CO binding to the FeMo cofactor of CO-inhibited nitrogenase: ¹³CO and ¹H Q-band ENDOR investigation

The resting state of nitrogenase shows an S = 3/2 electron paramagnetic resonance (EPR) signal resulting from the FeMo-cofactor (MoFe₇S₉:homocitrate) 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; 8.08 atm) and the other under high pressure of CO (denoted hi-CO; 0.5 atm). Our recent Q-band (35 GHz) ¹³C and ⁵⁷Fe electron nuclear double resonance (ENDOR) studies demonstrated that one CO is bound to the FeMo-cofactor of lo-CO and two to the cofactor of hi-CO. [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. 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.] In the present report, we examine the GO-bound FeMo-cofactor in both the lo- and hi-CO forms of the MoFe protein from Azotobacter vinelandii by complete orientation-selective ¹³C and ¹H ENDOR measurements. ¹H ENDOR studies reveal that well-resolved signals from a solvent-exchangeable proton seen in the resting state FeMo-cofactor are lost in both of the GO-inhibited forms, indicating a loss in hydrogen bonding as compared to the resting state. This supports the hypothesis that CO binds near the 'waist' of the cofactor. Determination of ¹³C hyperfine tensors of bound ¹³CO to lo-CO and hi-CO leads to the suggestion that the single CO bound to the FeMo-cofactor of lo-CO may bridge or semibridge two iron ions, while each of the two CO bound to hi-CO is a terminal ligand. These ENDOR measurements and recent FTIR results of Thorneley and co-workers [George, S. J.; Ashby, G. A.; Wharton, C. W.; Thorneley, R. N. F. J. Am. Chem. Sec. 1997, 119, 6450-6451] provide strong mutual support.