CATALYTIC FORMATION OF A NITROGENASE IRON-SULFUR CLUSTER

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme co mprised of two component proteins called the Fe protein and the MoFe p rotein. Both nitrogenase component proteins contain metalloclusters. T he Azotobacter vinelandii nifS gene product (NifS), which is required for full activation of the nitrogenase component proteins, is a pyrido xal phosphate enzyme and is able to catalyze the desulfurization of L- cysteine to yield sulfur and L-alanine (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). An enzyme-bound persulfide that was identified a s an intermediate in the cysteine desulfurization reaction catalyzed b y NifS has been suggested as a possible S-donor in formation of the ir on-sulfide cores of the nitrogenase metalloclusters. In the present wo rk it is shown that NifS is able to effectively catalyze activation of an apo-form of the Fe protein that was prepared by removal of its Fe4 S4 cluster using the chelator, alpha,alpha'-dipyridyl. The reconstitut ion reaction includes apo-Fe protein, NifS, L-cysteine, ferrous ion, d ithiothreitol, and MgATP. Reconstitution of the inactive apo-Fe protei n catalyzed by NifS results in 80-95% recovery of the original activit y and yields an Fe protein having the normal electron paramagnetic res onance spectral properties associated with the Fe protein's Fe4S4 clus ter. An altered NifS protein, NifS-Ala(325) which lacks the desulfuras e activity and is unable to form the NifS bound persulfide, is not abl e to catalyze reactivation of the apo-Fe protein. These in vitro resul ts support the proposal that NifS activity provides the inorganic sulf ide necessary for in vivo formation of the nitrogenase metalloclusters . Moreover, because NifS has recently been shown to be a member of a h ighly homologous gene family, it appears that pyridoxal phosphate chem istry might play a general role in iron-sulfur cluster assembly.