Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction.
The effects of Tinopals (cationic benzoxazoles) AMS-GX and 5BM-GX on NADH-oxidase, NADH:ferricyanide reductase, and NADH --> APAD+ transhydrogenase reactions and energy-linked NAD+ reduction by succinate, catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in submitochondrial particles (SMP), were investigated. AMS-GX competes with NADH in NADH-oxidase and NADH:ferricyanide reductase reactions (K(i) = 1 micro M). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K(i) = 5 micro M) mechanism. Neither compound affects reverse electron transfer from succinate to NAD+. The type of the Tinopals' effect on the NADH --> APAD+ transhydrogenase reaction, occurring with formation of a ternary complex, suggests the ordered binding of nucleotides by the enzyme during the reaction: AMS-GX and 5BM-GX inhibit this reaction uncompetitively just with respect to one of the substrates (APAD+ and NADH, correspondingly). The competition between 5BM-GX and APAD+ confirms that NADH is the first substrate bound by the enzyme. Direct and reverse electron transfer reactions demonstrate different specificity for NADH and NAD+ analogs: the nicotinamide part of the molecule is significant for reduced nucleotide binding. The data confirm the model suggesting that during NADH --> APAD+ reaction, occurring with ternary complex formation, reduced nucleotide interacts with the center participating in NADH oxidation, whereas oxidized nucleotide reacts with the center binding NAD+ in the reverse electron transfer reaction.
Zakharova
N V
NV
Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119992 Russia. adv@biochem.bio.msu.su
Zharova
T V
TV
D001667:Binding, Competitive; D004579:Electron Transport; D042967:Electron Transport Complex I; D004583:Electrons; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008956:Models, Chemical; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D013379:Substrate Specificity
D009711:Nucleotides; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D042967:Electron Transport Complex I