Theoretical investigations on Azotobacter vinelandii ferredoxin I: effects of electron transfer on protein dynamics.
ABSTRACT: Structural, energetic, and dynamical studies of Azotobacter vinelandii ferredoxin I are presented for native and mutant forms. The protein contains two iron-sulfur clusters, one of which ([3Fe-4S]) is believed to play a central role in the electron-coupled proton transfer. Different charge sets for the [3Fe-4S] cluster in its reduced and oxidized state are calculated with broken symmetry ab initio density functional theory methods and used in molecular dynamics (MD) simulations. The validity of the ab initio calculations is assessed by comparing partially optimized structures of the [3Fe-4S] clusters with x-ray structures. Possible proton transfer pathways between the protein and the iron-sulfur cluster are examined by both MD simulations and ab initio calculations. The MD simulations identify three main-chain hydrogen atoms--HN(13), HN(14), and HN(16)--that are within H-bonding distance of the [3Fe-4S] cluster throughout the MD simulations. They could thus play a role in the proton transfer from the protein to the iron-sulfur cluster. By contrast, the HD2(15) atom of the Asp-15 is seldom close enough to the [3Fe-4S] cluster to transfer a proton. Poisson-Boltzmann calculations indicate that there is a low, but nonzero probability, that Asp-15 is protonated at pH 7; this is a requirement for it to serve as a proton donor. Ab initio calculations with a fragment model for the protein find similar behavior for the transfer of a proton from the OH of the protonated side chain and the main-chain NH of Asp-15. The existence of a stable salt bridge between Asp-15 and Lys-84 in the D15E mutant, versus its absence in the wild-type, has been suggested as the cause of the difference in the rate of proton transfer. Extensive MD simulations were done to test this idea; the results do not support the proposal. The present findings, together with the available data, serve as the basis for an alternative proposal for the mechanism of the coupled electron-proton transfer reaction in ferredoxin I.
Project description:The role of water molecules in assisting proton transfer (PT) is investigated for the proton-pumping protein ferredoxin I (FdI) from Azotobacter vinelandii. It was shown previously that individual water molecules can stabilize between Asp(15) and the buried [3Fe-4S](0) cluster and thus can potentially act as a proton relay in transferring H(+) from the protein to the μ(2) sulfur atom. Here, we generalize molecular mechanics with proton transfer to studying proton transfer reactions in the condensed phase. Both umbrella sampling simulations and electronic structure calculations suggest that the PT Asp(15)-COOH + H(2)O + [3Fe-4S](0) → Asp(15)-COO(-) + H(2)O + [3Fe-4S](0) H(+) is concerted, and no stable intermediate hydronium ion (H(3)O(+)) is expected. The free energy difference of 11.7 kcal/mol for the forward reaction is in good agreement with the experimental value (13.3 kcal/mol). For the reverse reaction (Asp(15)-COO(-) + H(2)O + [3Fe-4S](0)H(+) → Asp(15)-COOH + H(2)O + [3Fe-4S](0)), a larger barrier than for the forward reaction is correctly predicted, but it is quantitatively overestimated (23.1 kcal/mol from simulations versus 14.1 from experiment). Possible reasons for this discrepancy are discussed. Compared with the water-assisted process (ΔE ≈ 10 kcal/mol), water-unassisted proton transfer yields a considerably higher barrier of ΔE ≈ 35 kcal/mol.
Project description:The reaction of HNO3 with hydrated electrons (H2O)n- (n = 35-65) in the gas phase was studied using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry and ab initio molecular dynamics simulations. Kinetic analysis of the experimental data shows that OH-(H2O)m is formed primarily via a reaction of the hydrated electron with HNO3 inside the cluster, while proton transfer is not observed and NO3-(H2O)m is just a secondary product. The reaction enthalpy was determined using nanocalorimetry, revealing a quite exothermic charge transfer with -241 ± 69 kJ mol-1. Ab initio molecular dynamics simulations indicate that proton transfer is an allowed reaction pathway, but the overall thermochemistry favors charge transfer.
