The One-Electron Reduced Active-Site FeFe-Cofactor of Fe-Nitrogenase Contains a Hydride Bound to a Formally Oxidized Metal-Ion Core.

The nitrogenase active-site cofactor must accumulate 4e/4H (E(4H) state) before N can bind and be reduced. Earlier studies demonstrated that this E(4H) state stores the reducing-equivalents as two hydrides, with the cofactor metal-ion core formally at its resting-state redox level. This led to the understanding that N binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H. The state having acquired 2e/2H (E(2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e/H (E(H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E(H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E(H) state of Fe-nitrogenase shows an = 1/2 EPR spectrum with = [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic , 450 nm photolysis converts to a new and likewise photoactive = 1/2 state (denoted E(H)) with = [2.009, 1.950, 1.860], which results in a photostationary state, with E(H) relaxing to E(H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E(H)/E(H)* photointerconversion. These observations indicate that the addition of the first e/H to the FeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core is one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.