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.

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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

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...