PNNL

Abstract

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe-9S-C-Mo-homocitrate] contained within the MoFe protein ?-subunit. The cofactor contains a CFe6 core: a carbide centered within a trigonal prism of six Fe. The role of this CFe6 core in nitrogenase catalysis is unknown. A procedure for targeted 13C labeling the carbide enables ENDOR spectroscopy to sensitively and precisely monitor the electronic properties of the carbide-Fe bonds and the spin-coupling scheme adopted by FeMo-cofactor metal-ions. This report compares 13C-carbide ENDOR measurements for (i) wild-type protein resting state (E0) to those of (ii) a-Ile70, (iii) a-Ala70 substituted proteins, (iv) the crystallographically characterized CO-inhibited ‘hi-CO’ state; (v) the E4(4H) Janus intermediate, activated for N2 binding/reduction by accumulation of 4[e-/H+]; (vi) the E4(2H)* state containing doubly-reduced FeMo-cofactor without Fe-bound substrates; and (vii) a propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, whether S = ½ or 3/2, exhibit 13C isotropic hyperfine coupling constants, Ca ~ 0, consistent with persistence of a [3spin-up/3spin-down] spin-exchange configuration of the CFe6 Fe-ion spins that produces spin-cancellation on carbide. These results are illuminated by density functional theory computations and natural bond orbital analysis of the C-Fe bonds. Present findings indicate that in all these states a largely geometrically-invariant CFe6 core acts as ‘a heart of steel’ stabilizing the structure of FeMo-co, and further suggest this core acts to stabilize the FeMo-cofactor active site throughout the nitrogenase catalytic cycle.