Abstract
We have shown that the key state in N-2 reduction to two NH3 molecules by the enzyme nitrogenase is E-4(4H), the "Janus" intermediate, which has accumulated four [e(-)/H+] and is poised to undergo reductive elimination of H-2 coupled to N-2 binding and activation. Initial H-1 and Mo-95 ENDOR studies of freeze-trapped E-4(4H) revealed that the catalytic multimetallic cluster (FeMo-co) binds two Fe-bridging hydrides, [Fe-H-Fe]. However, the analysis failed to provide a satisfactory picture of the relative spatial relationships of the two [Fe-H-Fe]. Our recent density functional theory (DFT) study yielded a lowest-energy form, denoted as E-4(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 E-4(4H)((b)), with one bridging and one terminal hydride, and E-4(4H)((c)), with one pair of anchor Fe supporting two bridging hydrides, were not beyond the uncertainties in the calculation. Moreover, a structure of V-dependent nitrogenase resulted in a proposed structure analogous to E-4(4H)((c)), and additional structures have been proposed in the DFT studies of others. To resolve the nature of hydride binding to the Janus intermediate, we performed exhaustive, high-resolution CW-stochastic H-1-ENDOR experiments using improved instrumentation, Mims H-2 ENDOR, and a recently developed 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 is the freeze-trapped, lowest energy Janus intermediate structure, E-4(4H)((a)).