Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN.

The maturation and installation of the active site metal cluster (FeMo-co, FeSCMo--homocitrate) in Mo-dependent nitrogenase requires the protein product of the gene for production of the FeS cluster precursor (NifB-co, [FeSC]) and the action of the maturase complex composed of the protein products from the and genes. However, some putative diazotrophic bacteria, like sp. RS-1, lack the genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the NifH, NifB, and apo-NifDK proteins produced in are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The expressed NifDK contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDK). Apo-NifDK could be activated for N reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDK plus produced NifB and NifH were sufficient to yield active NifDK when incubated with the necessary substrates (homocitrate, molybdate, and -adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The produced NifH and NifB proteins were independently shown to be functional. The reconstituted NifDK demonstrated reduction of N, protons, and acetylene in ratios observed for NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifH, NifB, and apo-NifDK.