A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N2 to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H 2N-NH2) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N2 and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of α-70Val that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when α-195His, a putative proton donor near the active site, is substituted by glutamine in combination with substitution of α-70 Val by alanine, and the resulting doubly substituted MoFe protein (α-70Ala/α-195Gln) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = 1/2 state in high yield (∼70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using 15N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g1 this species gives a single observed 15N signal, A(g1) = 1.5 MHz.
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