Kinetic Mechanism of Mitochondrial NADH:Ubiquinone Oxidoreductase Interaction with Nucleotide Substrates of the Transhydrogenase Reaction

N. V. Zakharova^* and T. V. Zharova

Department of Biochemistry, School of Biology, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 939-3955; E-mail: adv@biochem.bio.msu.su

^* To whom correspondence should be addressed.

Received December 19, 2001; Revision received February 13, 2002

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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 µM). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K_i = 5 µM) 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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KEY WORDS: NADH:ubiquinone oxidoreductase, Complex I, nucleotide-binding centers

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