Hydride Conformers of the Nitrogenase FeMo-cofactor Two-Electron Reduced State E₂(2H), Assigned Using Cryogenic Intra Electron Paramagnetic Resonance Cavity Photolysis

Abstract

Early studies in which nitrogenase was freeze-trapped during enzymatic turnover revealed the presence of high-spin (S = 3/2) electron paramagnetic resonance (EPR) signals from the active-site FeMo-cofactor (FeMo-co) in electron-reduced intermediates of the MoFe protein. Historically denoted as 1b and 1c, each of the signals is describable as a fictitious spin system, S′ = 1/2, with anisotropic g′ tensor, 1b with g′ = [4.21, 3.76, ?] and 1c with g′ = [4.69, ∼3.20, ?]. A clear discrepancy between the magnetic properties of 1b and 1c and the kinetic analysis of their appearance during pre-steady-state turnover left their identities in doubt, however. We subsequently associated 1b with the state having accumulated 2[e–/H+], denoted as E2(2H), and suggested that the reducing equivalents are stored on the catalytic FeMo-co cluster as an iron hydride, likely an [Fe–H–Fe] hydride bridge. Intra-EPR cavity photolysis (450 nm; temperature-independent from 4 to 12 K) of the E2(2H)/1b state now corroborates the identification of this state as storing two reducing equivalents as a hydride. Photolysis converts E2(2H)/1b to a state with the same EPR spectrum, and thus the same cofactor structure as pre-steady-state turnover 1c, but with a different active-site environment. Upon annealing of the photogenerated state at temperature T = 145 K, it relaxes back to E2(2H)/1b. This implies that the 1c signal comes from an E2(2H) hydride isomer of E2(2H)/1b that stores its two reducing equivalents either as a hydride bridge between a different pair of iron atoms or an Fe–H terminal hydride.

Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N 2 reduction

“…Spectroscopic investigations have provided the spin state of various intermediates in the Lowe-Thorneley kinetic model, notably for this report, E 0 , E 2 , and both E 4 (4H) and E 4 (2N2H). As recently established (47), not only E 0 but also the E 2 intermediates have a total spin S = 3/2, while the two E 4 intermediates have S = 1/2 (6). Therefore, calculations were performed for the observed S = 3/2 spin state (E 0 and E 2 ) and the two S = 1/2 (E 4 ) states within the BS approach (16).…”

An S = 1/2 Iron Complex Featuring N2, Thiolate, and Hydride Ligands: Reductive Elimination of H2 and Relevant Thermochemical Fe–H Parameters

“…Two 1 H nuclei (H 1 : a iso = 24.3 MHz, T = [−13.3, 0.7, 12.7] MHz; H 2 : a iso = 22.3 MHz, T = [10.7, −12.3,1.7] MHz) are assigned as Fe−( μ -H)−Fe moieties. 9a The 1 H signals are believed to arise from bridging, as opposed to terminal hydrides, due to the approximate rhombic symmetry of the dipolar tensor; a point−dipole model predicts a dipolar tensor of approximate axial symmetry for a terminally bound hydride. 49…”

The E2 state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H2 Evolution

“…[76] It seems not inconceivable that these two distinct EPR signals, 1b and 1c, could correspond to the E 2 -hyd and E 2 -nonhyd models suggested in this work. The study by Lukoyanov et al [26] demonstrated that the state with the 1b EPR signal likely to a hydride state at the E 2 redox level based on the known photoreactivity of metal hydrides. It was furthermore shown by photolysis of 1b that a photogenerated version of 1c (1c*) can form that was suggested to be a different hydride isomer.…”