An Organometallic Intermediate during Alkyne Reduction by Nitrogenase

Abstract

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-70^Val 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-70^Ala 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 (H_a) and one weakly coupled (H_b); H_a^c originates as the C3 proton of PA, while H_a^s and H_b are solvent-derived. The two H_a protons have identical hyperfine tensors, despite having different origins. The equality of the (H_a^s, H_a^c) 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 H_b.