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

Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N-2 to 2NH(3). Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HCequivalent toC-CH2OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid alpha-70(Val). Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HCequivalent toCH), 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 (alpha-70(Val)) MoFe protein. Substitution of the alpha-70(Val) residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of alpha-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.