Identification of the NADH-NAD+ transhydrogenase peptide of the mitochondrial NADH-CoQ reductase (Complex I). A photodependent labeling study utilizing arylazido-beta-alanyl NAD+.
Incubation of Complex I (NADH-CoQ reductase) of ox heart mitochondria at 4 degrees C in the presence of 0.5 M NaClO4 followed by ammonium sulfate fractionation of the solubilized proteins results in the isolation of a resolved preparation still capable of catalyzing NADH-NAD+ transhydrogenation but having only low levels of NADH dehydrogenase activity. A number of NAD(H) analogues, including the photoaffinity probes, arylazido-beta-alanyl NAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]NAD+ and arylazido-beta-alanyl AcPyAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]AcPyAD+ can be utilized as substrates for transhydrogenation in this preparation. A further incubation (10 min) of the resolved NADH-NAD+ transhydrogenase in the presence of 0.5 M NaClO4, but now at 30 degrees C, results in the complete loss of this transhydrogenase activity. Photoaffinity labeling experiments utilizing arylazido-[3-3H]beta-alanyl NAD+ and arylazido-[3-3H]beta-alanyl AcPyAD+ with the resolved NADH-NAD+ transhydrogenase preparation prior to and following NaClO4 (30 degrees C) treatment indicates that the 42,000 molecular weight component of Complex I is the pyridine nucleotide binding site responsible for the major NADH-NAD+ (DD) transhydrogenase activity of Complex I.
D000818:Animals; D001386:Azides / Q000494:pharmacology*; D002417:Cattle; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives* / Q000494:pharmacology; D016660:NAD(P)H Dehydrogenase (Quinone); D009245:NADH Dehydrogenase / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010777:Photochemistry; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001386:Azides; C020606:arylazido-beta-alanyl-NAD; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C021842:NADH-NAD transhydrogenase; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D009245:NADH Dehydrogenase