Reduction of short chain alkynes by a nitrogenase α-70Ala-substituted MoFe protein

The Mo-dependent nitrogenase comprises two component proteins, called the Fe protein and MoFe protein, which together catalyze the nucleotide-dependent reduction of N2 to NH3. Nitrogenase also catalyzes the reduction of a variety of other multiply bonded substrates including the short chain alkyne acetylene. Substrate reduction is known to occur at an Fe7S9Mo : homocitrate cluster, designated FeMo-cofactor. Despite 40 years of intensive research and knowledge of the macromolecular structures of the nitrogenase component proteins from several different sources, the mechanism for the binding, activation and reduction of N2 is still unclear. Based on amino acid substitution studies for those residues that provide the first shell of non-covalent interactions with FeMo-cofactor we previously targeted a specific 4Fe-4S face of FeMo-cofactor approached by the MoFe protein α-subunit residues α-70Val and α-96Arg as providing the substrate binding and reduction site. In the present work, support for this hypothesis was obtained by showing that substitution of the α-70Val residue by α-70Ala relaxes constraints within the substrate-binding pocket so that effective reduction of the short chain alkynes, propargyl alcohol and propyne, which are not effectively reduced by the wild type enzyme, is now permitted.