Metal-ion valencies of the femo cofactor in CO-inhibited and resting state nitrogenase by ⁵⁷Fe Q-band ENDOR

The resting state of nitrogenase shows an S = 3/2 electron paramagnetic resonance (EPR) signal resulting from the FeMo-cofactor (M-center, inorganic portion, [Mo, Fe₇, S₉]) of the MoFe-protein. When the enzyme undergoes turnover under a CO atmosphere, this signal disappears and two new ones appear: one under low pressure of CO (denoted to-CO; 0.08 atm) with g = [2.09, 1.97, 1.93] and the other under high pressure of CO (denoted hi-CO; 0.5 atm) with g = [2.06, 2.06, 2.17]. Our recent Q-band (35 GHz) ⁵⁷Fe and ¹³C electron nuclear double resonance (ENDOR) studies clearly identified [FeMo-cofactor][CO](n), as the origin of the EPR signals from both lo-CO (n = 1) and hi-CO (n = 2) [Christie, P. D.; Lee, H. I.; Cameron, L. M.; Hales, B. L; Orme-Jolmson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1991L 118, 8707- 8709 and PoUack, R. C.; Lee, H. I.; Cameron, L. M.; Derose, V. J.; Hales, B. J.; Orme-J hns W.H.; Hoffman, B. M. J. Am. Chem. Soc. 1995, 117, 8686-8687], and a previous paper discusses CO binding in detail [Lee, H. I.; Cameron, L. M.; Hales, B. J.; Hoffman, B. M. J. Am. Chem. Soc. 1997, 119, 10121 - 10126]. We now present complete orientation-selective S7Fe ENDOR measurements of the CO-bound FeMo-cofactor in both lo- and hi-CO forms of the MoFe-protein from Azotobacter vlnelandii. The 57Fe ENDOR signals associated with the seven Fe ions of the FeMo-cofactor of 1o-CO can be completely assigned and interpreted in terms of four magnetically distinct iron signals. Analysis of these signals following the procedures of Monesea et al. [Mouesca, J.-M.; Noodleman, L.; Case, D. A.; Lamotte, B. Inorg. Chem. 1995, 34, 4347-4359] has led us to propose valence assignments and charges for the cofactor cluster, [Mo, Fe₇, S₉]⁺ = [Mo⁴⁺, Fe³⁺₁, Fe²⁺₆, S^2-₉]⁺, organized into one Fe².⁵ pair and five Fe²⁺ ions, [Mo⁴⁺, (2Fe².⁵)1, Fe²⁺₅, S^2- ₉]⁺. The result is a formal d-electron count of 43. ENDOR and functional studies indicate that the lo-CO, hi-CO, and resting states of the M-center are all at the same oxidation level. Hence, the proposed valency assignments apply to all three states.