FeMoco Research Articles

Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN

The maturation and installation of the active site metal cluster (FeMo-co, Fe7S9CMo-R-homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe8S9C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. However, some putative diazotrophic bacteria, like Roseiflexus sp. RS-1, lack the nifEN genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the Roseiflexus NifH, NifB, and apo-NifDK proteins produced in Escherichia coli are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The E. coli expressed NifDKRS contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDKRS). Apo-NifDKRS could be activated for N2 reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDKRS plus E. coli produced NifBRS and NifHRS were sufficient to yield active NifDKRS when incubated with the necessary substrates (homocitrate, molybdate, and S-adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The E. coli produced NifHRS and NifBRS proteins were independently shown to be functional. The reconstituted NifDKRS demonstrated reduction of N2, protons, and acetylene in ratios observed for Azotobacter vinelandii NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifHRS, NifBRS, and apo-NifDKRS.

Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

The Azotobacter vinelandii molybdenum nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires an existing [Fe-S] cluster to form a [Mo-Fe3-S4] group, which acts as a specific molybdenum donor during nitrogenase FeMo-co biosynthesis. Here, we show biochemical evidence supporting the role of NifU as the [Fe-S] cluster donor. Protein-protein interaction studies involving apo-NifQ and as-isolated NifU demonstrated their interaction, which was only effective when NifQ lacked its [Fe-S] cluster. Incubation of apo-NifQ with [Fe4-S4]-loaded NifU increased the iron content of the former, contingent on both proteins being able to interact with one another. As a result of this interaction, a [Fe4-S4] cluster was transferred from NifU to NifQ. In A. vinelandii, NifQ was preferentially metalated by NifU rather than by the [Fe-S] cluster scaffold protein IscU. These results indicate the necessity of co-expressing NifU and NifQ to efficiently provide molybdenum for FeMo-co biosynthesis when engineering nitrogenase in plants.

Tensor Network State Algorithms on AI Accelerators.

We introduce novel algorithmic solutions for hybrid CPU-multiGPU tensor network state algorithms utilizing non-Abelian symmetries building on AI-motivated state-of-the-art hardware and software technologies. The presented numerical simulations on the FeMo cofactor, which plays a crucial role in converting atmospheric nitrogen to ammonia, are far beyond the scope of traditional approaches. Our large-scale SU(2) spin adapted density matrix renormalization group calculations up to bond dimension D = 216 on complete active space (CAS) size of 18 electrons in 18 orbitals [CAS(18, 18)] demonstrate that the current limit of exact solution, i.e. full-CI limit, can be achieved in fraction of time. Furthermore, benchmarks up to CAS(113, 76) demonstrate the utilization of NVIDIA's highly specialized AI accelerators via NVIDIA Tensor Cores, leading to performance around 115 TFLOPS on a single node supplied with eight NVIDIA A100 devices. As a consequence of reaching 71% of the full capacity of the hardware, the cubic scaling of computational time with bond dimension can be reduced to a linear form for a broad range of D values; thus, breaking the current computational limits of small CAS spaces in ab initio quantum chemistry and material science is becoming a reality. In comparison to strict U(1) implementations with matching accuracy, our solution has an estimated effective performance of 300-500 TFLOPS, which emphasizes the mutual need for both algorithmic and technological developments to push current frontiers on classical computation.

Nature's Catalyst: Investigating the Mechanisms behind Nitrogenase's N2rr Activity

