AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV‐cofactor

Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo‐dependent, V‐dependent, or Fe‐only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo‐cofactor, FeV‐cofactor, and FeFe‐cofactor. Fe‐only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo‐dependent and V‐dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe‐only nitrogenase a gene product designated AnfO preserves the fidelity of Fe‐only nitrogenase by preventing the misincorporation of FeV‐cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2. These results are interpreted to indicate that AnfO controls the fidelity of Fe‐only nitrogenase maturation during the physiological transition from conditions that favor V‐dependent nitrogenase utilization to Fe‐only nitrogenase utilization to support diazotrophic growth. Azotobacter vinelandii produces three nitrogenase isozymes designated Mo‐dependent, V‐dependent, and Fe‐only on the basis of the heterometal contained in their corresponding active site cofactors. Inactivation of anfO results in misincorporation of the active site cofactor associated with V‐dependent nitrogenase into the Fe‐only nitrogenase. This observation indicates that AnfO preserves the fidelity of Fe‐only nitrogenase by preventing incorrect active site cofactor insertion during the transition phase to conditions that favor Fe‐only nitrogenase utilization.