Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: Trapping propargyl alcohol with an α-70-substituted MoFe protein

Substitution of the MoFe protein α-70Val residue with Ala or Gly expands the substrate range of nitrogenase, allowing the reduction of larger alkynes, including propargyl alcohol (HC⋮CCH2OH). Herein, we report characterization of the α-70Val→Ala MoFe protein with propargyl alcohol trapped at the active site. The α-70Ala variant MoFe protein was rapidly frozen during reduction of propargyl alcohol, resulting in the conversion of the resting-state FeMo-cofactor EPR signal (S = 3/2 and g = [4.41, 3.60, 2.00]) to a new state (S = 1/2 and g = [2.123, 1.998, 1.986]). This EPR signal of the new state increased in intensity with increasing propargyl alcohol concentration, consistent with the binding of a single substrate. The EPR signal of the propargyl alcohol state showed temperature and microwave power dependencies markedly different from those of the classic FeMo-cofactor EPR signal, consistent with the difference in spin. The new state is analogous to that induced by the binding of the inhibitor CO (“lo CO” ...