Electrochemical nitrogen reduction reaction (eNRR), one of the most studied problems in catalysis, has the potential to replace the century-old Haber-Bosch process for ammonia synthesis. However, a low-cost, highly selective and efficient catalyst for eNRR is yet to be discovered. Nitrogenase, a naturally occurring biocatalyst responsible for nitrogen conversion to ammonia under ambient conditions, is the most efficient catalyst for eNRR. The Iron-Molybdenum cofactor (FeMo-co) in the enzyme is identified as the catalytic part with bridging irons being active sites. Despite decades of work the exact mechanism of its operation and various intermediates along the pathway are still debated. We revisit eNRR on FeMo-co and decipher the key mechanisms at play.Most studies on FeMo-co tend to ignore the magnetic nature of transition metals (TMs) in the cofactor. Proper treatment of these TMs (Fe and Mo) is necessary to pinpoint the intermediates and potential limiting step along the eNRR pathway. We start with an iterative optimization of the Hubbard U parameter to capture the correct magnetic structure of the cofactor. We also explore the effect of local chemical environment including, H-coverage and various ligands attached to the cofactor, on its activity and selectivity. The potential limiting step is found to be sensitive to the magnetic orientation of the TMs in the cofactor and it is thus imperative to provide the U correction to capture the true bottleneck of eNRR. The cofactor also shows an affinity towards a medium H-coverage under eNRR operating conditions. These findings provide a comprehensive and elaborate view of nitrogen reduction on FeMo-co and insights from this study paves the path towards designing heterogeneous counterparts to the catalyst.Acknowledgments:This work was supported by the U.S. Department of Energy, via Grant DE-FOA-0002676 to the SUNCAT Center for Interface Science and Catalysis.

Low-Coordinate Iron Hydride Chemistry at an N,N,C-Heteroscorpionate Platform.

Locally high-spin iron hydrides are proposed to play a critical role as intermediates in iron-molybdenum cofactor (FeMoco)-catalyzed N2 fixation. Inspired by these biological systems, we report herein our initial investigations into low-coordinate iron hydride chemistry supported by our N,N,C-heteroscorpionate ligands. Those ligands with smaller steric profiles are unable to completely suppress the formation of a binuclear [Fe(μ2-H)]2 complex; however, the incorporation of more substantial steric bulk allows for the isolation of a rare example of a terminal, high-spin (S = 2) Fe2+ hydride. Fourier transform infrared spectroscopy suggests an unusually weak Fe-H bond in the latter molecule. Mössbauer spectroscopies, coupled with density functional theory calculations, highlights the substantial influence of the terminal hydride ligand on 57Fe isomer shift.

Nitride‐Bridged Mo‐Fe‐S Double‐Cubanes: Syntheses, Redox Behaviors and Comparison with their W Analogs

The FeMo cofactor (FeMoco) of the Mo‐dependent nitrogenase was identified as a Mo‐Fe‐S cluster incorporating a 2p atom in the core. The edge‐bridged double‐cubane (EBDC, [M2Fe6(µ3‐S)6(µ4‐S)2]z (M = Mo/V; z denotes charge)) clusters were demonstrated to undergo rearrangement reactions to generate the apex‐fused double‐cubane clusters ([M2Fe6(μ2‐S)2(μ3‐S)6(μ6‐S)]z) as mimics of the PN‐clusters. The introduction of 2p atoms into the core of EBDC clusters will furnish the possibility of mimicking the FeMoco structure through rearrangement. In this work, an EBDC Mo‐Fe‐S cluster with core nitrides, namely [(Tp*)2Mo2Fe6(μ4‐N)2S6Cl4]2− (Tp* = tris(3,5‐dimethyl‐1‐pyrazolyl)hydroborate), has been synthesized. Subsequent terminal ligand substitution has produced a series of EBDC clusters with isostructural cores. Further studies of the series of nitride‐containing EBDC clusters are focused on their structures and redox properties, as well as comparisons with their W analogs. The results indicate that the core structures of these clusters are affected by distinct hetero‐metal centers and different terminal ligands to a limited extent, but the redox behavior and redox potentials of these clusters differ significantly. This work provides us with a viable method for regulating the properties of the nitride‐containing EBDC clusters, which may be enlightening for further studies of the rearrangement reactions towards mimicking the FeMoco structure.

Terminal Hydride Complex of High-Spin Mn.

The iron-molybdenum cofactor of nitrogenase (FeMoco) catalyzes fixation of N2 via Fe hydride intermediates. Our understanding of these species has relied heavily on the characterization of well-defined 3d metal hydride complexes, which serve as putative spectroscopic models. Although the Fe ions in FeMoco, a weak-field cluster, are expected to adopt locally high-spin Fe2+/3+ configurations, synthetically accessible hydride complexes featuring d5 or d6 electron counts are almost exclusively low-spin. We report herein the isolation of a terminal hydride complex of four-coordinate, high-spin (d5; S = 5/2) Mn2+. Electron paramagnetic resonance and electron-nuclear double resonance studies reveal an unusually large degree of spin density on the hydrido ligand. In light of the isoelectronic relationship between Mn2+ and Fe3+, our results are expected to inform our understanding of the valence electronic structures of reactive hydride intermediates derived from FeMoco.

Synthesis and crystal structure of the cluster (Et4N)[(Tp*)MoFe3S3(μ3-NSiMe3)(N3)3

The title compound, tetraethylammonium triazidotri-μ3-sulfido-[μ3-(trimethylsilyl)azanediido][tris(3,5-dimethylpyrazol-1-yl)hydroborato]triiron(+2.33)molybdenum(IV), (C8H20N)[Fe3MoS3(C15H22BN6)(C3H9NSi)(N3)3] or (Et4N)[(Tp*)MoFe3S3(μ3-NSiMe3)(N3)3] [Tp* = tris(3,5-dimethylpyrazol-1-yl)hydroborate(1−)], crystallizes as needle-like black crystals in space group P\overline{1}. In this cluster, the Mo site is in a distorted octahedral coordination model, coordinating three N atoms on the Tp* ligand and three μ3-bridging S atoms in the core. The Fe sites are in a distorted tetrahedral coordination model, coordinating two μ3-bridging S atoms, one μ3-bridging N atom from Me3SiN2−, and another N atom on the terminal azide ligand. This type of heterometallic and heteroleptic single cubane cluster represents a typical example within the Mo–Fe–S cluster family, which may be a good reference for understanding the structure and function of the nitrogenase FeMo cofactor. The residual electron density of disordered solvent molecules in the void space could not be reasonably modeled, thus the SQUEEZE [Spek (2015). Acta Cryst. C71, 9–18] function was applied. The solvent contribution is not included in the reported molecular weight and density.

Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra

Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E0. Two of these species were preliminary assigned to hydrogenated E2 states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of 33S and 77Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.

Theoretical insights into Mo cluster modified Fe5C2 catalysts for electrocatalytic nitrogen reduction

The pursuit of green ammonia synthesis via electrocatalytic nitrogen reduction (NRR) faces challenges in developing high-performance, selective electrocatalysts. Inspired by the nitrogenase Fe-Mo cofactor, this study explores the catalytic efficiency of Mo clusters anchored on Fe5C2 for NRR. We investigate structural stability, N2 adsorption, and Gibbs free energy, revealing that Mon clusters on Fe5C2 demonstrate robust NRR selectivity and notable catalytic activity. Each of the three cluster structures demonstrated superior adsorption capacity in the "side-on" configuration. Among the studied cluster configurations, the Mo5-Fe5C2 shows the lowest limiting potential (-0.24 V), and the Mo4-Fe5C2 exhibits heightened selectivity for NRR, minimizing interference from the hydrogen evolution reaction (HER). This enhanced performance is attributed to the unique structure and interactive dynamics of Mon-Fe5C2 and the electron orbital interactions facilitating direct nitrogen charge transfer. Our findings underscore the potential of Mo cluster-modified Fe5C2 as a pioneering catalyst for electrocatalytic nitrogen reduction.

Highly Activated Terminal Carbon Monoxide Ligand in an Iron-Sulfur Cluster Model of FeMco with Intermediate Local Spin State at Fe.

Nitrogenases, the enzymes that convert N2 to NH3, also catalyze the reductive coupling of CO to yield hydrocarbons. CO-coordinated species of nitrogenase clusters have been isolated and used to infer mechanistic information. However, synthetic FeS clusters displaying CO ligands remain rare, which limits benchmarking. Starting from a synthetic cluster that models a cubane portion of the FeMo cofactor (FeMoco), including a bridging carbyne ligand, we report a heterometallic tungsten-iron-sulfur cluster with a single terminal CO coordination in two oxidation states with a high level of CO activation (νCO = 1851 and 1751 cm-1). The local Fe coordination environment (2S, 1C, 1CO) is identical to that in the protein making this system a suitable benchmark. Computational studies find an unusual intermediate spin electronic configuration at the Fe sites promoted by the presence the carbyne ligand. This electronic feature is partly responsible for the high degree of CO activation in the reduced cluster.

Chemical and redox non-innocence in low-valent molybdenum β diketonate complexes: novel pathways for CO2 and CS2 activation.

