Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach

Mo-dependent nitrogenase catalyzes the biological reduction of N₂ to two NH₃ molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N₂, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N₂ from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding

Manganese-Based Molecular Electrocatalysts for Oxidation of Hydrogen

Oxidation of H₂ (1 atm) is catalyzed by the manganese electrocatalysts [(P₂N₂)­Mn^I(CO)­(bppm)]⁺ and [(PNP)­Mn^I(CO)­(bppm)]⁺ (P₂N₂ = 1,5-dibenzyl-3,7-diphenyl-1,5-diaza-3,7-diphosphacyclooctane; PNP = (Ph₂PCH₂)₂NMe); bppm = (PAr^F₂)₂CH₂; Ar^F = 3,5-(CF₃)₂C₆H₃). In fluorobenzene solvent using 2,6-lutidine as the exogeneous base, the turnover frequency for [(P₂N₂)­Mn^I(CO)­(bppm)]⁺ is 3.5 s^–1, with an estimated overpotential of 700 mV. For [(PNP)­Mn^I(CO)­(bppm)]⁺ in fluorobenzene solvent using <i>N</i>-methylpyrrolidine as the exogeneous base, the turnover frequency is 1.4 s^–1, with an estimated overpotential of 880 mV. Density functional theory calculations suggest that the slow step in the catalytic cycle is proton transfer from the oxidized 17-electron manganese hydride [(P₂N₂)­Mn^IIH­(CO)­(bppm)]⁺ to the pendant amine. The computed activation barrier for intramolecular proton transfer from the metal to the pendant amine is 20.4 kcal/mol for [(P₂N₂)­Mn^IIH­(CO)­(bppm)]⁺ and 21.3 kcal/mol for [(PNP)­Mn^IIH­(CO)­(bppm)]⁺. The high barrier appears to result from both the unfavorability of the metal to nitrogen proton transfer (thermodynamically uphill by 9 kcal/mol for [(P₂N₂)­Mn^IIH­(CO)­(bppm)]⁺ due to a mismatch of 6.6 p<i>K</i>_a units) and the relatively long manganese–nitrogen separation in the Mn^IIH complexes

Evaluation of the Role of Water in the H₂ Bond Formation by Ni(II)-Based Electrocatalysts

We investigate the role of water in the H–H bond formation by a family of nickel molecular catalysts that exhibit high rates for H₂ production in acetonitrile solvent. A key feature leading to the high reactivity is the Lewis acidity of the Ni­(II) center and pendant amines in the diphosphine ligand that function as Lewis bases, facilitating H–H bond formation or cleavage. Significant increases in the rate of H₂ production have been reported in the presence of added water. Our calculations show that molecular water can displace an acetonitrile solvent molecule in the first solvation shell of the metal. One or two water molecules can also participate in shuttling a proton that can combine with a metal hydride to form the H–H bond. However the participation of the water molecules does not lower the barrier to H–H bond formation. Thus these calculations suggest that the rate increase due to water in these electrocatalysts is not associated with the elementary step of H–H bond formation or cleavage but rather with the proton delivery steps. We attribute the higher barrier in the H–H bond formation in the presence of water to a decrease in direct interaction between the protic and hydridic hydrogen atoms forced by the water molecules

Protonation state space exploration.

<p>(A) GB corrected energy (E_corr) as a function of MC step for the MC/MD sampling on hIns₂ at the main charge state (q = 6+). (B) Prediction of the main charge state of hIns₂. GB_app values (in kJ/mol) were calculated for the lowest energy protonation states of hIns₂ (black line and cycle symbols). Standard deviation from the average is given as error bars. When not visible, the standard deviation is smaller than the symbol size. The red horizontal line indicates the GB of water (660.3 kJ/mol taken from ref. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003838#pcbi.1003838-Hunter1" target="_blank">[101]</a>). The experimental main charge state <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003838#pcbi.1003838-Salbo1" target="_blank">[52]</a> is shown by red solid diamond.</p

