Q cycle, and this mechanism appears to be universal to all bc1 complexes. Experimentation is currently focused on understanding selected structure-function relationships prerequisite for these redox proteins to participate in the Q-cycle mechanism. The cytochrome bc1 complexes of mitochondria differ from those of bacteria, in that the former contain six to eight supernumerary polypeptides, in addition to the three redox proteins common to bacteria and mitochondria. These extra polypeptides are encoded in the nucleus and do not contain redox prosthetic groups. The functions of the supernumerary polypeptides of the mitochondrial bc1 complexes are generally not known and are being actively explored by genetically manipulating these proteins in Saccharomyces cerevisiae.     

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.     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

Changes to the length of the flexible linker region of the Rieske protein impair the interaction of ubiquinol with the cytochrome bc1 complex.

Crystal structures of the cytochrome bc1 complex indicate that the catalytic domain of the Rieske iron-sulfur protein, which carries the [2Fe-2S] cluster, is connected to a transmembrane anchor by a flexible linker region. This flexible linker allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c1. Addition of an alanine residue to the flexible linker region of the Rieske protein lowers the ubiquinol-cytochrome c reductase activity of the mitochondrial membranes by one half and causes the apparent Km for ubiquinol to decrease from 9.3 to 2.6 microM. Addition of two alanine residues lowers the activity by 90% and the apparent Km decreases to 1.9 microM. Deletion of an alanine residue lowers the activity by approximately 40% and the apparent Km decreases to 5.0 microM. Addition or deletion of an alanine residue also causes a pronounced decrease in efficacy of inhibition of ubiquinol-cytochrome c reductase activity by stigmatellin, which binds analogous to reaction intermediates of ubiquinol oxidation. These results indicate that the length of the flexible linker region is critical for interaction of ubiquinol with the bc1 complex, consistent with electron transfer mechanisms in which ubiquinol must simultaneously interact with the iron-sulfur protein and cytochrome b.     

Purification of a reconstitutively active iron-sulfur protein (oxidation factor) from succinate . cytochrome c reductase complex of bovine heart mitochondria.

Oxidation factor, a protein required for electron transfer from succinate to cytochrome c in the mitochondrial respiratory chain, has been purified from isolated succinate . cytochrome c reductase complex. Purification of the protein has been followed by a reconstitution assay in which restoration of ubiquinol . cytochrome c reductase activity is proportional to the amount of oxidation factor added back to depleted reductase complex. The purified protein is a homogeneous polypeptide on acrylamide gel electrophoresis in sodium dodecyl sulfate and migrates with an apparent Mr = 24,500. Purified oxidation factor restores succinate . cytochrome c reductase and ubiquinol . cytochrome c reductase activities to depleted reductase complex. It is not required for succinate dehydrogenase nor for succinate . ubiquinone reductase activities of the reconstituted reductase complex. Oxidation factor co-electrophoreses with the iron-sulfur protein polypeptide of ubiquinol . cytochrome c reductase complex. The purified protein contains 56 nmol of nonheme iron and 36 nmol of acid-labile sulfide/mg of protein and possesses an EPR spectrum with the characteristic "g = 1.90" signal identical to that of the iron-sulfur protein of the cytochrome b . c1 complex. In addition, the optimal conditions for extraction of oxidation factor, including reduction with hydrosulfite and treatment of the b . c1 complex with antimycin, are identical to those which facilitate extraction of the iron-sulfur protein from the b . c1 complex. These results indicate that oxidation factor is a reconstitutively active form of the iron-sulfur protein of the cytochrome b . c1 complex first discovered by Rieske and co-workers (Rieske, J.S., Maclennan, D.H., and Coleman, R. (1964) Biochem. Biophys. Res. Commun. 15, 338-344) and thus demonstrate that this iron-sulfur protein is required for electron transfer from ubiquinol to cytochrome c in the mitochondrial respiratory chain.     

Reconstitution of the ubiquinol: cytochrome c reductase from a bc1 subcomplex and the 'Rieske' iron-sulfur protein isolated by a new method

1. A method for preparing the 'Rieske' iron-sulfur protein and the bc1 subcomplex of complex III was developed. The new method is advantageous over the published ones in that: (a) the final yield and amount exceeds by far those obtained when employing the hitherto published methods; (b) the iron-sulfur protein as well as the bc1 subcomplex are obtained by one and the same preparation procedure from a common source; and (c) the preparation method is easier than the published ones. 2. The iron-sulfur protein obtained represents the first reconstitutively active preparation present in a monodisperse state. 3. The reconstitution of the ubiquinol:cytochrome c reductase from the two components is a reversible dissociation process. Full activity of ubiquinol:cytochrome c reductase is reached after saturation of the binding site of the bc1 subcomplex for iron-sulfur protein. 4. Full reduction of the constituent cytochrome c1 of the bc1 subcomplex can already be obtained with substoichiometric amounts of iron-sulfur protein, however. 5. The question might be raised whether the observed dissociation equilibrium represents merely a phenomenon occurring specifically with the proteins isolated in Triton X-100 and investigated in a Triton-containing buffer, or whether dissociation of the iron-sulfur protein also takes place in the mitochondrial membrane in the course of the electron-transfer reaction sequence.     

Three molecules of ubiquinone bind specifically to mitochondrial cytochrome bc1 complex

Bifurcated electron flow to high potential "Rieske" iron-sulfur cluster and low potential heme b(L) is crucial for respiratory energy conservation by the cytochrome bc(1) complex. The chemistry of ubiquinol oxidation has to ensure the thermodynamically unfavorable electron transfer to heme b(L). To resolve a central controversy about the number of ubiquinol molecules involved in this reaction, we used high resolution magic-angle-spinning nuclear magnetic resonance experiments to show that two out of three n-decyl-ubiquinones bind at the ubiquinol oxidation center of the complex. This substantiates a proposed mechanism in which a charge transfer between a ubiquinol/ubiquinone pair explains the bifurcation of electron flow.     

Inter- and intra-molecular electron transfer in the cytochrome bc(1) complex

In this review, we compare the intra-molecular and inter-molecular electron transfer rate constants of the high-potential branch of the cytochrome bc(1) complex. Several methods such as the conventional stopped-flow spectroscopy, pH-induced electron transfer, photoactivated ruthenium complex induced electron transfer and photoreleaseable caged quinol, have been used to determine reaction rates between redox centers in an attempt to elucidate the reaction mechanism of this vital energy conserving complex. Since the most active pure cytochrome bc(1) complex has a turnover number of 800 s(-1), any step with a rate constant much larger than this will not be rate-limiting. The most likely rate-limiting step is the cytochrome b redox state governed movement of the head domain of iron-sulfur protein from its electron-accepting site ("fixed" or "b-state" position) to its electron donating site ("c(1)-state" position).     

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.     

Use of a photoactivated ruthenium dimer complex to measure electron transfer between the Rieske iron-sulfur protein and cytochrome c(1) in the cytochrome bc(1) complex

Electron transfer between the Rieske iron-sulfur protein (Fe(2)S(2)) and cytochrome c(1) was studied using the ruthenium dimer, Ru(2)D, to either photoreduce or photooxidize cytochrome c(1) within 1 micros. Ru(2)D has a charge of +4, which allows it to bind with high affinity to the cytochrome bc(1) complex. Flash photolysis of a solution containing beef cytochrome bc(1), Ru(2)D, and a sacrificial donor resulted in reduction of cytochrome c(1) within 1 micros, followed by electron transfer from cytochrome c(1) to Fe(2)S(2) with a rate constant of 90,000 s(-1). Flash photolysis of reduced beef bc(1), Ru(2)D, and a sacrificial acceptor resulted in oxidation of cytochrome c(1) within 1 micros, followed by electron transfer from Fe(2)S(2) to cytochrome c(1) with a rate constant of 16,000 s(-1). Oxidant-induced reduction of cytochrome b(H) was observed with a rate constant of 250 s(-1) in the presence of antimycin A. Electron transfer from Fe(2)S(2) to cytochrome c(1) within the Rhodobacter sphaeroides cyt bc(1) complex was found to have a rate constant of 60,000 s(-1) at 25 degrees C, while reduction of cytochrome b(H) occurred with a rate constant of 1000 s(-1). Double mutation of Ala-46 and Ala-48 in the neck region of the Rieske protein to prolines resulted in a decrease in the rate constants for both cyt c(1) and cyt b(H) reduction to 25 s(-1), indicating that a conformational change in the Rieske protein has become rate-limiting.     

Observations concerning the quinol oxidation site of the cytochrome bc1 complex

A direct hydrogen bond between ubiquinone/quinol bound at the QO site and a cluster-ligand histidine of the iron-sulfur protein (ISP) is described as a major determining factor explaining much experimental data on position of the ISP ectodomain, electron paramagnetic resonance (EPR) lineshape and midpoint potential of the iron-sulfur cluster, and the mechanism of the bifurcated electron transfer from ubiquinol to the high and low potential chains of the bc1 complex.     

Effect of mutations in the cytochrome b ef loop on the electron-transfer reactions of the Rieske iron-sulfur protein in the cytochrome bc1 complex

Long-range movement of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c1 redox centers plays a key role in electron transfer within the cyt bc1 complex. A series of 21 mutants in the cyt b ef loop of Rhodobacter sphaeroides cyt bc1 were prepared to examine the role of this loop in controlling the capture and release of the ISP from cyt b. Electron transfer in the cyt bc1 complex was studied using a ruthenium dimer to rapidly photo-oxidize cyt c1 within 1 mus and initiate the reaction. The rate constant for electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c1 was k1 = 60 000 s-1. Famoxadone binding to the Qo site decreases k1 to 5400 s-1, indicating that a conformational change on the surface of cyt b decreases the rate of release of the ISP from cyt b. The mutation I292A on the surface of the ISP-binding crater decreased k1 to 4400 s-1, while the addition of famoxadone further decreased it to 3000 s-1. The mutation L286A at the tip of the ef loop decreased k1 to 33 000 s-1, but famoxadone binding caused no further decrease, suggesting that this mutation blocked the conformational change induced by famoxadone. Studies of all of the mutants provide further evidence that the ef loop plays an important role in regulating the domain movement of the ISP to facilitate productive electron transfer and prevent short-circuit reactions.