The Functional Role of Thiol Groups in Protease-Solubilized NADPH-Cytochrome c Reductase from Pork-Liver Microsomes

The total —SH content of protease-solubilized NADPH—cytochrome c reductase (EC 1.6.2.4) from pork liver microsomes was determined to be 5.9 ± 0.3 mol thiol groups per mol of protein. Only three —SH groups of the protease-solubilized NADPH – cytochrome c reductase could be modified by 0.34 or 1.0 mM 5,5′-dithio-bis(2-nitrobenzoate) at pH 7.5 and + 4°C. More than 95% of the original enzymatic activity was lost during this treatment. In the presence of 1 mM NADP+ only two —SH groups of the NADPH – cytochrome c reductase reacted with 5,5′-dithio-bis(2-nitrobenzoate). After removal of the competitive inhibitor NADP+, an enzyme derivative with the specific activity of the unmodified enzyme was obtained, containing two cysteinyl residues as mixed disulfides with 2-nitro-5-thiobenzoate. Renewed treatment of the modified reductase with 0.34 or 1.0 mM 5,5′-dithio-bis(2-nitrobenzoate) resulted in the modification of one additional thiol group under complete inactivation (k for modification = 0.027 min−1, k for inactivation = 0.039 min−1 at 0.34 mM Nbs2, pH 7.5, + 4°C). Kinetic analysis confirmed the suggestion that a single thiol group of the NADPH—cytochrome c reductase was protected by NADP+ against the reaction with 5,5′-dithio-bis(2-nitrobenzoate). Cytochrome c (58 μM) apparently enhanced the effect of NADP+. The modification of the accessible thiol groups by 1 mM 5,5′-dithio-bis(2-nitrobenzoate) did not affect the half-reduced state of the NADPH–cytochrome c reductase.