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) Fe-57 and C-13 electron nuclear double resonance (ENDOR) studies clearly identified [FeMo-cofactor][CO],, 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. Sec. 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. Sec. 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. Sec. 1997, 119, 10121-10126]. We now present complete orientation-selective Fe-57 ENDOR measurements of the GO-bound FeMo-cofactor in both Io-and hi-CO forms of the MoFe-protein from Azotobacter vinelandii. The Fe-57 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](+) = [Mo4+, Fe-1(3+), Fe-6(2+), S-9(2-)](+), organized into one Fe2.5+ pair and five Fe2+ ions, [Mo4+, (2Fe(2.5+))(1), Fe-5(2+), S-9(2-)](+). 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.