Metal-Ion Valencies of the FeMo Cofactor in CO-Inhibited and Resting State Nitrogenase by 57 Fe Q-Band ENDOR Hong-In Lee, † Brian J. Hales,* ,‡ and Brian M. Hoffman* ,† Contribution from the Departments of Chemistry, Northwestern UniVersity, EVanston, Illinois 60208, and Louisiana State UniVersity, Baton Rouge, Louisiana 70803 ReceiVed May 9, 1997 X Abstract: 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. Introduction Nitrogenase, which is comprised of the electron-transfer Fe- protein and the catalytic MoFe-protein, catalyzes the reduction of dinitrogen to ammonia, a key reaction of the biological nitrogen cycle. 1-3 X-ray diffraction of the MoFe-protein from Azotobacter Vinelandii (AV) 4 and Clostridium pasteurianum (Cp) disclosed the structures of two clusters in the MoFe-protein, P-cluster (Fe 8 S 7 ), and FeMo-cofactor (MoFe 7 S 9 :homocitrate), the site of substrate reduction. 5-9 This paper presents a proposal for the valencies of the metal ions and the charge on the cofactor. The resting state of nitrogenase shows an S ) 3 / 2 electron paramagnetic resonance (EPR) signal resulting from the FeMo- cofactor; 10-16 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 † Northwestern University. ‡ Louisiana State University X Abstract published in AdVance ACS Abstracts, November 1, 1997. (1) Burgess, B. K.; Lowe, D. J. Chem. ReV. 1996, 96, 2983-3011. (2) Burgess, B. K. Chem. ReV. 1990, 90, 1377-1406. (3) Newton, W. E. In Biological Nitrogen Fixation; Stacey, G., Burris, R. H., Evans, H. J., Eds.; Chapman and Hall: New York, 1992; pp 877- 929. 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