Exploring The Role Of The Central Carbide Of The Nitrogenase Active-Site Femo-Cofactor Through Targeted C-13 Labeling And Endor Spectroscopy

Mo-dependent nitrogenase is a major contributor to global biological N-2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4-) centered within a trigonal prismatic CFe6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with C-13, which enables its detailed examination by ENDOR/ESEEM spectroscopies. C-13-carbide ENDOR of the S = 3/2 resting state (E-0) is remarkable, with an extremely small isotropic hyperfine coupling constant, (C)a = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the C-13 carbide isotropic hyperfine-coupling constant essentially unchanged, (C)a = -1.30 MHz. This indicates that both the E-0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E-0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E-0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide-Fe bonds.