Kinetics of the Transhydrogenase Reaction Catalyzed by Mitochondrial NADH:Ubiquinone Oxidoreductase (Complex I)

N. V. Zakharova

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

Received April 11, 2001

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The kinetics of the NADH-->3´-acetylpyridine adenine dinucleotide (APAD⁺) transhydrogenase reaction (DD-reaction) catalyzed by different preparations of mitochondrial NADH-dehydrogenase (submitochondrial particles (SMP), purified Complex I, and three-subunit fragment of Complex I (FP)) have been studied. Complex I (in SMP or in purified preparation) catalyzes two NADH-->APAD⁺ reactions with different rates and nucleotide affinities. Reaction 1 has high affinity to APAD⁺ (K_m = 7 µM, for SMP) and low rate (V_m = 0.2 µmol/min per mg protein, for SMP) and occurs with formation of a ternary complex. Reaction 2 has much higher rate and considerably lower affinity for oxidized nucleotide (V_m = 1.7 µmol/min per mg protein and K_m = 160 µM, for SMP). FP catalyzes only reaction 1. ADP-ribose inhibits reaction 1 with mixed type inhibition (competitive with non-competitive) with respect to NADH and APAD⁺. Rhein competes with both substrates. The results suggest that at least two nucleotide-binding sites exist in Complex I.

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KEY WORDS: NADH:ubiquinone oxidoreductase, transhydrogenase reaction, kinetics, Complex I, submitochondrial particles, three-subunit flavoprotein

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