PNNL

Abstract

We have shown that the key state in N2 reduction to two NH3 by the enzyme nitrogenase is the E4(4H), ‘Janus’ intermediate, which has accumulated four [e-/H+] and is poised to undergo reductive elimination of H2 coupled to N2 binding and activation. Initial 1H and 95Mo ENDOR studies of freeze-trapped E4(4H) revealed the catalytic multi-metallic cluster (FeMo-co) binds two Fe-bridging hydrides, [Fe-H-Fe]. However, the analysis failed to provide a satisfactory picture of structure - the relative spatial relationships of the two [Fe-H-Fe]. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted E4(4H)(a), with two parallel Fe-H-Fe planes bridging pairs of ‘anchor’ Fe on the Fe2,3,6,7 face of FeMo-co. However, the relative energies of structures E4(4H)(b), with one bridging and one terminal hydride, and E4(4H)(c), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties of the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E4(4H)(c), and additional structures have been proposed by DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic 1H-ENDOR experiments using improved instrumentation, Mims 2H ENDOR, and a pulsed-ENDOR protocol (‘PESTRE’) to obtain absolute hyperfine-interaction signs. These measurements are coupled to DFT structural models through an analytical point-dipole Hamiltonian for the hydride electron-nuclear dipolar coupling to its ‘anchoring’ Fe ions, an approach that overcomes limitations inherent in both experimental interpretation and computational accuracy. The result: the freeze-trapped, lowest-energy Janus intermediate structure is E4(4H)(a).