Kinetics of the transhydrogenase reaction catalyzed by mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Abstract

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+ (Km = 7 microM, for SMP) and low rate (Vm = 0.2 micromol/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 (Vm = 1.7 micromol/min per mg protein and Km = 160 microM, 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.