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

H. I. Lee, L. M. Cameron, B. J. Hales*, B. M. Hoffman

*Corresponding author for this work

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104 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)10121-10126
Number of pages6
JournalJournal of the American Chemical Society
Volume119
Issue number42
DOIs
StatePublished - Jan 1 1997

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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