Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase

Dance, Ian G.

doi:10.1002/cbic.201900636

Cited by 44 publications

(109 citation statements)

References 229 publications

(429 reference statements)

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“…It is also possible that proton tunneling, which is generally thought to have a very large KIE (but also see references 96 and 97 ) and has been proposed to occur in nitrogenase ( 79 ), could be contributing to the KIE observed here, although we note that the temperature effect observed here is opposite the predicted effect for tunneling ( 80 , 81 ). Computational models, which can distinguish the rates of hydrogenation based on 1 H and 2 H, and might be able to shed light on the mechanism responsible for the observed fractionation and whether the currently proposed, multistep mechanisms of hydrogenation by nitrogenase ( 82 – 85 ) are compatible with the measured KIE of 2.1. The clumped isotopic composition of methane produced by nitrogenase could also provide additional constraints.…”

Section: Resultsmentioning

confidence: 99%

“…Computational models can distinguish the rates of hydrogenation based on 1 H and 2 H and might be able to shed light on the mechanism responsible for the observed fractionation and whether the currently proposed, multi-step mechanisms of hydrogenation by nitrogenase (85)(86)(87)(88) are compatible with the measured KIE of ~2. The clumped isotopic composition of methane produced by nitrogenase could also provide additional constraints.…”

Section: Mechanistic Implications For Nitrogenasementioning

confidence: 99%

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Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

Luxem

Leavitt

Zhang

2020

Appl Environ Microbiol

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Biological nitrogen fixation is catalyzed by the enzyme nitrogenase. Two forms of this metalloenzyme, the vanadium (V) and iron (Fe)-only nitrogenases, were recently found to reduce small amounts of carbon dioxide (CO2) into the potent greenhouse gas methane (CH4). Here we report carbon (13C/12C) and hydrogen (2H/1H) stable isotopic compositions and fractionations of methane generated by V- and Fe-only nitrogenases in the metabolically versatile nitrogen fixer Rhodopseudomonas palustris. The stable carbon isotope fractionation imparted by both forms of alternative nitrogenase are within the range observed for hydrogenotrophic methanogenesis (13αCO2/CH4 = 1.051 ± 0.002 for V-nitrogenase and 1.055 ± 0.001 for Fe-only nitrogenase, mean ± SE). In contrast, the hydrogen isotope fractionations (2αH2O/CH4 = 2.071 ± 0.014 for V-nitrogenase and 2.078 ± 0.018 for Fe-only nitrogenase) are the largest of any known biogenic or geogenic pathway. The large 2αH2O/CH4 shows that the reaction pathway nitrogenases use to form methane strongly discriminates against 2H, and that 2αH2O/CH4 distinguishes nitrogenase-derived methane from all other known biotic and abiotic sources. These findings on nitrogenase-derived methane will help constrain carbon and nitrogen flows in microbial communities and the role of the alternative nitrogenases in global biogeochemical cycles. Importance All forms of life require nitrogen for growth. Many different kinds of microbes living in diverse environments make inert nitrogen gas from the atmosphere bioavailable using a special enzyme, nitrogenase. Nitrogenase has a wide substrate range, and in addition to producing bioavailable nitrogen, some forms of nitrogenase also produce small amounts of the greenhouse gas methane. This is different from other microbes that produce methane to generate energy. Until now, there was no good way to determine when microbes with nitrogenases are making methane in nature. Here, we present an isotopic fingerprint that allows scientists to distinguish methane from microbes making it for energy versus those making it as a byproduct of nitrogen acquisition. With this new fingerprint, it will be possible to improve our understanding of the relationship between methane production and nitrogen acquisition in nature.

