Characterization of an Intermediate in the Reduction of Acetylene by the Nitrogenase α-Gln195 MoFe Protein by Q-band EPR and 13C,1H ENDOR

Abstract

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 SEPR1, SEPR2, and SEPR3. 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 SEPR1 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 C2Hx. 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 SEPR2, 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.