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 His 195 residue has been substituted by Gln(α-Gln 195 MoFe protein) revealed that it exhibits three new S = 1 / 2 EPR signals when incubated under turnover conditions in the presence of acetylene (C 2 H 2 ). These three signals are designated S EPR1, S EPR2, and S EPR3 . We now report Q-band EPR and 13 C and 1 H ENDOR of the α-Gln 195 MoFe protein when incubated under turnover conditions in either H 2 O or D 2 O buffers with 12 C 2 H 2, 13 C 2 H 2, or C 2 D 2 as the substrate. ENDOR measurements from S EPR1 prepared with 13 C 2 H 2 reveal interactions with three distinct 13 C nuclei, indicating that at least two C 2 H 2 -derived species are bound to the cofactor of the α-Gln 195 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 g 1 . 1 H ENDOR measurements further show strongly coupled proton signals ( A ∼ 12 MHz) that are associated with bound C 2 H x . The observation of this signal from the C 2 H 2 /D 2 O sample indicates that this proton is not exchangeable with solvent in this cluster-bound state. Conversely, the absence of a signal in the C 2 D 2 /H 2 O sample indicates that there is no strongly coupled proton derived from solvent. We propose that we are monitoring a C 2 H 2 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 13 C signals with enriched substrate, confirming an earlier suggestion that this signal is not derived from C 2 H 2 .