QM/MM Study of the Nitrogenase MoFe Protein Resting State: Broken-Symmetry States, Protonation States, and QM Region Convergence in the FeMoco Active Site

Nitrogenase is one of the most fascinating enzymes in nature, being responsible for all biological nitrogen reduction. Despite decades of research, it is among the enzymes in bioinorganic chemistry whose mechanism is the most poorly understood. The MoFe protein of nitrogenase contains an iron–molybdenum–sulfur cluster, FeMoco, where N2 reduction takes place. The resting state of FeMoco has been characterized by crystallography, multiple spectroscopic techniques, and theory (broken-symmetry density functional theory), and all heavy atoms are now characterized. The cofactor charge, however, has been controversial, the electronic structure has proved enigmatic, and little is known about the mechanism. While many computational studies have been performed on FeMoco, few have taken the protein environment properly into account. In this study, we put forward QM/MM models of the MoFe protein from Azotobacter vinelandii, centered on FeMoco. By a detailed analysis of the FeMoco geometry and comparison to the atomic resolution crystal structure, we conclude that only the [MoFe7S9C]1– charge is a possible resting state charge. Further, we find that of the three lowest energy broken-symmetry solutions of FeMoco, the BS7-235 spin isomer (where 235 refers to Fe atoms that are “spin-down”) is the only one that can be reconciled with experiment. This is revealed by a comparison of the metal–metal distances in the experimental crystal structure, a rare case of spin-coupling phenomena being visible through the molecular structure. This could be interpreted as the enzyme deliberately stabilizing a specific electronic state of the cofactor, possibly for tuning specific reactivity on specific metal atoms. Finally, we show that the alkoxide group on the Mo-bound homocitrate must be protonated under resting state conditions, the presence of which has implications regarding the nature of FeMoco redox states as well as for potential substrate reduction mechanisms.We thank Albert Th. Thórhallsson for useful discussions. R.B. acknowledges support from the Icelandic Research Fund, grant nos. 141218051 and 141218052, and the University of Iceland Research Fund. R.B. thanks Hannes Jónsson and Egill Skúlason for support.Peer Reviewe

Proton Shuttling and Reaction Paths in Dissociative Electron Attachment to o- and p-Tetrafluorohydroquinone, an Experimental and Theoretical Study

Here we present a combined experimental and theoretical study on the fragmentation of o- and p-tetrafluorohydroquinone upon low energy electron attachment. Despite an identical ring-skeleton and identical functional groups in these constitutional isomers, they show distinctly different fragmentation patterns, a phenomenon that cannot be explained by distinct resonances or different thermochemistry. Using high-level quantum chemical calculations with the computationally affordable domain localized pair natural orbital approach, DLPNO–CCSD(T), we are able to provide a complete and accurate description of the respective reaction dynamics, revealing proton shuttling and transition states for competing channels as the explanation for the different behavior of these isomers. The results represent a “schoolbook example” of how the combination of experiment and modern high-level theory may today provide a thorough understanding of complex reaction dynamics by computationally affordable means.(This work was supported by the Icelandic Center of Research (RANNIS) Grant No. 13049305 and 141218051 and the University of Iceland Research Fund.Peer Reviewe

Revisiting the Mössbauer Isomer Shifts of the FeMoco Cluster of Nitrogenase and the Cofactor Charge

Despite decades of research, the structure–activity relationship of nitrogenase is still not understood. Only recently was the full molecular structure of the FeMo cofactor (FeMoco) revealed, but the charge and metal oxidation states of FeMoco have been controversial. With the recent identification of the interstitial atom as a carbide and the more recent revised oxidation-state assignment of the molybdenum atom as Mo(III), here we revisit the Mössbauer properties of FeMoco. By a detailed error analysis of density functional theory-computed isomer shifts and computing isomer shifts relative to the P-cluster, we find that only the charge of [MoFe7S9C]1– fits the experimental data. In view of the recent Mo(III) identification, the charge of [MoFe7S9C]1– corresponds to a formal oxidation-state assignment of Mo(III)3Fe(II)4Fe(III), although due to spin delocalization, the physical oxidation state distribution might also be interpreted as Mo(III)1Fe(II)4Fe(2.5)2Fe(III), according to a localized orbital analysis of the MS = 3/2 broken symmetry solution. These results can be reconciled with the recent spatially resolved anomalous dispersion study by Einsle et al. that suggests the Mo(III)3Fe(II)4Fe(III) distribution, if some spin localization (either through interactions with the protein environment or through vibronic coupling) were to take place.We thank E. Bill for valuable discussions and comments on the manuscript. S.D. and F.N. acknowledge the Max Planck Society for funding. This work was supported by the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP/2007-2013) ERC Grant Agreement No. 615414 (S.D.). R.B. acknowledges support from the Icelandic Research Fund, Grant Nos. 141218051 and 162880051 and the Univ. of Iceland Research Fund.Peer Reviewe

