High-Resolution ENDOR Spectroscopy Combined with Quantum Chemical Calculations Reveals the Structure of Nitrogenase Janus Intermediate E(4H).

We have shown that the key state in N reduction to two NH molecules by the enzyme nitrogenase is E(4H), the "Janus" intermediate, which has accumulated four [e/H] and is poised to undergo reductive elimination of H coupled to N binding and activation. Initial H and Mo ENDOR studies of freeze-trapped E(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(4H), 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(4H), with one bridging and one terminal hydride, and E(4H), 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(4H), 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-ENDOR experiments using improved instrumentation, Mims H 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(4H).