title: Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles.
abstract: A kinetic study on ubiquinol-cytochrome c reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c1 complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a Km for ubiquinol-1 of 13.4 microM in mitochondria and of 24.6 microM in the isolated cytochrome b-c1 complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent Km values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the Vmax and the Km for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.
mesh_terms: D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology; D014450:Electron Transport Complex III; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*
chemical_list: D003574:Cytochrome c Group; D009097:Multienzyme Complexes; D003840:Deoxycholic Acid; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III