Photoinduced Reductive Elimination of H₂ from the Nitrogenase Dihydride (Janus) State Involves a FeMo-cofactor‑H₂ Intermediate

N₂ reduction by nitrogenase involves the accumulation of four reducing equivalents at the active site FeMo-cofactor to form a state with two [Fe–H–Fe] bridging hydrides (denoted E₄(4H), the Janus intermediate), and we recently demonstrated that the enzyme is activated to cleave the NN triple bond by the reductive elimination (<i>re</i>) of H₂ from this state. We are exploring a photochemical approach to obtaining atomic-level details of the <i>re</i> activation process. We have shown that, when E₄(4H) at cryogenic temperatures is subjected to 450 nm irradiation in an EPR cavity, it cleanly undergoes photoinduced <i>re</i> of H₂ to give a reactive doubly reduced intermediate, denoted E₄(2H)*, which corresponds to the intermediate that would form if thermal dissociative <i>re</i> loss of H₂ preceded N₂ binding. Experiments reported here establish that photoinduced <i>re</i> primarily occurs in two steps. Photolysis of E₄(4H) generates an intermediate state that undergoes subsequent photoinduced conversion to [E₄(2H)* + H₂]. The experiments, supported by DFT calculations, indicate that the trapped intermediate is an H₂ complex on the ground adiabatic potential energy suface that connects E₄(4H) with [E₄(2H)* + H₂]. We suggest that this complex, denoted E₄(H₂; 2H), is a thermally populated intermediate in the catalytically central <i>re</i> of H₂ by E₄(4H) and that N₂ reacts with this complex to complete the activated conversion of [E₄(4H) + N₂] into [E₄(2N2H) + H₂]

Comparison of a local inter- and intra-molecular hydrogen bond network in water (A) and in the gas phase (B).

<p>The final snapshots obtained from the MD simulations in water and in the gas phase at the main charge were selected. The monomer I and II are indicated in cyan and green, respectively. The water oxygen atoms are indicated by yellow balls. Nitrogen, dark blue; oxygen, red; hydrogen, white. Hydrogen bonds are shown as dashed lines.</p

Correlation plots of the differences in energy for 60 protonation states of the hIns₂ relative to the lowest-energy protonation state.

<p>(A) Energy differences calculated with the OPLS/AA force field (ΔE_FF) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003838#pcbi.1003838-Jorgensen2" target="_blank">[63]</a> versus differences calculated with DFT (ΔE_QM) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003838#pcbi.1003838-Marchese2" target="_blank">[38]</a>. (B) Energy differences calculated with the OPLS/AA force field along with the GB correction (ΔE_corr) versus differences calculated with DFT (ΔE_QM). The correlation is much better with GB correction than with OPLS/AA (<i>R</i>² = 0.81 and 0.03, respectively), confirming the crucial role of GB for estimating the energies of the protonation states.</p

MD simulations in the gas phase of the [hIns₂]⁶⁺.

<p>(A) Models of [hIns₂]⁶⁺ obtained from MD simulations in the gas phase (from left to right, at 0 µs, 5.7 µs, 8.1 µs, 27.6 µs, 36.3 µs, 42.6 µs, 54.9 µs, and 75.0 µs). The monomer I and II are indicated in cyan (lower structure) and green (upper structure), respectively. The α-helices and β-sheets are highlighted in blue and red, respectively. Schematic representations of the complex models are shown below the complex structures, at corresponding positions on the simulation time axis. The backbone RMSD values of the models in respect to the one at 0 µs are 0.25 nm (5.7 µs), 0.54 nm (8.1 µs), 0.55 nm (27.6 µs), 0.49 nm (36.3 µs), 0.45 nm (42.6 µs), and 0.49 nm (54.9 µs), and 0.50 (75.0 µs). (B) Secondary structure analysis for [hIns₂]⁶⁺. (C) The angle between the center of mass (COM) of monomer I – β-sheet region – monomer II. (D) CCS values. The experimental value of 12.9 nm², as reported <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003838#pcbi.1003838-Salbo1" target="_blank">[52]</a>, at the main charge state is indicated by a red solid line and its 5% variations are indicated by the dashed lines. The average value from our MD simulation in the gas phase is 12.8±0.2 nm². (E) Number of contact pairs between the carbon atoms of the monomers within 0.60 nm.</p

