Comparison of iron-molybdenum cofactor-deficient nitrogenase MoFe proteins by X-ray absorption spectroscopy: implications for P-cluster biosynthesis.

Nitrogenase, the enzyme system responsible for biological nitrogen fixation, is believed to utilize two unique metalloclusters in catalysis. There is considerable interest in understanding how these metalloclusters are assembled in vivo. It has been presumed that immature iron-molybdenum cofactor-deficient nitrogenase MoFe proteins contain the P-cluster, although no biosynthetic pathway for the assembly of this complex cluster has been identified as yet. Through the comparison by iron K-edge x-ray absorption edge and extended fine structure analyses of cofactor-deficient MoFe proteins resulting from nifH and nifB deletion strains of Azotobacter vinelandii, a novel [Fe-S] cluster is identified in the DeltanifH MoFe protein. The iron-iron scattering displayed by the DeltanifH MoFe protein is more similar to that of a standard [Fe(4)S(4)]-containing protein than that of the DeltanifB MoFe protein, which is shown to contain a "normal" P-cluster. The iron-sulfur scattering of the DeltanifH MoFe protein, however, indicates differences in its cluster from an [Fe(4)S(4)](Cys)(4) site that may be consistent with the presence of either oxygenic or nitrogenic ligation. Based on these results, models for the [Fe-S] center in the DeltanifH MoFe protein are constructed, the most likely of which consist of two separate [Fe(4)S(4)] sites, each with some non-cysteinyl coordination. This type of model suggests that the P-cluster is formed by the condensation of two [Fe(4)S(4)] fragments, possibly concomitant with Fe protein (NifH)-induced conformational change.