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

Publisher's version (útgefin grein)The nitrogenase enzymes are responsible for all biological nitrogen reduction. How this is accomplished at the atomic level, however, has still not been established. The molybdenum-dependent nitrogenase has been extensively studied and is the most active catalyst for dinitrogen reduction of the nitrogenase enzymes. The vanadium-dependent form, on the other hand, displays different reactivity, being capable of CO and CO2 reduction to hydrocarbons. Only recently did a crystal structure of the VFe protein of vanadium nitrogenase become available, paving the way for detailed theoretical studies of the iron-vanadium cofactor (FeVco) within the protein matrix. The crystal structure revealed a bridging 4-atom ligand between two Fe atoms, proposed to be either a CO32- or NO3- ligand. Using a quantum mechanics/molecular mechanics model of the VFe protein, starting from the 1.35 Å crystal structure, we have systematically explored multiple computational models for FeVco, considering either a CO32- or NO3- ligand, three different redox states, and multiple broken-symmetry states. We find that only a [VFe7S8C(CO3)]2- model for FeVco reproduces the crystal structure of FeVco well, as seen in a comparison of the Fe-Fe and V-Fe distances in the computed models. Furthermore, a broken-symmetry solution with Fe2, Fe3, and Fe5 spin-down (BS7-235) is energetically preferred. The electronic structure of the [VFe7S8C(CO3)]2- BS7-235 model is compared to our [MoFe7S9C]- BS7-235 model of FeMoco via localized orbital analysis and is discussed in terms of local oxidation states and different degrees of delocalization. As previously found from Fe X-ray absorption spectroscopy studies, the Fe part of FeVco is reduced compared to FeMoco, and the calculations reveal Fe5 as locally ferrous. This suggests resting-state FeVco to be analogous to an unprotonated E1 state of FeMoco. Furthermore, V-Fe interactions in FeVco are not as strong compared to Mo-Fe interactions in FeMoco. These clear differences in the electronic structures of otherwise similar cofactors suggest an explanation for distinct differences in reactivity.R.B. acknowledges support from the Icelandic Research Fund (Grants 141218051 and 162880051) and University of Iceland Research Fund. Open Access funding was provided by the Max Planck Society.Peer Reviewe

Identification of a spin-coupled Mo(III) in the nitrogenase iron-molybdenum cofactor

International audienceNitrogenase is a complex enzyme that catalyzes the formation of ammonia utilizing a MoFe7S9C cluster. The presence of a central carbon atom was recently revealed, finally completing the atomic level description of the active site. However, important prerequisites for understanding the mechanism - the total charge, metal oxidation states and electronic structure are unknown. Herein we present high-energy resolution fluorescence detected Mo K-edge X-ray absorption spectroscopy of nitrogenase. Comparison to FeMo model complexes of known oxidation state indicates that the Mo in the FeMo cofactor of nitrogenase is best described as Mo(III), in contrast to the universally accepted Mo(IV) assignment. The oxidation state assignment is supported by theoretical calculations, which reveal the presence of an unusual spin-coupled Mo(III) site. Although so far Mo(III) was not reported to occur in biology the suggestion raises interesting parallels with the known homogenous Mo catalysts for N-2 reduction, where a Mo(III) compound is the N-2-binding species. It also requires a reassignment of the Fe oxidation states in the cofacto

Molecular structure of 1,2-bis(trifluoromethyl)-1,1,2,2-tetramethyldisilane in the gas, liquid, and solid phases : Unusual conformational changes between phases

The molecular structure of 1,2-bis(trifluoromethyl)-1,1,2,2-tetramethyldisilane has been determined in three different phases (solid, liquid, and gas) using various spectroscopic and diffraction techniques. Both the solid-state and gas-phase investigations revealed only one conformer to be present in the sample analyzed, whereas the liquid phase revealed the presence of three conformers. The data have been reproduced using computational methods and a rationale is presented for the observation of three conformers in the liquid state

X-ray Absorption Spectroscopy Combined with Time-Dependent Density Functional Theory Elucidates Differential Substitution Pathways of Au(I) and Au(III) with Zinc Fingers

