High resolution ENDOR spectroscopy combined with quantum chemical calculations reveals the structure of the nitrogenase Janus intermediate E4(4H)

We have shown that the key state in N2 reduction to two NH3 molecules by the enzyme nitrogenase is E4(4H), the “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 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 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 in the calculation. Moreover, a structure of V-dependent nitrogenase...