The investigation of fundamental properties of low-valent molybdenum complexes bearing anionic ligands is crucial for elucidating the molybdenum's role in critical enzymatic systems involved in the transformation of small molecules, including the nitrogenase's iron molybdenum cofactor, FeMoco. The β-diketonate ligands in [Mo(acac)3] (acac = acetylacetonate), one of the earliest low-valent Mo complexes reported, provide a robust anionic platform to stabilize Mo in its +III oxidation state. This complex played a key role in demonstrating the potential of low-valent molybdenum for small molecule activation, serving as the starting material for the preparation of the first reported molybdenum dinitrogen complex. Surprisingly however, given this fact and the widespread use of β-diketonate ligands in coordination chemistry, only a very limited number of low-valent Mo β-diketonate complexes have been reported. To address this gap, we explored the redox behavior of homoleptic molybdenum tris-β-diketonate complexes, employing a tertiary butyl substituted diketonate ligand (dipivaloylmethanate, tBudiket) to isolate and fully characterize the corresponding Mo complexes across three consecutive oxidation states (+IV, +III, +II). We observed marked reactivity of the most reduced congener with heterocumulenes CE2 (E = O, S), yet with very distinct outcomes. Specifically, CO2 stoichiometrically carboxylates one of the β-diketonate ligands, while in the presence of excess CS2, catalytic reductive dimerization to tetrathiooxalate occurs. Through the isolation and characterization of reaction products and intermediates, we demonstrate that the observed reactivity results from the chemical non-innocence of the β-diketonate ligands, which facilitates the formation of a common ligand-bound intermediate, [Mo( tBudiket)2( tBudiket·CE2)]1- (E = O, S). The stability of this proposed intermediate dictates the specific reduction products observed, highlighting the relevance of the chemically non-innocent nature of β-diketonate ligands.

Structural Insights and Mechanistic Understanding of Iron-Molybdenum Cofactor Biosynthesis by NifB in Nitrogenase Assembly Process.

NifB, a radical S-adenosylmethionine (SAM) enzyme, is pivotal in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), commonly referred to as the M-cluster. This cofactor, located within the active site of nitrogenase, is essential for the conversion of dinitrogen (N2) to NH3. Recognized as the most intricate metallocluster in nature, FeMo-co biosynthesis involves multiple proteins and a sequence of steps. Of particular significance, NifB directs the fusion of two [Fe4S4] clusters to assemble the 8Fe core, while also incorporating an interstitial carbide. Although NifB has been extensively studied, its molecular mechanisms remain elusive. In this review, we explore recent structural analyses of NifB and provide a comprehensive overview of the established catalytic mechanisms. We propose prospective directions for future research, emphasizing the relevance to biochemistry, agriculture, and environmental science. The goal of this review is to lay a solid foundation for future endeavors aimed at elucidating the atomic details of FeMo-co biosynthesis.

Valence Delocalization and Metal-Metal Bonding in Carbon-Bridged Mixed-Valence Iron Complexes.

The carbide ligand in the iron-molybdenum cofactor (FeMoco) in nitrogenase bridges iron atoms in different oxidation states, yet it is difficult to discern its ability to mediate magnetic exchange interactions due to the structural complexity of the cofactor. Here, we describe two mixed-valent diiron complexes with C-based ketenylidene bridging ligands, and compare the carbon bridges with the more familiar sulfur bridges. The ground state of the [Fe2 (μ-CCO)2 ]+ complex with two carbon bridges (4) is S= , and it is valence delocalized on the Mössbauer timescale with a small thermal barrier for electron hopping that stems from the low Fe-C force constant. In contrast, one-electron reduction of the [Fe2 (μ-CCO)] complex with one carbon bridge (2) affords a mixed-valence species with a high-spin ground state (S= ), and the Fe-Fe distance contracts by 1 Å. Spectroscopic, magnetic, and computational studies of the latter reveal an Fe-Fe bonding interaction that leads to complete valence delocalization. Analysis of near-IR intervalence charge transfer transitions in 5 indicates a very large double exchange constant (B) in the range of 780-965 cm-1 . These results show that carbon bridges are extremely effective at stabilizing valence delocalized ground states in mixed-valent iron dimers.

The structural principles underlying molybdenum insertase complex assembly.

Within the cell, the trace element molybdenum (Mo) is only biologically active when complexed either within the nitrogenase specific FeMo cofactor or within the molybdenum cofactor (Moco). Moco consists of an organic part, called molybdopterin (MPT) and an inorganic part, i.e. the Mo-center. The enzyme which catalyses the Mo-center formation is the molybdenum insertase (Mo-insertase). Mo-insertases consist of two functional domains called G- and E-domain. The G-domain catalyses the formation of adenylated MPT (MPT-AMP), which is the substrate for the E-domain, that catalyses the actual molybdate insertion reaction. Though the functions of E- and G-domain have been elucidated to great structural and mechanistic detail, their combined function is poorly characterized. In this work, we describe a structural model of the eukaryotic Mo-insertase Cnx1 complex that was generated based on cross-linking mass spectrometry combined with computational modelling. We revealed Cnx1 to form an asymmetric hexameric complex which allows the E- and G-domain active sites to align in a catalytic productive orientation towards each other. This article is protected by copyright. All rights reserved.

How thermal fluctuations influence the function of the FeMo cofactor in nitrogenase enzymes

The catalytic mechanism of N2 fixation by nitrogenase remains unresolved in how the strong N≡N bond is activated and why the reductive elimination of H2 is required. Here, we use density functional theory and physiologically relevant thermal simulations to elucidate the mechanism of the complete nitrogenase catalytic cycle. Over the accumulation of four reducing equivalents, we find that protons and electrons transfer to the FeMo cofactor to weaken and break its bridge Fe–S bond, leading to temporary H2S formation that exposes the Fe sites to weakly bind N2. Remarkably, we find that subsequent H2 formation is responsible for chemical activation to an N=N double bond accompanied by a low barrier for H2 release. We emphasize that finite temperature effects smooth out mechanistic differences between DFT functionals observed at 0 K, thus leading to a consistent understanding as to why H formation is an obligatory step in N2 adsorption and activation.

Statistical analyses of the oxidized P-clusters in MoFe proteins using the bond-valence method: towards their electron transfer in nitrogenases.

26 well selected oxidized P-clusters (P2+) from the crystallographic data deposited in the Protein Data Bank have been analysed statistically by the bond-valence sum method with weighting schemes for MoFe proteins at different resolutions. Interestingly, the oxidation states of P2+ clusters correspond to Fe23+Fe62+ with high electron delocalization, showing the same oxidation states as the resting states of P-clusters (PN) in nitrogenases. The previously uncertain reduction of P2+ to PN clusters by two electrons was assigned as a double protonation of P2+, in which decoordination of the serine residue and the peptide chain of cysteine take place, in MoFe proteins. This is further supported by the obviously shorter α-alkoxy C-O bond (average of 1.398 Å) in P2+ clusters and longer α-hydroxy C-O bond (average of 1.422 Å) in PN clusters, while no change is observed in the electronic structures of Fe8S7 Fe atoms in P-clusters. Spatially, the calculations show that Fe3 and Fe6, the most oxidized and most reduced Fe atoms, have the shortest distances of 9.329 Å from the homocitrate in the FeMo cofactor and 14.947 Å from the [Fe4S4] cluster, respectively, and may well function as important electron-transport sites.

Understanding the Electronic Structure Basis for N2 Binding to FeMoco: A Systematic Quantum Mechanics/Molecular Mechanics Investigation.

