Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups

Abstract

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).

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase: chemical modification with N-ethylmaleimide and 2,4-pentanedione

“…These data do not allow a conclusion as to whether potentiation of inactivation results from a NADPH-induced conformational change in the membrane component that, in turn, is coupled to a conformational change in the soluble factor or if NADPH binding to the complexed soluble factor directly alters its conformation. These observations are consistent with the recent demonstration that NADP* prevents, whereas NADPH promotes, MalNEt modification of a peripheral sulfhydryl group outside the NADP-binding site of bovine heart mitochondrial transhydrogenase (Earle et al, 1978b). Similar results have been observed for MalNEt inactivation of E. coli transhydrogenase (Houghton et al, 1976).…”

“…N-Ethylmaleimide has been employed to localize sulfhydryl groups in both the NADand NADP-binding sites of Escherichia coli transhydrogenase (Houghton et al, 1976). However, the active-site sulfhydryl group in bovine heart transhydrogenase (O'Neal & Fisher, 1977) is not susceptible to MalNEt modification (Earle et al, 1978b). Inactivation with p-chloromercuribenzoate demonstrated the presence of an essential sulfhydryl group on the R. rubrum transhydrogenase soluble factor (Koenings & Guillory, 1973).…”

Reconstitution of bovine heart mitochondrial transhydrogenase: a reversible proton pump

“…As can be seen in Figure 4A, the pH profiles for the soluble and uncoupled reconstituted enzymes are similar in the reverse direction, with pH optima at about 6.25. These results are comparable to those of the submitochondrial particle enzyme (Earle et al, 1978b). However, the pH optima for forward transhydrogenation with soluble enzyme and submitochondrial particles are 5.5 (Figure 4B) and 6-6.5 (Earle et al, 1978b), respectively, whereas the rate of the uncoupled Reconstitution of transhydrogenase (13 gg) with dioleoylphosphatidylcholine and assays were performed as described under reconstituted reaction continued to increase to the lowest pH tested (5.0).…”

“…These results are comparable to those of the submitochondrial particle enzyme (Earle et al, 1978b). However, the pH optima for forward transhydrogenation with soluble enzyme and submitochondrial particles are 5.5 (Figure 4B) and 6-6.5 (Earle et al, 1978b), respectively, whereas the rate of the uncoupled Reconstitution of transhydrogenase (13 gg) with dioleoylphosphatidylcholine and assays were performed as described under reconstituted reaction continued to increase to the lowest pH tested (5.0). Although the soluble transhydrogenase is active in the forward reaction up to pH 9.0, reconstituted enzyme is inactive above pH 7.5.…”

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

“…The amino acid sequences of the two subunits of the Escherichia coli transhydrogenase have also been reported (Clarke et al, 1986). The bovine transhydrogenase is inhibited by a large number of protein modifying reagents, including thiol group modifiers (O'Neal & Fisher, 1977;Earle et al, 1978;Persson & Rydstrom, 1987), dicyclohexylcarbodiimide (DCCD) (Phelps & Hatefi, 1981, 1984aPennington & Fisher, 1981;Wakabayashi & Hatefi, 1987b), 7V-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) (Phelps & Hatefi, 1984b), [(p-fluorosulfonyl)benzoyl]-5'-adenosine (FSBA) (Phelps & Hatefi, 1985; Wakabayashi & Hatefi, 1987a), ethoxyformic anhydride (Yamaguchi & Hatefi, 1985), dansyl chloride (Yamaguchi & Hatefi, 1985), tetranitromethane (Wu & Fisher, 1982), pyridoxal phosphate (Yamaguchi & Hatefi, 1985), butanedione and phenylglyoxal (Djavadi-Ohaniance & Hatefi, 1975), and 4-chloro-7-nitrobenzofurazan (Nbf-Cl) (Persson et al, 1988). In addition, the enzyme activity is highly sensitive to trypsin (Juntti et al, 1970; Djavadi-Ohaniance & Hatefi, 1975;Blazyk et al, 1976).…”