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-70^Val and α-96^Arg 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-70^Val residue by α-70^Ala 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.