No abstract

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...

Quinol oxidation by the bc 1 complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q o site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c 1 . Effects of mutations to the ISP that change the redox potential and͞or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or ␤-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H ؉ transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.The bc 1 complex family of enzymes plays a central role in all the main pathways of energy conversion, and the photosynthetic apparatus of Rhodobacter sphaeroides exemplifies one of the simplest of these (1-5). This system is convenient experimentally because of the ease with which electron transfer can be initiated by illumination (6). X-ray crystallographic structures of mitochondrial complexes (7-10) contain at their core the three catalytic subunits, cytochrome (cyt) b, cyt c 1 , and the Rieske iron-sulfur protein (ISP), common to the bacterial enzymes (11-13). Homology models of these show that the catalytic superstructure is highly conserved, as had been expected from studies of the mechanism, which is essentially the same in the two systems (1-5). The bc 1 complex catalyzes the oxidation of quinol and the reduction of cyt c (or c 2 ) through a modified Q cycle (1-6, 14-17). Two separate internal electron transfer chains connect three catalytic sites. At one site, heme c 1 is oxidized by cyt c 2 . Two catalytic sites in cyt b are involved in oxidation or reduction of ubiquinone. In the bifurcated reaction at the quinol-oxidizing site (the Q o site), one electron from quinol is passed to the ISP, which transfers it to cyt c 1 , while the semiquinone produced is oxidized by another chain consisting of the two b hemes of cyt b. At the quinone-reducing site (Q i -site), electrons fr...