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 o f acetylpyridine adenine dinucleotide (APAD(+)) bJ NADH, DD transhydrogenas e) catalyzed by bovine heart submitochondrial particles (SMP), purified Com plex I, and by tbe soluble three-subunit NADH dehydrogenase (FP) were studi ed 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 vel ocities were not linear when the substrate concentrations were varied in a wide range, No binary complex (ping-pong) mechanism las expected for a sing le substrate-binding site enzyme) was operating within any range of the var iable substrates, ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (K-i = 40 mu M) than with APAD( +) (K-i = 150 mu M) in the transhydrogenase reaction. FMN redox cycling-dep endent, FP catalyzed DD transhydrogenase reaction was shown to proceed thro ugh a ternary complex mechanism, The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess m ore than one nucleotide-binding sites operating in the transhydrogenase rea ction, (C) 1999 Federation of European Biochemical Societies.