A model for dinitrogen binding in the E₄state of nitrogenase

Abstract

Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic ironmolybdenum-sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N 2 -binding E 4 state is not known. In this study we present theoretical QM/ MM calculations of new structural models of the E 4 state of molybdenum-dependent nitrogenase and compare to previously suggested models for this enigmatic redox state. We propose two models as possible candidates for the E 4 state. Both models feature two hydrides on the FeMo cofactor, bridging atoms Fe 2 and Fe 6 with a terminal sulfhydryl group on either Fe 2 or Fe 6 (derived from the S2B bridge) and the change in coordination results in local lower-spin electronic structure at Fe 2 and Fe 6 . These structures appear consistent with the bridging hydride proposal put forward from ENDOR studies and are calculated to be lower in energy than other proposed models for E 4 at the TPSSh-QM/MM level of theory. We critically analyze the DFT method dependency in calculations of FeMoco that has resulted in strikingly different proposals for this state. Importantly, dinitrogen binds exothermically to either Fe 2 or Fe 6 in our models, contrary to others, an effect rationalized via the unique ligand field (from the hydrides) at the Fe with an empty coordination site. A low-spin Fe site is proposed as being important to N 2 binding. Furthermore, the geometries of these states suggest a feasible reductive elimination step that could follow, as experiments indicate. Via this step, two electrons are released, reducing the cofactor to yield a distorted 4-coordinate Fe 2 or Fe 6 that partially activates N 2 . We speculate that stabilization of an N 2 -bound Fe(I) at Fe 6 (not found for Fe 2 model) via reductive elimination is a crucial part of N 2 activation in nitrogenases, possibly aided by the apical heterometal ion (Mo or V). By using protons from the sulfhydryl group (to regenerate the sulfide bridge between Fe 2 and Fe 6 ) and the nearby homocitrate hydroxy group, we calculate a plausible route to yield a diazene intermediate. This is found to be more favorable with the Fe 6 -bound model than the Fe 2 -bound model; however, this protonation is uphill in energy, suggesting protonation of N 2 might occur later in the catalytic cycle or via another mechanism. Electronic supplementary information (ESI) available: Further information on all broken-symmetry solutions calculated for all models. Details of the QM/MM model preparation. Spin populations of all models. Localized orbital analysis of selected models. Geometry analysis of E 0 state calculated with different functionals and electronic structure analysis. Cartesian coordinates for the QM regions of all optimized structures available as XYZ les. See

Quantum Mechanics/Molecular Mechanics Study of Resting-State Vanadium Nitrogenase: Molecular and Electronic Structure of the Iron–Vanadium Cofactor

“…This problem of high HF exchange functionals deteriorating the electronic structure of nitrogenase cofactors has been noted previously by us and others for FeMoco. 51 , 98 …”

“…Our protocol accounts for the protein environment via a systematically improvable QM/MM model and describes the electronic structure via broken-symmetry DFT (BS-DFT) calculations using the TPSSh exchange-correlation functional, which we have found to describe the complex electronic structure of the cofactor better than other functionals. 51 Furthermore, we have shown that the calculated structures are highly sensitive to the redox state of the cofactor and that the charge state of FeMoco could be unambiguously determined by the structural comparison. The analysis furthermore indicated a specific electronic state (BS determinant) to be in better agreement with the experimental structure than the other low-lying states.…”

The active E4 structure of nitrogenase studied with different DFT functionals

“…In one of these, Ryde et al 2 got results suggesting that the TPSS functional 3 does reproduce the experimental findings for E4, the key species in N 2 activation by nitrogenase. In the other one, Björnsson et al 4 showed that the TPSSh functional 5 might also work. The suggested E4 structures were obtained after only four reductions.…”

“…This result suggests that all DFT functionals should have a similar problem for the suggested E4 structure. In the two recent theoretical studies by Ryde et al 2 and Björnsson et al 4 of the E4 state, the energetics of that step was not investigated.…”

Assignment of protonated R-homocitrate in extracted FeMo-cofactor of nitrogenase via vibrational circular dichroism spectroscopy

“…But ≡C-O(H) coordination with molybdenum of chelated R-homocitrate is still suspicious in view of protons are almost always invisible in limited resolutions of crystal structures, and severe lack of experimental evidence in addition to computational researches 25,26 . The protonation step confers a certain degree of lability to the homocitrate ligation to the cofactor 18,[27][28][29] , and thus allows structural flexibility that is important for the mechanism of N 2 reduction. Recent capture of N 2 species in FeMo-protein confirmed proton translocation and facilitate a serial of hydrogenation like α-hydroxy group 30 .…”