Catalytic formation of a nitrogenase iron-sulfur cluster.

Abstract

Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme comprised of two component proteins called the Fe protein and the MoFe protein. Both nitrogenase component proteins contain metalloclusters. The Azotobacter vinelandii nifS gene product (NifS), which is required for full activation of the nitrogenase component proteins, is a pyridoxal 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 as an intermediate in the cysteine desulfurization reaction catalyzed by NifS has been suggested as a possible S-donor in formation of the iron-sulfide cores of the nitrogenase metalloclusters. In the present work 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 Fe4S4 cluster using the chelator, alpha,alpha'-dipyridyl. The reconstitution reaction includes apo-Fe protein, NifS, L-cysteine, ferrous ion, dithiothreitol, and MgATP. Reconstitution of the inactive apo-Fe protein catalyzed by NifS results in 80-95% recovery of the original activity and yields an Fe protein having the normal electron paramagnetic resonance spectra properties associated with the Fe protein's Fe4S4 cluster. An altered NifS protein, NifS-Ala325, which lacks the desulfurase activity and is unable to from the NifS-bound persulfide, is not able to catalyze reactivation of the apo-Fe protein. These in vitro results support the proposal that NifS activity provides the inorganic sulfide necessary for in vivo formation of the nitrogenase metalloclusters. Moreover, because NifS has recently been shown to be a member of a highly homologous gene family, it appears that pyridoxal phosphate chemistry might play a general role in iron-sulfur cluster assembly.