Identification of the CO-Binding Cluster in Nitrogenase MoFe Protein by ENDOR of ⁵⁷Fe Isotopomers

The X-ray structure of the nitrogenase MoFe protein has established the organization and architecture of its multimetallic cofactors, the P-cluster (Fe 8 S 7 - 8 ) and the FeMo-cofactor (MoFe 7 S 9:homocitrate). Nonetheless, until recently it has not been possible to detect or characterize a substrate or inhibitor interacting with the functioning enzyme. In the present study we have used 57 Fe ENDOR to study the CO-inhibited turnover states of a novel suite of 56,57 Fe isotopomers of the MoFe protein, including those in which these two clusters are selectively, as well as uniformly, labeled. CO-inhibited MoFe protein exhibits two distinct EPR signals, one under low and another under high CO pressure. The 57 Fe measurements, along with an earlier 13 C ENDOR study of bound 13 CO (Pollock, 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), show that the two EPR signals arise from CO-bound FeMo-cofactor, in one case with one bound CO and in the other with two bound CO, and they further provide initial insights into the properties of the inhibitor-bound cluster.