Exploring the Role of the Central Carbide of the Nitrogenase Active-Site FeMo-cofactor through Targeted C Labeling and ENDOR Spectroscopy.

Mo-dependent nitrogenase is a major contributor to global biological N reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (FeMoSC-homocitrate) contains a carbide (C) centered within a trigonal prismatic CFe 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, which enables its detailed examination by ENDOR/ESEEM spectroscopies. C-carbide ENDOR of the = 3/2 resting state (E) is remarkable, with an extremely small isotropic hyperfine coupling constant, = +0.86 MHz. Turnover under high CO partial pressure generates the = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small of the C carbide isotropic hyperfine-coupling constant essentially unchanged, = -1.30 MHz. This indicates that both the E and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to 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 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe core structures of E 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.