Mechanism for the Desulfurization of L-Cysteine Catalyzed by the nifS Gene Product

The nifS gene product (NIFS) is a pyridoxal phosphate binding enzyme that catalyzes the desulfurization Of L-cysteine to yield L-alanine and sulfur. In Azotobacter vinelandii this activity is required for the full activation of the nitrogenase component proteins. Because the nitrogenase component proteins, Fe protein and MoFe protein, both contain metalloclusters which are required for their respective activities, it is suggested that NIFS participates in the biosynthesis of the nitrogenase metalloclusters by providing the inorganic sulfur required for Fe-S core formation [Zheng, L., White, R.H., Cash, V.L., Jack, R.F., and Dean, D.R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2754-2758]. In the present study the mechanism for the desulfurization of L-cysteine catalyzed by NIFS was determined in the following ways. First, the substrate analogs, L-allylglycine and vinylglycine, were shown to irreversibly inactivate NIFS by formation of a gamma-methylcystathionyl or cystathionyl residue, respectively, through nucleophilic attack by an active site cysteinyl residue on the corresponding analog-pyridoxal phosphate adduct. Second, this reactive cysteinyl residue, which is required for L-cysteine desulfurization activity, was identified as Cys325 by the specific alkylation of that residue and by site-directed mutagenesis experiments. Third, the formation of an enzyme-bound cysteinyl persulfide was identified as an intermediate in the NIFS-catalyzed reaction. Fourth, evidence was obtained for an enamine intermediate in the formation of L-alanine. All of these results support a mechanism for NIFS-catalyzed desulfurization of L-cysteine which involves formation of a substrate cysteine-pyridoxal phosphate ketimine adduct and subsequent nucleophilic attack by the thiolate anion of Cys325 on the sulfur of the substrate cysteine. These events result in formation of a protein-bound persulfide, which is the proposed sulfur donor in Fe-S core formation, and a pyridoxal phosphate-bound