Substrate interaction at an iron-sulfur face of the FeMo-cofactor during nitrogenase catalysis

Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N₂ to 2NH₃. Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HC≡C-CH ₂OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid α-70^Val. Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HC≡CH), the physiological substrate dinitrogen, and its semi-reduced form hydrazine (H₂N-NH₂) interact with the same Fe-S face of the FeMo-cofactor. Hydrazine is a relatively poor substrate for the wild-type (α-70^Val) MoFe protein. Substitution of the α-70 ^Val residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of α-70^Val by an amino acid having a larger side chain (isoleucine) significantly lowered the capacity of the MoFe protein to reduce dinitrogen, hydrazine, or acetylene.