Mechanism of Ubiquinol Oxidation by the bc₁ Complex:  Different Domains of the Quinol Binding Pocket and Their Role in the Mechanism and Binding of Inhibitors

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...

Effect of Mutations of Arginine 94 on Proton Pumping, Electron Transfer, and Superoxide Anion Generation in Cytochrome b of the bc1 Complex from Rhodobacter sphaeroides

“…The histidine ligands take up a proton from the substrate, ubiquinol, upon reduction of the [2Fe-2S] cluster and release it to the intermembrane space when oxidized by cytochrome c 1 , as was suggested recently (31). Glu-295 in cytochrome b is important for the release of the second proton, as proposed previously (31)(32)(33). Glu-295 is completely conserved in mitochondrial cytochrome b (34), and the importance of the residue in proton transfer is indicated by mutagenesis studies because alteration of glutamine abolishes ubiquinol oxidation in R. sphaeroides (35).…”

Mechanism of Ubiquinol Oxidation by the bc1 Complex: Role of the Iron Sulfur Protein and Its Mobility

“…In the native structure the site is vacant. The position of the ISP in our myxothiazole or MOA-stilbenecontaining crystals (23) is the same as in the native structure (7,15,23). A number of features of interest to an understanding of the mechanism of the Q o site should be noted.…”

“…We have suggested that the g x ) 1.800 signal reflects the close interaction between quinone bound at the end of the pocket distal from heme b L and the reduced ISP docked at the interface on cytochrome b (15,23). This simple interpretation allows us to use the g x ) 1.800 signal as diagnostic of this interaction and provides a tool to explore the effects of mutation.…”

“…This rotation facilitates access to the inhibitor, which is also aided by the widening on displacement of the -PEWYloop. Changes deeper in the pocket, including the rotation of Glu-272 into the pocket noted above, are discussed in a separate paper (23). Impaired function upon site-directed mutagenesis of several of these residues has demonstrated their importance to function.…”

“…In solid-state systems, equivalent rates are achieved by a fixed intermediate redox carrier (64). Although the main evolutionary advantage of the more flexible arrangement probably pertains to the peculiar mechanism of the bifurcated reaction (22,23), the tethered head mechanism might provide other useful options. By constraining the local diffusional domain, the mechanism avoids the entropic loss of free diffusion and the slower rate of the second-order reaction resulting from dilution in a larger volume.…”