Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites.

The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD+) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I-associated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (Ki = 40 microM) than with APAD+ (Ki = 150 microM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction.