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-70^Val 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-70^Val→Ala MoFe protein with propargyl alcohol trapped at the active site. The α-70^Ala 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" state) to FeMo-cofactor in the wild-type MoFe protein. The ¹³C ENDOR spectrum of the α-70^Ala MoFe protein with trapped ¹³C-labeled propargyl alcohol exhibited three well-resolved ¹³C doublets centered at the ¹³C Larmor frequency with isotropic hyperfine couplings of ∼3.2, ∼1.4, and ∼0.7 MHz, indicating that the alcohol (or a fragment) is coordinated to the cofactor. The results presented here localize the binding site of propargyl alcohol to one [4Fe-4S] face of FeMo-cofactor and indicate roles for the α-70^Val residue in controlling FeMo-cofactor reactivity.