Project description:Many processes important to chemistry, materials science, and biology cannot be described without considering electronic and nuclear-level dynamics and their coupling to slower, cooperative motions of the system. These inherently multiscale problems require computationally efficient and accurate methods to converge statistical properties. In this paper, a method is presented that uses data directly from condensed phase ab initio simulations to develop reactive molecular dynamics models that do not require predefined empirical functions. Instead, the interactions used in the reactive model are expressed as linear combinations of interpolating functions that are optimized by using a linear least-squares algorithm. One notable benefit of the procedure outlined here is the capability to minimize the number of parameters requiring nonlinear optimization. The method presented can be generally applied to multiscale problems and is demonstrated by generating reactive models for the hydrated excess proton and hydroxide ion based directly on condensed phase ab initio molecular dynamics simulations. The resulting models faithfully reproduce the water-ion structural properties and diffusion constants from the ab initio simulations. Additionally, the free energy profiles for proton transfer, which is sensitive to the structural diffusion of both ions in water, are reproduced. The high fidelity of these models to ab initio simulations will permit accurate modeling of general chemical reactions in condensed phase systems with computational efficiency orders of magnitudes greater than currently possible with ab initio simulation methods, thus facilitating a proper statistical sampling of the coupling to slow, large-scale motions of the system.
Project description:Direct molecular dynamics (MD) simulation with ab initio quantum mechanical and molecular mechanical (QM/MM) methods is very powerful for studying the mechanism of chemical reactions in a complex environment but also very time-consuming. The computational cost of QM/MM calculations during MD simulations can be reduced significantly using semiempirical QM/MM methods with lower accuracy. To achieve higher accuracy at the ab initio QM/MM level, a correction on the existing semiempirical QM/MM model is an attractive idea. Recently, we reported a neural network (NN) method as QM/MM-NN to predict the potential energy difference between semiempirical and ab initio QM/MM approaches. The high-level results can be obtained using neural network based on semiempirical QM/MM MD simulations, but the lack of direct MD samplings at the ab initio QM/MM level is still a deficiency that limits the applications of QM/MM-NN. In the present paper, we developed a dynamic scheme of QM/MM-NN for direct MD simulations on the NN-predicted potential energy surface to approximate ab initio QM/MM MD. Since some configurations excluded from the database for NN training were encountered during simulations, which may cause some difficulties on MD samplings, an adaptive procedure inspired by the selection scheme reported by Behler [ Behler Int. J. Quantum Chem. 2015 , 115 , 1032 ; Behler Angew. Chem., Int. Ed. 2017 , 56 , 12828 ] was employed with some adaptions to update NN and carry out MD iteratively. We further applied the adaptive QM/MM-NN MD method to the free energy calculation and transition path optimization on chemical reactions in water. The results at the ab initio QM/MM level can be well reproduced using this method after 2-4 iteration cycles. The saving in computational cost is about 2 orders of magnitude. It demonstrates that the QM/MM-NN with direct MD simulations has great potentials not only for the calculation of thermodynamic properties but also for the characterization of reaction dynamics, which provides a useful tool to study chemical or biochemical systems in solution or enzymes.
Project description:Human DNA Pol ? is a polymerase enzyme, specialized for near error-free bypass of certain bulky chemical lesions to DNA that are derived from environmental carcinogens present in tobacco smoke, automobile exhaust and cooked food. By employing ab initio QM/MM-MD (Quantum Mechanics/Molecular Mechanics-Molecular Dynamics) simulations with umbrella sampling, we have determined the entire free energy profile of the nucleotidyl transfer reaction catalyzed by Pol ? and provided detailed mechanistic insights. Our results show that a variant of the Water Mediated and Substrate Assisted (WMSA) mechanism that we previously deduced for Dpo4 and T7 DNA polymerases is preferred for Pol ? as well, suggesting its broad applicability. The hydrogen on the 3'-OH primer terminus is transferred through crystal and solvent waters to the ?-phosphate of the dNTP, followed by the associative nucleotidyl transfer reaction; this is facilitated by a proton transfer from the ?-phosphate to the ?,?-bridging oxygen as pyrophosphate leaves, to neutralize the evolving negative charge. MD simulations show that the near error-free incorporation of dCTP opposite the major benzo[a]pyrene-derived dG lesion is compatible with the WMSA mechanism, allowing for an essentially undisturbed pentacovalent phosphorane transition state, and explaining the bypass of this lesion with little mutation by Pol ?.
