Oxidation process of bovine heart ubiquinol-cytochrome c reductase as studied by stopped-flow rapid-scan spectrophotometry and simulations based on the mechanistic Q cycle model.
Stopped-flow rapid-scan spectrophotometry was employed to study complicated oxidation processes of ubiquinol-cytochrome c reductase (QCR) that was purified from bovine heart mitochondria and maximally contained 0.36 mol of ubiquinone-10/mol of heme c1. When fully reduced QCR was allowed to react with dioxygen in the presence of cytochrome c plus cytochrome c oxidase, the oxidation of b-type hemes accompanied an initial lag, apparently low potential heme bL was oxidized first, followed by high potential heme bH. Antimycin A inhibited the oxidation of both b-type hemes. The oxidation of heme c1 was triphasic and became biphasic in the presence of antimycin A. On the other hand, starting from partially reduced QCR that was poised at a higher redox potential with succinate and succinate-cytochrome c reductase, the b-type hemes were oxidized immediately without a lag. When the ubiquinone content in QCR was as low as 0.1 mol/mol heme c1 the oxidation of the b-type hemes was almost suppressed. As the Q-deficient QCR was supplemented with ubiquinol-2, the rapid oxidation of b-type hemes was restored to some extent. These results indicate that a limited amount of ubiquinone-10 found in purified preparations of QCR is obligatory for electron transfer from the b-type hemes to iron-sulfur protein (ISP) and heme c1. The characteristic oxidation profiles of heme bL, heme bH, and heme c1 were simulated successfully based on a mechanistic Q cycle model. According to the simulations the two-electron oxidation of ubiquinol-10 via the ISP and heme c1 pathway, which is more favorable thermodynamically than the bifurcation of electron flow into both ISP and heme bL, does really occur as long as heme bL is in the reduced state and provides ubiquinone-10 at center i. Mechanistically this process takes time, thus explaining the initial lag in the oxidation of the b-type hemes. With the partially reduced QCR, inherent ubisemiquinone at center i immediately oxidizes reduced heme bH thus eliminating the lag. The mechanistic Q cycle model consists of 56 reaction species, which are interconnected by the reaction paths specified with microscopic rate constants. The simulations further indicate that the rate constants for electron transfer between the redox centers can be from 10(5) to 10(3) s-1 and are rarely rate-limiting. On the other hand, a shuttle of ubiquinone or ubiquinol between center o and center i and the oxidation of heme c1 can be rate-limiting. The interplay of the microscopic rate constants determines the actual reaction pathway that is shown schematically by the "reaction map." Most significantly, the simulations support the consecutive oxidation of ubiquinol in center o as long as both heme bL and heme bH are in the reduced state. Only when heme bL is oxidized and ISP is reduced can SQo donate an electron to heme bL. Thus, we propose that a kinetic control mechanism, or "a kinetic switch," is significant for the bifurcation of electron flow.
Orii
Y
Y
Department of Public Health, Graduate School of Medicine, Kyoto University, Kyoto 606, Japan. yorii@mbox.kyoto-inet.or.jp
Miki
T
T
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000968:Antimycin A / Q000494:pharmacology; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D014450:Electron Transport Complex III / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009206:Myocardium / Q000201:enzymology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D003574:Cytochrome c Group; D000968:Antimycin A; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III; D010100:Oxygen