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

The nitrogenase active-site cofactor must accumulate 4e-/4H+(E4(4H) state) before N2can bind and be reduced. Earlier studiesdemonstrated that this E4(4H) state stores the reducing-equivalents as twohydrides, with the cofactor metal-ion core formally at its resting-stateredox level. This led to the understanding that N2binding ismechanistically coupled to reductive-elimination of the two hydridesthat produce H2. The state having acquired 2e-/2H+(E2(2H))correspondingly contains one hydride with a resting-state core redoxlevel. How the cofactor accommodates addition of thefirst e-/H+(E1(H)state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to addressthis question because it is EPR-active in the E1(H) state, unlike the FeMo-cofactor of Mo-nitrogenase, thus allowing characterization by EPRspectroscopy. The freeze-trapped E1(H) state of Fe-nitrogenase showsanS= 1/2 EPR spectrum withg= [1.965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenicintracavity, 450 nmphotolysis converts to a new and likewise photoactiveS= 1/2 state (denoted E1(H)*) withg= [2.009, 1.950, 1.860], which resultsin a photostationary state, with E1(H)*relaxing to E1(H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4accompanies the 12 K E1(H)/E1(H)*photointerconversion. These observations indicate that the addition of thefirst e-/H+to theFeFe-cofactor of Fe-nitrogenase produces an Fe-bound hydride, not a sulfur-bound proton. As a result, the cluster metal-ion core isformallyone-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes