Reaction Mechanism Catalyzed by the Dissimilatory Sulfite Reductase - The Role of the Siroheme-[4FeS4] Cofactor.

The present work is another part of our investigation on the pathway of dissimilatory sulfate reduction and covers a theoretical study on the reaction catalyzed by dissimilatory sulfite reductase (dSIR). dSIR is the terminal enzyme involved in this metabolic pathway, which uses the siroheme-[4Fe4S] cofactor for six-electron reduction of sulfite to sulfide. In this study we use a large cluster model containing siroheme-[4Fe4S] cofactor and protein residues involved in the direct interactions with the substrate, to get insight into the most feasible reaction mechanism and to understand the role of each considered active site component. In combination with earlier studies reported in the literature, our results lead to several interesting insights. One of the most important conclusions is that the reaction mechanism consists of three steps of two-electron reduction of sulfur and the probable role of the siroheme-[4Fe4S] cofactor is to ensure the delivery of packages of two electrons to the reactant.

Identification of hydrogen bonding network for proton transfer at the quinol oxidation site of Rhodobacter capsulatus cytochrome bc1.

Cytochrome bc1 catalyzes electron transfer from quinol (QH2) to cytochrome c in reactions coupled to proton translocation across the energy-conserving membrane. Energetic efficiency of the catalytic cycle is secured by a two-electron and two-proton bifurcation reaction leading to oxidation of QH2 and reduction of the Rieske cluster and heme bL. The proton paths associated with this reaction remain elusive. Here, we used site-directed mutagenesis and quantum mechanical calculations to analyze the contribution of protonable side chains located at the heme bL side of the QH2 oxidation site in Rhodobacter capsulatus cytochrome bc1. We observe that the proton path is effectively switched off when H276 and E295 are simultaneously mutated to the nonprotonable residues in the H276F/E295V double mutant. The two single mutants, H276F or E295V, are less efficient but still transfer protons at functionally relevant rates. Natural selection exposed two single mutations, N279S and M154T, that restored the functional proton transfers in H276F/E295V. Quantum mechanical calculations indicated that H276F/E295V traps the side chain of Y147 in a position distant from QH2, whereas either N279S or M154T induce local changes releasing Y147 from that position. This shortens the distance between the protonable groups of Y147 and D278 and/or increases mobility of the Y147 side chain, which makes Y147 efficient in transferring protons from QH2 toward D278 in H276F/E295V. Overall, our study identified an extended hydrogen bonding network, build up by E295, H276, D278, and Y147, involved in efficient proton removal from QH2 at the heme bL side of QH2 oxidation site.

Probing molecular interactions of semiquinone radicals at quinone reduction sites of cytochrome bc1 by X-band HYSCORE EPR spectroscopy and quantum mechanical calculations.

Quinone redox reactions involve a semiquinone (SQ) intermediate state. The catalytic sites in enzymes stabilize the SQ state via various molecular interactions, such as hydrogen bonding to oxygens of the two carbonyls of the benzoquinone ring. To understand how these interactions contribute to SQ stabilization, we examined SQ in the quinone reduction site (Qi) of cytochrome bc1 using electron paramagnetic resonance (ESEEM, HYSCORE) at the X-band and quantum mechanical (QM) calculations. We compared native enzyme (WT) with a H217R mutant (replacement of histidine that interacts with one carbonyl of the occupant of Qi to arginine) in which the SQ stability has previously been shown to markedly increase. The 14N region of the HYSCORE 2D spectrum for SQi in WT had a shape typical of histidine residue, while in H217R, the spectrum shape changed significantly and appeared similar to the pattern described for SQ liganded natively by arginine in cytochrome bo3. Parametrization of hyperfine and quadrupolar interactions of SQi with surrounding magnetic nuclei (1H, 14N) allowed us to assign specific nitrogens of H217 or R217 as ligands of SQi in WT and H217R, respectively. This was further substantiated by qualitative agreement between the experimental (EPR-derived) and theoretical (QM-derived) parameters. The proton (1H) region of the HYSCORE spectrum in both WT and H217R was very similar and indicative of interactions with two protons, which in view of the QM calculations, were identified as directly involved in the formation of a H-bond with the two carbonyl oxygens of SQ (interaction of H217 or R217 with O4 and D252 with O1). In view of these assignments, we explain how different SQ ligands effectively influence SQ stability. We also propose that the characteristic X-band HYSCORE pattern and parameters of H217R are highly specific to the interaction of SQ with the nitrogen of arginine. These features can thus be considered as potential markers of the interaction of arginine with SQ in other proteins.

