Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane.
Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP+, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH leads to AcPyAD+ reaction of reconstituted enzyme. NAD+ had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H+ translocation that was comparable to the decrease in the rate of NADPH leads to AcPyAD+ transhydrogenation. It is concluded that tetranitromethane modification controls the H+ translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H+-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5'-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.
D000818:Animals; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008697:Methane / Q000031:analogs & derivatives*; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*; D013379:Substrate Specificity; D013774:Tetranitromethane / Q000494:pharmacology*
D004228:Dithionitrobenzoic Acid; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D013774:Tetranitromethane; D008697:Methane