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