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, Fe 7, S 9 ]) 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 lo-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) 57 Fe and 13 C 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. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 8707−8709 and Pollack, R. C.; Lee, H. I.; Cameron, L. M.; Derose, V. J.; Hales, B. J.; Orme-Johnson, 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 57 Fe ENDOR measurements of the CO-bound FeMo-cofactor in both lo- and hi-CO forms of the MoFe-protein from Azotobacter vinelandii . The 57 Fe ENDOR signals associated with the seven Fe ions of the FeMo-cofactor of lo-CO can be completely assigned and interpreted in terms of four magnetically distinct iron signals. Analysis of these signals following the procedures of Mouesca 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, Fe 7, S 9 ] + = [Mo 4+, Fe 3+ 1, Fe 2+ 6, S 2 - 9 ] +, organized into one Fe 2.5+ pair and five Fe 2+ ions, [Mo 4+, (2Fe 2.5+ ) 1, Fe 2+ 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.