Characterization of an intermediate in the reduction of acetylene by the nitrogenase α-Gln¹⁹⁵ MoFe protein by Q-band EPR and ¹³C,¹H ENDOR

Recent X-band EPR investigations of an altered nitrogenase MoFe protein for which the α-subunit His¹⁹⁵ residue has been substituted by Gln(α- Gln¹⁹⁵ MoFe protein) revealed that it exhibits three new S = 1/2 EPR signals when incubated under turnover conditions in the presence of acetylene (C₂H₂). These three signals are designated S(EPR1), S(EPR2), and S(EPR3). We now report Q-band EPR and ¹³C and ¹H ENDOR of the α-Gln¹⁹⁵ MoFe protein when incubated under turnover conditions in either H₂O or D₂O buffers with ¹²C₂H₂, ¹³C₂H₂, or C₂D₂ as the substrate. ENDOR measurements from S(EPR1) prepared with ¹³C₂H₂ reveal interactions with three distinct ¹³C nuclei, indicating that at least two C₂H₂-derived species are bound to the cofactor of the α-Gln¹⁹⁵ MoFe protein under turnover conditions. Although distinct, two of these species have approximately isotropic hyperfine tensors, with hyperfine splittings of A(C1,C2) ~ 2.4 MHz; the third has a smaller hyperfine splitting, A(C3) ≤ 0.5 MHz at g₁. ¹H ENDOR measurements further show strongly coupled proton signals (A ~ 12 MHz) that are associated with bound C₂H(x). The observation of this signal from the C₂H₂/D₂O sample indicates that this proton is not exchangeable with solvent in this cluster-bound state. Conversely, the absence of a signal in the C₂D₂/H₂O sample indicates that there is no strongly coupled proton derived from solvent. We propose that we are monitoring a C₂H₂ species that is bound to the FeMo-cofactor by bridging two Fe ions of a 4Fe4S 'face', thereby stabilizing the S = 1/2 cluster state. Q-band EPR also resolves rhombic features in the spectrum of S(EPR2), giving g = [2:007, 2.000, 1.992], but ENDOR showed no ¹³C signals with enriched substrate, confirming an earlier suggestion that this signal is not derived from C₂H₂.