Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction

• Published in: Biochemistry (Moscow) Vol. 67; no. 12; pp. 1395 - 1404
• Main Authors: Zakharova, N V, Zharova, T V
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01.12.2002
• Subjects: Binding, Competitive; Electron Transport; Electron Transport Complex I; Electrons; Enzymes; Kinetics; Mitochondria - enzymology; Models, Chemical; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - metabolism; NADP Transhydrogenases - chemistry; Nucleotides - chemistry; Substrate Specificity
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1023/A:1021818312040

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.