The FeMo cofactor (FeMoco) of Mo nitrogenase is responsible for reducing dinitrogen to ammonia, but it requires the addition of 3-4 e-/H+ pairs before N2 even binds. A binding site at the Fe2/Fe3/Fe6/Fe7 face of the cofactor has long been suggested based on mutation studies, with Fe2 or Fe6 nowadays being primarily discussed as possibilities. However, the nature of N2 binding to the cofactor is enigmatic as the metal ions are coordinatively saturated in the resting state with no obvious binding site. Furthermore, the cofactor consists of high-spin Fe(II)/Fe(III) ions (antiferromagnetically coupled but also mixed-valence delocalized), which are not known to bind N2. This suggests that an Fe binding site with a different molecular and electronic structure than the resting state must be responsible for the experimentally known N2 binding in the E4 state of FeMoco. We have systematically studied N2 binding to Fe2 and Fe6 sites of FeMoco at the broken-symmetry QM/MM level as a function of the redox-, spin-, and protonation state of the cofactor. The local and global electronic structure changes to the cofactor taking place during redox events, protonation, Fe-S cleavage, hydride formation, and N2 coordination are systematically analyzed. Localized orbital and quasi-restricted orbital analysis via diamagnetic substitution is used to get insights into the local single Fe ion electronic structure in various states of FeMoco. A few factors emerge as essential to N2 binding in the calculations: spin-pairing of dxz and dyz orbitals of the N2-binding Fe ion, a coordination change at the N2-binding Fe ion aided by a hemilabile protonated sulfur, and finally hydride ligation. The results show that N2 binding to E0, E1, and E2 models is generally unfavorable, likely due to the high-energy cost of stabilizing the necessary spin-paired electronic structure of the N2-binding Fe ion in a ligand environment that clearly favors high-spin states. The results for models of E4, however, suggest a feasible model for why N2 binding occurs experimentally in the E4 state. E4 models with two bridging hydrides between Fe2 and Fe6 show much more favorable N2 binding than other models. When two hydrides coordinate to the same Fe ion, an increased ligand-field splitting due to octahedral coordination at either Fe2 or Fe6 is found. This altered ligand field makes it easier for the Fe ion to acquire a spin-paired electronic structure with doubly occupied dxz and dyz orbitals that backbond to a terminal N2 ligand. Within this model for N2 binding, both Fe2 and Fe6 emerge as possible binding site scenarios. Due to steric effects involving the His195 residue, affecting both the N2 ligand and the terminal SH- group, distinguishing between Fe2 and Fe6 is difficult; furthermore, the binding depends on the protonation state of His195.

Connecting the geometric and electronic structures of the nitrogenase iron-molybdenum cofactor through site-selective 57Fe labelling.

Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the 57Fe-based spectroscopic data-although rich with information-combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.

The E3 state of FeMoco: one hydride, two hydrides or dihydrogen?

Hydrides are present in the reduced states of the iron-molybdenum cofactor (FeMoco) of Mo nitrogenase and are believed to play a key mechanistic role in the dinitrogen reduction reaction catalyzed by the enzyme. Two hydrides are present in the E4 state according to 1H ENDOR and there is likely a single hydride in the E2 redox state. The 2-hydride E4 state has been experimentally observed to bind N2 and it has been speculated that E3 may bind N2 as well. However, the E3 state has not been directly observed and very little is known about its molecular and electronic structure or reactivity. In recent computational studies, we have explored the energy surfaces of the E2 and E4 by QM/MM modelling, and found that the most stable hydride isomers contain bridging or partially bridging hydrides with an open protonated belt sulfide-bridge. In this work we systematically explore the energy surface of the E3 redox state, comparing single hydride and two-hydride isomers with varying coordination and bridging vs. terminal sulfhydryl groups. We also include a model featuring a triply protonated carbide. The results are only mildly dependent on the QM-region size. The three most stable E3 isomers at the r2SCAN level of theory have in common: an open belt sulfide-bridge (terminal sulfhydryl group on Fe6) and either 2 bridging hydrides (between Fe2 and Fe6), 1 bridging-1-terminal hydride (around Fe2 and Fe6) or a dihydrogen ligand bound at the Fe2 site. Analyzing the functional dependency of the results, we find that functionals previously found to predict accurate structures of spin-coupled Fe/Mo dimers and FeMoco (TPSSh, B97-D3, r2SCAN, and B3LYP*) are in generally good agreement about the stability of these 3 E3 isomers. However, B3LYP*, similar to its parent B3LYP method, predicts a triply protonated carbide isomer as the most stable isomer, an unlikely scenario in view of the lack of experimental evidence for carbide protonation occurring in reduced FeMoco states. Distinguishing further between the 3 hydride isomers is difficult and this flexible coordination nature of hydrides suggests that multiple hydride isomers could be present during experimental conditions. N2 binding was explored and resulted in geometries with 2 bridging hydrides and N2 bound to either Fe2 or Fe6 with a local low-spin state on the Fe. N2 binding is predicted to be mildly endothermic, similar to the E2 state, and it seems unlikely that the E3 state is capable of binding N2.