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

FEBS Letters

Vinogradov, A.D.¹  Zakharova, N.V.¹  Zharova, T.V.¹ 

[1] Department of Biochemistry, School of Biology, Moscow State University, Moscow 119899, Russian Federation

关键词: NADH-ubiquinone reductase;    Complex I;    Nicotinamide nucleotide;    Transhydrogenation;    (Bovine heart mitochondria);    SMP;    submitochondrial particles;    FP;    three-subunit iron-sulfur flavoprotein;    APAD+;    3-acetylpyridine adenine dinucleotide;    FMN;    flavine mononucleotide;    DCIP;    dichlorophenolindophenol;   

DOI  :  10.1016/S0014-5793(99)00062-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 (K _i=40 μM) than with APAD⁺ (K _i=150 μM) 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.

【 授权许可】

Unknown