The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM show it is not an exchangeable nitrogen

Hong In Lee*, Paul M.C. Benton, Mikhail Laryukhin, Robert Y. Igarashi, Dennis R. Dean, Lance C. Seefeldt, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

99 Scopus citations


A recent high-resolution X-ray crystallographic study (1.16 Å) of the Azotobacter vinelandii nitrogenase MoFe protein revealed a previously undetected electron density associated with the active site FeMo-cofactor. The density is located inside the cluster at the center of the "trigonal prism" of six irons and is assigned to a species "X". The identity of species X was not resolved, although the electron density is consistent with a single N, O, or C atom. One proposal is that X is an N atom that derives from and exchanges with N from N2 during catalysis. In the present study, we have examined this possibility by employing 14N and 15N isotopes of N2 along with ENDOR and ESEEM spectroscopies. The WT MoFe protein and α-359Arg→Lys and α-381Phe→Leu variants were allowed to turn over in the presence of 14N2 or 15N2, and then were examined as resting enzymes by ENDOR and ESEEM at X- and Q-bands to look for all 14N and 15N signals coupled to the electron spin of the FeMo-cofactor and to determine if any exchanged during turnover. We have found five peaks in Q-band pulsed ENDOR spectra that appear to arise not only from previously reported N1/N2, which give rise to the ESEEM, but also from one or two additional coupled nitrogens. None of the ENDOR and ESEEM signals vanish or are altered by catalytic turnover with 15N2, and no new 15N signal is detected, leading to the conclusion that if species X is a nitrogen atom, it does not exchange during dinitrogen reduction.

Original languageEnglish (US)
Pages (from-to)5604-5605
Number of pages2
JournalJournal of the American Chemical Society
Issue number19
StatePublished - May 14 2003

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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