A combination of two elements’ (Au, Zn) X-ray absorption spectroscopy (XAS) and time-dependent density functional theory (TD-DFT) allowed the elucidation of differential substitution pathways of Au(I) and Au(III) compounds reacting with biologically relevant zinc fingers (ZnFs). Gold L3-edge XAS probed the interaction of gold and the C-terminal Cys2HisCys finger of the HIV-1 nucleocapsid protein NCp7, and the Cys2His2 human transcription factor Sp1. The use of model compounds helped assign oxidation states and the identity of the gold-bound ligands. The computational studies accurately reproduced the experimental XAS spectra and allowed the proposition of structural models for the interaction products at early time points. The direct electrophilic attack on the ZnF by the highly thiophilic Au(I) resulted in a linear P–Au–Cys coordination sphere after zinc ejection whereas for the Sp1, loss of PEt3 results in linear Cys–Au–Cys or Cys–Au–His arrangements. Reactions with Au(III) compounds, on the other hand, showed multiple binding modes. Prompt reaction between [AuCl(dien)]2+ and [Au(dien)(DMAP)]3+ with Sp1 showed a partially reduced Au center and a final linear His–Au–His coordination. Differently, in the presence of NCp7, [AuCl(dien)]2+ readily reduces to Au(I) and changes from square-planar to linear geometry with Cys–Au–His coordination, while [Au(dien)(DMAP)]3+ initially maintains its Au(III) oxidation state and square-planar geometry and the same first coordination sphere. The latter is the first observation of a “noncovalent” interaction of a Au(III) complex with a zinc finger and confirms early hypotheses that stabilization of Au(III) occurs with N-donor ligands. Modification of the zinc coordination sphere, suggesting full or partial zinc ejection, is observed in all cases, and for [Au(dien)(DMAP)]3+ this represents a novel mechanism for nucleocapsid inactivation. The combination of XAS and TD-DFT presents the first direct experimental observation that not only compound reactivity, but also ZnF core specificity, can be modulated on the basis of the coordination sphere of Au(III) compounds.This work was supported by National Science Foundation NSF CHE-1413189, and São Paulo Research Foundation (FAPESP) 2013/20334-0 and 2015/9905-1, Brazilian Federal Agency, for the Support and Evaluation of Graduate Education (CAPES), CAPES/PVES 154/2012 and 0580/2013. We thank the Brazilian Synchrotron Light Laboratory for the beamtime and the XAFS1 and XAFS2 staff for the support. We especially thank Anna Paula Sotero and Carlos Doro Neto for the technical support at the synchrotron beamlines. F.A.L. acknowledges the National Council for Scientific and Technological Development (CNPq) for the productivity grant 311270/2015-8″. C.A. acknowledges support from FAPESP 2013/20334-0 and Brazilian Synchrotron Light Laboratory LNLS 1-XAFS1-17707 and LNLS2 research project 2015 0089. R.B. acknowledges support from the Icelandic Research Fund, Grant No. 141218051, and the University of Iceland Research Fund

Conformational properties of 1-cyano-1-silacyclohexane, C5H10SiHCN: Gas electron diffraction, low-temperature NMR and quantum chemical calculations

The conformational preference of the cyano group of the 1-cyano-1-silacyclohexane was studied experimentally by means of gas electron diffraction (GED) and dynamic nuclear magnetic resonance (DNMR) as well as by quantum chemical (QC) calculations applying high-level coupled cluster methods as well as DFT methods. According to the GED experiment, the compound exists in the gas-phase as a mixture of two conformers possessing the chair conformation of the six-membered ring and Cs symmetry while differing in the axial or equatorial position of the substituent (axial = 84(12) mol %/equatorial = 16(12) mol %) at T = 279(3) K, corresponding to an A value (Gax – Geq) of −1.0(4) kcal mol−1. Gas-phase CCSD(T) calculations predict an A value of −0.72 kcal mol−1 at 279 K. In contrast, the low-temperature 13C NMR experiments resulted in an axial/equatorial ratio of 35/65 mol % at 120 K corresponding to an A value of 0.14 kcal mol−1. An average value for ΔG#e→a = 5.6 ± 0.1 kcal mol−1 was obtained for the temperature range 110–145 K. The dramatically different conformational behaviour in the gas-phase (GED) compared to the liquid phase (DNMR) suggests a strong solvation effect. According to natural bond orbital analysis the axial conformer of the title compound is an example of stabilization of a form, which is not favored by electrostatic effects and is favored predominantly by steric and conjugation effects.A.V.B. and Yu.F.S. are grateful to the Ministry of Education and Science of Russia (State Contracts N 14.B25.31.0013) for financial support. S.A.Sh. thanks the Russian Foundation for Basic Research (Grant 14-03-0023-a). I.A., S.O.W., and N.R.J. thank the Icelandic Centre for Research (RANNIS) for financial support, Grants No 080038021 and 100040022. R.B. acknowledges support from the Icelandic Research Fund, grant no. 141218051

Electron interactions with the heteronuclear carbonyl precursor H2FeRu3(CO)13 and comparison with HFeCo3(CO)12: from fundamental gas phase and surface science studies to focused electron beam induced deposition