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Section: Resultsmentioning

confidence: 99%

Section: Mechanistic Implications For Nitrogenasementioning

confidence: 99%

Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

Luxem

Leavitt

Zhang

2020

Appl Environ Microbiol

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“…The crystallographers suggested that it shows the E 6 (NH − ) or E 7 ( ) reaction intermediates in the Lowe–Thorneley scheme [ 10 – 12 ]. This would be quite sensational as it would settle some important aspects of the highly controversial [ 1 , 9 ] reaction mechanism, viz. that one of the µ 2 sulphide ligands (S2B) dissociates to form a binding site of the substrate between the Fe 2 and Fe 6 ions, and that it is stored in a binding site, close to the FeV cluster.…”

Section: Discussionmentioning

confidence: 99%

“…It is notable that H 2 is a compulsory by-product. The mechanism of the nitrogenases has been extensively studied by biochemical, kinetic, spectroscopic and computational methods [ 1 , 5 , 7 – 9 ]. However, many details of the mechanism are still unknown.…”

Section: Introductionmentioning

confidence: 99%

Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement

2020

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Recently, a crystal structure of V-nitrogenase was presented, showing that one of the µ2 sulphide ions in the active site (S2B) is replaced by a lighter atom, suggested to be NH or NH2, i.e. representing a reaction intermediate. Moreover, a sulphur atom is found 7 Å from the S2B site, suggested to represent a storage site for this ion when it is displaced. We have re-evaluated this structure with quantum refinement, i.e. standard crystallographic refinement in which the empirical restraints (employed to ensure that the final structure makes chemical sense) are replaced by more accurate quantum–mechanical calculations. This allows us to test various interpretations of the structure, employing quantum–mechanical calculations to predict the ideal structure and to use crystallographic measures like the real-space Z-score and electron-density difference maps to decide which structure fits the crystallographic raw data best. We show that the structure contains an OH−-bound state, rather than an N2-derived reaction intermediate. Moreover, the structure shows dual conformations in the active site with ~ 14% undissociated S2B ligand, but the storage site seems to be fully occupied, weakening the suggestion that it represents a storage site for the dissociated ligand. Graphic abstract

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“…The calculations using the QM/MM approach were performed with the Gaussian 09 D.01 software package. 18 Hybrid density&orbital two-electron energy functionals are known not to be the best choice for iron-sulfur clusters, 19,20 as compared to density-gradient functionals (GGA) such as those of Becke 21 and Perdew. 22 We here use the BP86 functional for the QM part along with 6-311+g* basis sets 23 S1d).…”

Section: S12 Computational Methodsmentioning

confidence: 99%

H2 Formation Holds the Key to Opening the Fe Coordination Sites of Nitrogenase FeMo-cofactor for Dinitrogen Activation

Li¹,

Li²,

Liu³

et al. 2020

Preprint

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The present quantum-mechanical and molecular-mechanics study reveals the crucial roles of H2 formation, of H2S shift and of N2 bond expansion in the nitrogenase process of the reduction of N2 to <a href="https://en.wikipedia.org/wiki/Ammonia">NH3</a>. Proton and electron transfers to the Fe(C@Fe6S9)Mo unit of the FeMo-co complex weaken the Fe-S and Fe-H bonds and expose the Fe coordination sites, coupled with energy release due to H2 generation. Thereby the two sites Fe2 and Fe6 become prepared for stronger N2 adsorption, expanding and attenuating the ǀN≡Nǀ bond. After subsequent detachment of H2S from its Fe binding site into a holding site of the rearranged protein residue, the Fe6 site becomes completely unfolded, and the N2 triple bond becomes completely activated to an ‑N=N- double bond for easy subsequent hydrogenation to NH3. We explain in particular, why the obligatory H2 formation is an essential step in N2 adsorption and activation

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Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase

Abstract: Scheme3.Aconcerted proton slide from O6 to O5 via W3, W2, and W1 with as ingle transition state:distances []a nd relative energies [kcal mol À1 ]a re marked.A nterior and posterior refer to positions of Ho nW ,a long the proton wire in the direction of the protont ransfer.

Cited by 44 publications

References 229 publications

Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement

H2 Formation Holds the Key to Opening the Fe Coordination Sites of Nitrogenase FeMo-cofactor for Dinitrogen Activation

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