An organometallic intermediate during alkyne reduction by nitrogenase

Hong In Lee*, Robert Y. Igarashi, Mikhail Laryukhin, Peter E. Doan, Patricia C. Dos Santos, Dennis R. Dean, Lance C. Seefeld, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

112 Scopus citations


Nitrogenase is the metalloenzyme that catalyzes the nucleotide-dependent reduction of N2, as well as reduction of a variety of other triply bonded substrates, including the alkyne, acetylene. Substitution of the α-70Val residue in the nitrogenase MoFe protein by alanine expands the range of substrates to include short-chain alkynes not reduced by the unaltered protein. Rapid freezing of the α-70Ala nitrogenase MoFe protein during reduction of the alkyne propargyl alcohol (HC≡C-CH2OH; PA) traps an S = 1/2 intermediate state of the active-site metal cluster, the FeMo-cofactor. We have combined CW and pulsed 13C ENDOR (electron-nuclear double resonance) with two quantitative 35 GHz 1,2H ENDOR techniques, Mims pulsed ENDOR and the newly devised "stochastic field-modulated" ENDOR, to study this intermediate prepared with isotopically substituted (13C, 1,2H) propargyl alcohol in H2O and D2O buffers. These measurements allow the first description of a trapped nitrogenase reduction intermediate. The S = 1/2 turnover intermediate generated during the reduction of PA contains the 3-carbon chain of PA and exhibits resolved 1,2H ENDOR signals from three protons, two strongly coupled (Ha) and one weakly coupled (Hb); Hac originates as the C3 proton of PA, while Has and Hb are solvent-derived. The two Ha protons have identical hyperfine tensors, despite having different origins. The equality of the (Ha s, Hac) hyperfine tensors strongly constrains proposals for the structure of the cluster-bound reduced PA. Through consideration of model structures found in the Cambridge Structural Database, we propose that the intermediate contains a novel bio-organometallic complex in which a reduction product of propargyl alcohol binds as a metalla-cyclopropane ring to a single Fe atom of the Fe-S face of the FeMo-cofactor that is composed of Fe atoms 2, 3, 6, and 7. Of the two most attractive structures, one singly reduced at C3 (4), the other being the doubly reduced allyl alcohol product (6), we tentatively favor 6 because of the "natural" assignment it affords for Hb.

Original languageEnglish (US)
Pages (from-to)9563-9569
Number of pages7
JournalJournal of the American Chemical Society
Issue number31
StatePublished - Aug 11 2004

ASJC Scopus subject areas

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


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