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

Abstract

The nitrogenase active-site cofactor must accumulate 4e − / 4H + (E 4 (4H) state) before N 2 can bind and be reduced. Earlier studies demonstrated that this E 4 (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 2 binding is mechanistically coupled to reductive-elimination of the two hydrides that produce H 2 . The state having acquired 2e − /2H + (E 2 (2H)) correspondingly contains one hydride with a resting-state core redox level. How the cofactor accommodates addition of the first e − /H + (E 1 (H) state) is unknown. The Fe-nitrogenase FeFe-cofactor was used to address this question because it is EPR-active in the E 1 (H) state, unlike the FeMocofactor of Mo-nitrogenase, thus allowing characterization by EPR spectroscopy. The freeze-trapped E 1 (H) state of Fe-nitrogenase shows an S = 1/2 EPR spectrum with g = [1. 965, 1.928, 1.779]. This state is photoactive, and under 12 K cryogenic intracavity, 450 nm photolysis converts to a new and likewise photoactive S = 1/2 state (denoted E 1 (H)*) with g = [2.009, 1.950, 1.860], which results in a photostationary state, with E 1 (H)* relaxing to E 1 (H) at temperatures above 145 K. An H/D kinetic isotope effect of 2.4 accompanies the 12 K E 1 (H)/E 1 (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 formally one-electron oxidized relative to the resting state. It is proposed that this behavior applies to all three nitrogenase isozymes.

QM/MM Study of Partial Dissociation of S2B for the E2 Intermediate of Nitrogenase

“…A way to sort out these problems is to study simpler states with a lower number of possibilities. Most computational , and experimental , studies agree that the E 1 state involves the protonation of the S2B μ 2 -bridging sulfide ligand of the FeMo cluster (but a recent experimental study of Fe-nitrogenase instead suggested a hydride ion).…”

Can the E1state in nitrogenase tell if there is an activation process prior to catalysis?

“…The results are in line with a recent X-ray study, 9 but in strong disagreement with a recent EPR study. 10 The accuracy of the present calculations has been benchmarked on a set of redox enzymes indicating an accuracy of about 3 kcal mol À1 , also for the redox transitions, for all of them. 12 Also, the results for the N 2 activation in E 4 is in perfect agreement with observations by EPR.…”

“…Recent experiments have shown that the ground state for the cofactor of V-nitrogenase has the redox state (3Fe(II), 4Fe(III), V(III)), which is an S = 0 (or integer spin non-Kramers) state. 18 To repeat, the EPR study led to a suggested structure with a bridging hydride for E 1 , 10 while the X-ray study led to a suggested protonated belt sulfide. 9 Therefore, the suggested oxidation states were quite different with (2Fe(II), 5Fe(III), Mo(III)) for the EPR study and (4Fe(II), 3Fe(III), Mo(III)) for the X-ray study.…”

“…16 The cluster models for Mo-nitrogenase were taken from the previous studies. 6,10 For Mo-nitrogenase, besides the ligands of the cofactor, homocitrate, Cys275 and His442, the second sphere amino acids His195, Arg96, Arg359, Glu380, Phe381 and Gln191, were included in the model. For V-nitrogenase, besides the ligands of the cofactor, homocitrate, Cys257, His423, and a carbonate, the second sphere amino acids Gln176, Lys83, His180, Thr335, Arg339, Lys361 and Phe362 were included in the model.…”

Computational modeling of redox enzymes

“…The experiments concerned studies of a state obtained after only one reduction generally termed the E 1 state. In one study using X‐ray spectroscopy, it was concluded that there is a reduction of one of the irons and no hydride is present [34], while in an EPR study it was concluded that there should be a hydride present, implying that one of the irons is instead oxidized [35]. At present, model calculations suggest a hydride in the E 1 state, but no hydride in the A 1 state.…”