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 (Fe₈S_7-8) and the FeMo-cofactor (MoFe₇S₉: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 ⁵⁷Fe ENDOR to study the CO-inhibited turnover states of a novel suite of ^56,57Fe 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 ⁵⁷Fe measurements, along with an earlier ¹³C ENDOR study of bound ¹³CO (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.