In the current contribution we present a comprehensive study on the heteronuclear carbonyl complex H2FeRu3(CO)13 covering its low energy electron induced fragmentation in the gas phase through dissociative electron attachment (DEA) and dissociative ionization (DI), its decomposition when adsorbed on a surface under controlled ultrahigh vacuum (UHV) conditions and exposed to irradiation with 500 eV electrons, and its performance in focused electron beam induced deposition (FEBID) at room temperature under HV conditions. The performance of this precursor in FEBID is poor, resulting in maximum metal content of 26 atom % under optimized conditions. Furthermore, the Ru/Fe ratio in the FEBID deposit (≈3.5) is higher than the 3:1 ratio predicted. This is somewhat surprising as in recent FEBID studies on a structurally similar bimetallic precursor, HFeCo3(CO)12, metal contents of about 80 atom % is achievable on a routine basis and the deposits are found to maintain the initial Co/Fe ratio. Low temperature (≈213 K) surface science studies on thin films of H2FeRu3(CO)13 demonstrate that electron stimulated decomposition leads to significant CO desorption (average of 8–9 CO groups per molecule) to form partially decarbonylated intermediates. However, once formed these intermediates are largely unaffected by either further electron irradiation or annealing to room temperature, with a predicted metal content similar to what is observed in FEBID. Furthermore, gas phase experiments indicate formation of Fe(CO)4 from H2FeRu3(CO)13 upon low energy electron interaction. This fragment could desorb at room temperature under high vacuum conditions, which may explain the slight increase in the Ru/Fe ratio of deposits in FEBID. With the combination of gas phase experiments, surface science studies and actual FEBID experiments, we can offer new insights into the low energy electron induced decomposition of this precursor and how this is reflected in the relatively poor performance of H2FeRu3(CO)13 as compared to the structurally similar HFeCo3(CO)12.The authors acknowledge the fruitful and productive environment provided by the COST Action CELINA CM1301 and we would like to take the opportunity to extend our thanks to Prof. Petra Swiderek for running this Action exceptionally well. Marc Hanefeld and Michael Huth acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) through Priority Program SPP 1928, project HU 752/12-1. DHF thanks the National Science Foundation for support of this work through the linked collaborative grants CHE-1607621 and CHE-1607547. OI acknowledges supported from the Icelandic Center of Research (RANNIS) Grant No. 13049305(1-3) and the University of Iceland Research Fund. RKTP acknowledges a doctoral grant from the University of Iceland Research Fund and financial support from the COST Action CM1301; CELINA, for short term scientific missions (STSMs).Peer Reviewe

Comparative electronic structures of nitrogenase FeMoco and FeVco

An investigation of the active site cofactors of the molybdenum and vanadium nitrogenases (FeMoco and FeVco) was performed using high-resolution X-ray spectroscopy. Synthetic heterometallic iron–sulfur cluster models and density functional theory calculations complement the study of the MoFe and VFe holoproteins using both non-resonant and resonant X-ray emission spectroscopy. Spectroscopic data show the presence of direct iron–heterometal bonds, which are found to be weaker in FeVco. Furthermore, the interstitial carbide is found to perturb the electronic structures of the cofactors through highly covalent Fe–C bonding. The implications of these conclusions are discussed in light of the differential reactivity of the molybdenum and vanadium nitrogenases towards various substrates. Possible functional roles for both the heterometal and the interstitial carbide are detailed.This work was supported by the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP/2007–2013) ERC Grant Agreement number 615414 (S. D.) and the ERC N-ABLE project (O. E.). Funding was also provided by the Deutsche Forschungsgemeinschaft grants EI-520/7 and RTG1976 (O. E.), the NIH (R01-GM45881 to J. A. K.), and by the Max-Planck–Gesellschaft (S. D., R. B., J. K. K., and F. A. L.). J. A. R. was funded by a graduate study scholarship from the German Academic Exchange Service (DAAD). R. B. acknowledges support from the Icelandic Research Fund, Grant No. 141218051 and the University of Iceland Research Fund. Matthias Gschell and Florian Schneider are thanked for preparing the extracted FeMoco, and Tabea Hamann is thanked for providing samples of the molybdenum cubane. Stefan Hugenbruch, Benjamin Van Kuiken, Rebeca Gómez Castillo, and Anselm Hahn are thanked for assistance with data collection. The ESRF and CHESS are also acknowledged for providing beamtime, and Sara Lafuerza and Pieter Glatzel at beamline ID-26 (ESRF) and Kenneth D. Finkelstein at beamline C-1 (CHESS) are gratefully acknowledged for technical assistance with measurements. CHESS is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under NSF award DMR-133220. Open Access funding provided by the Max Planck Society.Peer Reviewe