Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups.
Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group with N-ethylmaleimide, or both classes with 5,5'-dithiobis(2-nitrobenzoic acid), completely inactivated transhydrogenase. NADP+ or NADPH nearly completely protected against 5,5'-dithiobis(2-nitrobenzoic acid) inactivation and modification of both classes of sulfhydryl groups, while NADP+ only partially protected against and NADPH substantially stimulated N-ethylmaleimide inactivation. Methyl methanethiolsulfonate treatment resulted in methanethiolation at both classes of sulfhydryl groups, and either NADP+ or NADPH protected only the NADP site group. S-Methanethio and S-cyano transhydrogenases were active derivatives with pH optima shifted about 1 unit lower than that of the native enzyme. These experiments indicate that neither class of sulfhydryl group is essential for transhydrogenation. Lack of involvement of either sulfhydryl group in energy coupling to transhydrogenation is suggested by the observations that S-methanethio transhydrogenase is functional in (a) energy-linked transhydrogenation promoted by phenazine methosulfate mediated ascorbate oxidation and (b) the generation of a membrane potential during the reduction of NAD+ by reduced nicotinamide adenine dinucleotide phosphate (NADPH).
D000818:Animals; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP; D010084:Oxidation-Reduction; D013367:Submitochondrial Particles / Q000201:enzymology*
D009243:NAD; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D005033:Ethylmaleimide; D004229:Dithiothreitol