PURIFICATION AND CHARACTERIZATION OF A FEMO COFACTOR-DEFICIENT MOFE PROTEIN

Previous studies have shown that the nifH gene product is required for FeMo cofactor biosynthesis and insertion and that a Delta nifH strain of Azotobacter vinelandii designated DJ54 accumulates a FeMo cofactor-deficient MoFe protein that is distinct from the FeMo cofactor-deficient protein synthesis by Nif B-, N-, or E(-) strains [Tal, S., Chun, T., Gavini, N., and Burgess, B. K. (1991) J. Biol. Chem. 266, 10654-10657]. Here we report the purification and activation of the MoFe protein from DJ54. The purified protein is an alpha(2) beta(2) tetramer that is indistinguishable from the wild-type MoFe protein by the criteria of SDS-polyacrylamide gel electrophoresis, native gel electrophoresis, and two-dimensional gel electrophoresis. It binds normally to its redox partner, the Fe protein, by the criterion of chemical cross-linking. It does not contain FeMo cofactor and does not catalyze significant C2H2 reduction or reductant-independent MgATP hydrolysis. It can, however, be activated with FeMo cofactor following the addition of the Fe protein and MgATP when an additional required component(s) is supplied by cell-free extracts from a Delta nifD strain of A. vinelandii. The purified DJ54 MoFe protein does contain P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the P-OX state. In the presence of dithionite it exhibits an axial S = 1/2 EPR signal that integrates to 0.1-0.3 spin per alpha(2) beta(2) tetramer. This signal has previously been observed for defective MoFe proteins from other organisms and for the VFe protein of A. vinelandii and may arise from a partially oxidized form of the P-clusters. The purified FeMoco-deficient MoFe protein can now be used to identify the spectral features specific for P-clusters in the MoFe and VFe proteins, for mechanistic studies, and as a starting material for in vitro assembly studies.