The importance of atomic partial charges in the reproduction of intermolecular interactions for the triacetin - a model of glycerol backbone.

Lipids play a central role within the cell. They not only encompass it but are also engaged in many processes such as cellular transport and energy production. Despite ongoing advances in experimental studies, computer simulations are a viable method to trace their behavior at the atomic level and on an elusive time scale. In molecular modeling studies, the quality of the obtained results is associated with the considered force field and its parameters. In the present work, the authors have investigated the procedure of partial charges fitting on the example of a triacetin molecule, containing chemical moieties present in the glycerol backbone. The goal of the study was to validate assigned partial charges based on the quality of the torsion profiles using optimally assigned torsional coefficients and reproduction of the condensed phase properties of triacetin. We applied various approaches and noticed a significant improvement in the parameterization of triacetin compared to the original one. The results showed that it is important to take into account the intermolecular interactions in the partial charges fitting procedure to obtain good quality validation results.

Optimal Solution to the Torsional Coefficient Fitting Problem in Force Field Parametrization.

Molecular modeling is an excellent tool for studying biological systems on the atomic scale. Depending on objects, which may be proteins, nucleic acids, or lipids, different force fields are recommended. The phospholipid bilayers constitute an example, in which behavior is extensively studied using molecular dynamics simulations due to limitations of experimental methods. The reliability of the results is strongly dependent on an appropriate description of these compounds. There are some deficiencies in the parametrization of intra- and intermolecular interactions that result in incorrect reproduction of phospholipid bilayer properties known from experimental studies, such as temperatures of phase transitions. Refinement of the force field parameters of nonbonded interactions present in the studied system is required to close these discrepancies. Such parameters as partial charges and torsional potential coefficients are crucial in this issue and not obtainable from experimental studies. This work presents a new fitting procedure for torsional coefficients that employs linear algebra theory and compares it with the Monte Carlo method. The proposed algebraic approach can be applied to any considered molecular system. In the manuscript, it is presented on the example of dimethyl phosphoric acid molecule. The advantages of our method encompass finding an optimal solution, the lack of additional parameters required by the algorithm, and significantly shorter computational time. Additionally, we indicate the importance of proper assignment of the partial charges.

Reaction mechanism catalyzed by the dissimilatory adenosine 5'-phosphosulfate reductase. Adenosine 5'-monophosphate inhibitor and key role of arginine 317 in switching the course of catalysis.

The present research is a continuation of our work on dissimilatory reduction pathway of sulfate - involved in biogeochemical sulfur turnover. Adenosine 5'-phosphosulfate reductase (APSR) is the second enzyme in the dissimilatory pathway of the sulfate to sulfide reduction. It reversibly catalyzes formation of the sulfite anion (HSO3-) from adenosine 5'-phosphosulfate (APS) - the activated form of sulfate provided by ATP sulfurylase (ATPS). Two electrons required for this redox reaction derive from reduced FAD cofactor, which is suggested to be involved directly in the catalysis by formation of FADH-SO3- intermediate. The present work covers quantum-mechanical (QM) studies on APSR reaction performed for eight models of APSR active site. The cluster models were constructed based on two crystal structures (PDB codes: 2FJA and 2FJB), differing in conformation of Arg317 active site residue. The described results indicated the most feasible mechanism of APSR forward reaction, including formation of FADHN-SO3- adduct (with proton on N5 atom of isoalloxazine), tautomerization of FADHN-SO3- to FADHO-SO3- (with proton on CO moiety of isoalloxazine), and its reductive cleavage to oxidized FAD and sulfite anion. The reverse reaction proceeds in the backward direction. It is suggested that it requires two AMP molecules, one acting as a substrate and another as an inhibitor of forward reaction, which forces change of Arg317 conformation from "arginine in" (2FJA) to "arginine out" (2FJB). Important role of Arg317 in switching the course of the APSR catalytic reaction is revealed by changing the direction of thermodynamic driving force. The presented research also shows the importance of the protonation pattern of the reduced FAD cofactor and protein residues within the active site.

Condensed phase properties of n-pentadecane emerging from application of biomolecular force fields.

