The cytochrome bc1 is one of the three major respiratory enzyme complexes residing in the inner mitochondrial membrane. Cytochrome bc1 transfers electrons from ubiquinol to cytochrome c and uses the energy thus released to form an electrochemical gradient across the inner membrane. Our X-ray crystal structures of the complex from chicken, cow and rabbit in both the presence and absence of inhibitors of quinone oxidation, reveal two different locations for the extrinsic domain of one component of the enzyme, an iron-sulphur protein. One location is close enough to the supposed quinol oxidation site to allow reduction of the Fe-S protein by ubiquinol. The other site is close enough to cytochrome c1 to allow oxidation of the Fe-S protein by the cytochrome. As neither location will allow both reactions to proceed at a suitable rate, the reaction mechanism must involve movement of the extrinsic domain of the Fe-S component in order to shuttle electrons from ubiquinol to cytochrome c1. Such a mechanism has not previously been observed in redox protein complexes.

Key Words oxidoreductase, respiratory chain, electron transfer, crystallography, membrane protein s Abstract The cytochrome bc complexes represent a phylogenetically diverse group of complexes of electron-transferring membrane proteins, most familiarly represented by the mitochondrial and bacterial bc 1 complexes and the chloroplast and cyanobacterial b 6 f complex. All these complexes couple electron transfer to proton translocation across a closed lipid bilayer membrane, conserving the free energy released by the oxidation-reduction process in the form of an electrochemical proton gradient across the membrane. Recent exciting developments include the application of site-directed mutagenesis to define the role of conserved residues, and the emergence over the past five years of X-ray structures for several mitochondrial complexes, and for two important domains of the b 6 f complex. CONTENTS

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.

Abstract(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c 2 oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c 1 and c 2 , the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a lowpotential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/ mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c 1 and c 2 , and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycinsensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/ mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 be...

Structures of mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc 1 complex) from several animal sources have provided a basis for understanding the functional mechanism at the molecular level. Using structures of the chicken complex with and without inhibitors, we analyze the effects of mutation on quinol oxidation at the Q o site of the complex. We suggest a mechanism for the reaction that incorporates two features revealed by the structures, a movement of the iron sulfur protein between two separate reaction domains on cytochrome c 1 and cytochrome b and a bifurcated volume for the Q o site. The volume identified by inhibitor binding as the Q o site has two domains in which inhibitors of different classes bind differentially; a domain proximal to heme b L , where myxothiazole and -methoxyacrylate-(MOA-) type inhibitors bind (class II), and a distal domain close to the iron sulfur protein docking interface, where stigmatellin and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiaole (UHDBT) bind (class I). Displacement of one class of inhibitor by another is accounted for by the overlap of their volumes, since the exit tunnel to the lipid phase forces the hydrophobic "tails" to occupy common space. We conclude that the site can contain only one "tailed" occupant, either an inhibitor or a quinol or one of their reaction products. The differential sensitivity of strains with mutations in the different domains is explained by the proximity of the affected residues to the binding domains of the inhibitors. New insights into mechanism are provided by analysis of mutations that affect changes in the electron paramagnetic resonance (EPR) spectrum of the iron sulfur protein, associated with its interactions with the Q o -site occupant. The structures show that all interactions with the iron sulfur protein must occur at the distal position. These include interactions between quinone, or class I inhibitors, and the reduced iron sulfur protein and formation of a reaction complex between quinol and oxidized iron sulfur protein. The step with high activation energy is after formation of the reaction complex, likely in formation of the semiquinone and subsequent dissociation of the complex into products. We suggest that further progress of the reaction requires a movement of semiquinone to the proximal position, thus mapping the bifurcated reaction to the bifurcated volume. We suggest that such a movement, together with a change in conformation of the site, would remove any semiquinone formed from further interaction with the oxidized [2Fe-2S] center and also from reaction with O 2 to form superoxide anion. We also identify two separate reaction paths for exit of the two protons released in quinol oxidation.The ubiquinol:cytochrome c oxidoreductases family of enzymes (the bc 1 complexes) 1 are central components of many electron-transfer systems, occurring ubiquitously in respiratory and photosynthetic chains of mitochondria and bacteria and (as the b 6 f complex) in the photosynthetic chain of oxygenic photosynth...