Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction |
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Authors: | Zakharova N V Zharova T V |
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Institution: | (1) Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119992, Russia |
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Abstract: | 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 M). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K
i = 5 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. |
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Keywords: | NADH:ubiquinone oxidoreductase Complex I nucleotide-binding centers |
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