Transhydrogenation reactions catalyzed by mitochondrial NADH-ubiquinone oxidoreductase (Complex I).
NADH-ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory electron transport chain in mitochondria. It conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the inner membrane. Complex I catalyzes NADPH oxidation, NAD+ reduction, and hydride transfers from reduced to oxidized nicotinamide nucleotides also. Here, we investigate the transhydrogenation reactions of complex I, using four different nucleotide pairs to encompass a range of reaction rates. Our experimental data are described accurately by a ping-pong mechanism with double substrate inhibition. Thus, we contend that complex I contains only one functional nucleotide binding site, in agreement with recent structural information, but in disagreement with previous mechanistic models which have suggested that two different binding sites are employed to catalyze the two half reactions. We apply the Michaelis-Menten equation to describe the productive states formed when the nucleotide and the active-site flavin mononucleotide have complementary oxidation states, and dissociation constants to describe the nonproductive states formed when they have the same oxidation state. Consequently, we derive kinetic and thermodynamic information about nucleotide binding and interconversion in complex I, relevant to understanding the mechanisms of coupled NADH oxidation and NAD+ reduction, and to understanding how superoxide formation by the reduced flavin is controlled. Finally, we discuss whether NADPH oxidation and/or transhydrogenation by complex I are physiologically relevant processes.
Yakovlev
Gregory
G
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.
Hirst
Judy
J
D000818:Animals; D002417:Cattle; D042967:Electron Transport Complex I / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000378:metabolism
D009243:NAD; C003451:thionicotinamide adenine dinucleotide; D009249:NADP; D006859:Hydrogen; C005941:3-acetylpyridine adenine dinucleotide; D042967:Electron Transport Complex I