Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites.
The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD+) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I-associated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (Ki = 40 microM) than with APAD+ (Ki = 150 microM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction.
Zakharova
N V
NV
Department of Biochemistry, School of Biology, Moscow State University, Russian Federation.
Zharova
T V
TV
Vinogradov
A D
AD
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D042967:Electron Transport Complex I; D005420:Flavoproteins / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism; D013367:Submitochondrial Particles / Q000378:metabolism
D005420:Flavoproteins; D009243:NAD; C005941:3-acetylpyridine adenine dinucleotide; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D042967:Electron Transport Complex I