The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM evidence that it is not a nitrogen

X-ray crystallographic study of the nitrogenase MoFe protein revealed electron density from an atom (denoted X) inside the active-site metal cluster, the [MoFe₇S₉:homocitrate] FeMo-cofactor. The electron density associated with X is consistent with a single N, O, or C atom. We now have tested whether X is an N or not by comparing the Q-band ENDOR and ESEEM signals from resting-state (S = 3/2) MoFe protein and NMF-extracted FeMo-co from bacteria grown with either ¹⁴N or ¹⁵N as the exclusive N source. All of the ¹⁴N or ¹⁵N signals associated with the protein are lost upon extraction of the FeMo-co. We interpret this as strong evidence that X is not an N.