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, Fe₇S₉CMo-R-homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe₈S₉C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. However, some putative diazotrophic bacteria, like Roseiflexus sp. RS-1, lack the nifEN genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the Roseiflexus NifH, NifB, and apo-NifDK proteins produced in Escherichia coli are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The E. coli expressed NifDK^RS contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDK^RS). Apo-NifDK^RS could be activated for N₂ reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDK^RS plus E. coli produced NifB^RS and NifH^RS were sufficient to yield active NifDK^RS when incubated with the necessary substrates (homocitrate, molybdate, and S-adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The E. coli produced NifH^RS and NifB^RS proteins were independently shown to be functional. The reconstituted NifDK^RS demonstrated reduction of N₂, protons, and acetylene in ratios observed for Azotobacter vinelandii NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifH^RS, NifB^RS, and apo-NifDK^RS.