Project description:Proton transport at water/membrane interfaces plays a fundamental role for a myriad of bioenergetic processes. Here we have performed ab initio molecular dynamics simulations of proton transfer along two phosphatidylcholine bilayers. As found in previous theoretical studies, the excess proton is preferably located at the water/membrane interface. Further, our simulations indicate that it interacts not only with phosphate head groups, but also with water molecules at the interfaces. Interfacial water molecules turn out to be oriented relative to the lipid bilayers, consistently with experimental evidence. Hence, the specific water-proton interaction may help explain the proton mobility experimentally observed at the membrane interface.
Project description:A previous study showed that Nitrogen-Fixing-subunit-U-type protein NFU3 may act an iron-sulfur scaffold protein in the assembly and transfer of 4Fe-4S and 3Fe-4S clusters in the chloroplast. Examples of 4Fe-4S and 3Fe-4S-requiring proteins and complexes include Photosystem I (PSI), NAD(P)H dehydrogenase, and ferredoxin-dependent glutamine oxoglutarate aminotransferases. In this paper, the authors provided additional evidence for the role of NFU3 in 4Fe-4S and 3Fe-4S cluster assembly and transfer, as well as its role in overall plant fitness. Confocal microscopic analysis of the fluorescently-tagged NFU3 protein confirmed the chloroplast localization of the NFU3 protein. Detailed analysis of chlorophyll fluorescence data revealed that a substantial increase in minimal fluorescence is the primary contributor to the decrease in PSII maximum photochemical efficiency observed in the nfu3 mutants. The substantial increase in minimal fluorescence in the nfu3 mutants is probably the result of an impaired PSI function, blockage of electron flow from PSII to PSI, and over-accumulation of reduced plastoquinone at the acceptor side of PSII. Analyses of seed morphology and germination showed that NFU3 is essential to seed development and germination, in addition to plant growth, development, and flowering. In summary, NFU3 has wide-ranging effects on many biologic processes and is therefore important to overall plant fitness. NFU3 may exert these effects by modulating the availability of 4Fe-4S and 3Fe-4S clusters to 4Fe-4S and 3Fe-4S-requiring proteins and complexes involved in various biologic processes.
Project description:Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S](2+/1+) cluster and one [3Fe-4S](1+/0) cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys(11)-Xaa-Xaa-Asp(14)-Xaa-Xaa-Cys(17)-Xaa(n)-Cys(51)-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S]<-->[4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg.l(-1)), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl(3) together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.
Project description:Single electron transfers have been examined in complex II (succinate:ubiquinone oxidoreductase) by the method of pulse radiolysis. Electrons are introduced into the enzyme initially at the [3Fe-4S] and ubiquinone sites followed by intramolecular equilibration with the b heme of the enzyme. To define thermodynamic and other controlling parameters for the pathways of electron transfer in complex II, site-directed variants were constructed and analyzed. Variants at SdhB-His207 and SdhB-Ile209 exhibit significantly perturbed electron transfer between the [3Fe-4S] cluster and ubiquinone. Analysis of the data using Marcus theory shows that the electronic coupling constants for wild-type and variant enzyme are all small, indicating that electron transfer occurs by diabatic tunneling. The presence of the ubiquinone is necessary for efficient electron transfer to the heme, which only slowly equilibrates with the [3Fe-4S] cluster in the absence of the quinone.
Project description:Two-dimensional (2D) graphene and graphene oxide (GO) offer great potential as a new type of cost-efficient proton-exchange membranes (PEM) for electrochemical devices. However, fundamental issues of proton transfer mechanism via 2D membranes are unclear and the transfer barrier for perfect graphene are too high for practical application. Using ab initio molecular dynamic simulations, we screened the proton transfer barrier for different un-doped and nitrogen doped GO membranes, and clarified the corresponding transfer mechanisms. More significantly, we further identify that N-mediated GO can be built into a highly efficient PEM with a proton transfer rate of seven orders of magnitude higher than an un-doped case via. a proton relay mechanism between a ketone-like oxygen and a pyridine-like nitrogen across the vacancy site. The N-doped 2D GO is also impermeable to small molecules, and hence a highly efficient PEM for practical applications.