An organometallic intermediate during alkyne reduction by nitrogenase.

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 turno...