CO₂ Reduction Catalyzed by Nitrogenase: Pathways to Formate, Carbon Monoxide, and Methane

The reduction of N₂ to NH₃ by Mo-dependent nitrogenase at its active-site metal cluster FeMo-cofactor utilizes reductive elimination of Fe-bound hydrides with obligatory loss of H₂ to activate the enzyme for binding/reduction of N₂. Earlier work showed that wild-type nitrogenase and a nitrogenase with amino acid substitutions in the MoFe protein near FeMo-cofactor can catalytically reduce CO₂ by two or eight electrons/protons to carbon monoxide (CO) and methane (CH₄) at low rates. Here, it is demonstrated that nitrogenase preferentially reduces CO₂ by two electrons/protons to formate (HCOO^–) at rates >10 times higher than rates of CO₂ reduction to CO and CH₄. Quantum mechanical calculations on the doubly reduced FeMo-cofactor with a Fe-bound hydride and S-bound proton (E₂(2H) state) favor a direct reaction of CO₂ with the hydride (“direct hydride transfer” reaction pathway), with facile hydride transfer to CO₂ yielding formate. In contrast, a significant barrier is observed for reaction of Fe-bound CO₂ with the hydride (“associative” reaction pathway), which leads to CO and CH₄. Remarkably, in the direct hydride transfer pathway, the Fe-H behaves as a hydridic hydrogen, whereas in the associative pathway it acts as a protic hydrogen. MoFe proteins with amino acid substitutions near FeMo-cofactor (α-70^Val→Ala, α-195^His→Gln) are found to significantly alter the distribution of products between formate and CO/CH₄

Achieving Reversible H₂/H⁺ Interconversion at Room Temperature with Enzyme-Inspired Molecular Complexes: A Mechanistic Study

Inspired by the contribution of the protein scaffold to the efficiency with which enzymes function, we used outer coordination sphere features to develop a molecular electrocatalyst for the reversible production/oxidation of H₂ at 25 °C: [Ni­(P^Cy₂N^Phe₂)₂]²⁺ (<b>CyPhe</b>; P^R₂N^{R^′}₂ = 1,5-diaza-3,7-diphosphacyclooctane, Cy = cyclohexyl, Phe = phenylalanine). Electrocatalytic reversibility is observed in aqueous, acidic methanol. The aromatic rings in the peripheral phenylalanine groups appear to be essential to achieving reversibility based on the observation that reversibility for arginine (<b>CyArg</b>) or glycine (<b>CyGly</b>) complexes is only achieved with elevated temperature (>50 °C) in 100% water. A complex with a hydroxyl group in the <i>para</i>-position of the aromatic ring, R′ = tyrosine (<b>CyTyr</b>), shows similar reversible behavior. NMR spectroscopy and molecular dynamics studies suggest that interactions between the aromatic groups as well as between the carboxylic acid groups limit conformational flexibility, contributing to reversibility. NMR spectroscopy studies also show extremely fast proton exchange along a pathway from the Ni–H through the pendant amine to the carboxyl group. Further, a complex containing a side chain similar to tyrosine but without the carboxyl group (<b>CyTym</b>; Tym = tyramine) does not display reversible catalysis and has limited proton exchange from the pendant amine, demonstrating an essential role for the carboxylic acid and the proton pathway in achieving catalytic reversibility. This minimal pathway mimics proton pathways found in hydrogenases. The influence of multiple factors on lowering barriers and optimizing relative energies to achieve reversibility for this synthetic catalyst is a clear indication of the intricate interplay between the first, second, and outer coordination spheres that begins to mimic the complexity observed in metalloenzymes