For over 20 years, the OPLS-All Atom (OPLS-AA) force field has been efficiently used in molecular modelling studies of proteins, carbohydrates and nucleic acids. OPLS-AA is successfully applied in computer modelling of many organic compounds, including decane and shorter alkanes, but it fails when employed for longer linear alkanes, whose chemical structure corresponds to hydrocarbon tails in phospholipids constituting cellular membranes. There have been several attempts to address this problem. In this work, we compare the ability to reproduce various condensed phase properties by six distinct sets of force field parameters which can be assigned to phospholipid hydrocarbon chains. In this comparison, we include three alternative sets of the OPLS-AA force field, as well as the commonly used CHARMM C36, Slipids, and Berger lipids' parameters.

Charge polarization imposed by the binding site facilitates enzymatic redox reactions of quinone.

Quinone reduction site (Qi) of cytochrome bc1 represents one of the canonical sites used to explore the enzymatic redox reactions involving semiquinone (SQ) states. However, the mechanism by which Qi allows the completion of quinone reduction during the sequential transfers of two electrons from the adjacent heme bH and two protons to C1- and C4-carbonyl remains unclear. Here we established that the SQ coupled to an oxidized heme bH is a dominant intermediate of catalytic forward reaction and, contrary to the long-standing assumption, represents a significant population of SQ detected across pH 5-9. The pH dependence of its redox midpoint potential implicated proton exchange with histidine. Complementary quantum mechanical calculations revealed that the SQ anion formed after the first electron transfer undergoes charge and spin polarization imposed by the electrostatic field generated by histidine and the aspartate/lysine pair interacting with the C4- and C1-carbonyl, respectively. This favors a barrierless proton exchange between histidine and the C4-carbonyl, which continues until the second electron reaches the SQi. Inversion of charge polarization facilitates the uptake of the second proton by the C1-carbonyl. Based on these findings we developed a comprehensive scheme for electron and proton transfers at Qi featuring the equilibration between the anionic and neutral states of SQi as means for a leak-proof stabilization of the radical intermediate. The key catalytic role of the initial charge/spin polarization of the SQ anion at the active site, inherent to the proposed mechanism, may also be applicable to the other quinone oxidoreductases.

Mechanism of Sulfate Activation Catalyzed by ATP Sulfurylase - Magnesium Inhibits the Activity.

ATPS Sulfurylase (ATPS) is the first of three enzymes in the sulfate reduction pathway - one of the oldest metabolic pathways on Earth, utilized by Sulfate Reducing Bacteria (SRB). Due to the low redox potential of the sulfate ion, its reduction requires activation via formation of adenosine 5'-phosphosulfate (APS), which is catalyzed by ATPS. Dispersion-corrected hybrid density functional theory (DFT/B3LYP-D3) was used to test three reaction mechanisms proposed for conversion of ATP to APS: two-step SN-1 reaction running through AMP anhydride intermediate, two-step reaction involving cyclic AMP intermediate and direct SN-2 conversion of ATP to APS molecule. The study employed five different cluster models of the ATPS active site: one containing magnesium cation and four without it, constructed based on the crystal structure (PDB code: 1G8H) solved for ATPS from Saccharomyces cerevisiae in complex with APS and pyrophosphate (PPi), where Mg2+ was not detected. The model with magnesium ion was constructed based on the representative structure obtained from trajectory analysis of the molecular dynamics simulations (MD) performed for the hexameric ATPS-APS-Mg2+-PPi complex. The results obtained for all considered models suggest that ATPS-AMP anhydride intermediate is a highly energetic and unstable complex, while formation of cyclic AMP molecule requires formation of unfavorable hypervalent geometry at the transition state. Among all tested mechanism, the energetically most feasible mechanism of the ATPS reaction is SN-2 one-step conversion of ATP to APS occurring via a pentavalent transition state. Interestingly, such a reaction is inhibited by the presence of Mg2+ in the ATPS active site. Magnesium cation forces unfavorable geometry of reactants for SN-2 mechanism and formation of pentavalent transition state. Such a reaction requires rearrangement of Mg2+ ligands, which raises the barrier from 11-14 kcal/mol for the models without Mg2+ to 48 kcal/mol for model with magnesium ion included.

Reaction mechanism of sterol hydroxylation by steroid C25 dehydrogenase - Homology model, reactivity and isoenzymatic diversity.

Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O2-independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[2H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH.