Alexander N. Morozov

Chloroperoxidase-Catalyzed Epoxidation of Cis-β-Methylstyrene:Distal Pocket Flexibility Tunes Catalytic Reactivity

Chloroperoxidase, the most versatile heme protein, has a hybrid active site pocket that shares structural features with peroxidases and cytochrome P450s. The simulation studies presented here show that the enzyme possesses a remarkable ability to efficiently utilize its hybrid structure, assuming structurally different peroxidase-like and P450-like distal pocket faces and thereby enhancing the inherent catalytic capability of the active center. We find that, during epoxidation of cis-β-methylstyrene (CBMS), the native peroxidase-like aspect of the distal pocket is diminished as the polar Glu183 side chain is displaced away from the active center and the distal pocket takes on a more hydrophobic, P450-like, aspect. The P450-like distal pocket provides a significant enthalpic stabilization of ∼4 kcal/mol of the 14 kcal/mol reaction barrier for gas-phase epoxidation of CMBS by an oxyferryl heme-thiolate species. This stabilization comes from breathing of the distal pocket. As until recently the active site of chloroperoxidase was postulated to be inflexible, these results suggest a new conceptual understanding of the enzymes versatility: catalytic reactivity is tuned by flexibility of the distal pocket.

Enantiospecificity of Chloroperoxidase-Catalyzed Epoxidation: Biased Molecular Dynamics Study of a Cis-β-Methylstyrene/Chloroperoxidase-Compound I Complex

Molecular dynamics simulations of an explicitly solvated cis-β-methylstyrene/chloroperoxidase-Compound I complex are performed to determine the cause of the high enantiospecificity of epoxidation. From the simulations, a two-dimensional free energy potential is calculated to distinguish binding potential wells from which reaction to 1S2R and 1R2S epoxide products may occur. Convergence of the free energy potential is accelerated with an adaptive biasing potential. Analysis of binding is followed by analysis of 1S2R and 1R2S reaction precursor structures in which the substrate, having left the binding wells, places its reactive double bond in steric proximity to the oxyferryl heme center. Structural analysis of binding and reaction precursor conformations is presented. We find that 1), a distortion of Glu(183) is important for CPO-catalyzed epoxidation as was postulated previously based on experimental results; 2), the free energy of binding does not provide significant differentiation between structures leading to the respective epoxide enantiomers; and 3), CPOs enantiospecificity toward cis-β-methylstyrene is likely to be caused by a specific group of residues which form a hydrophobic core surrounding the oxyferryl heme center.

A possible mechanism for redox control of human neuroglobin activity.

Neuroglobin (Ngb) promotes neuron survival under hypoxic/ischemic conditions. In vivo and in vitro assays provide evidence for redox-regulated functioning of Ngb. On the basis of X-ray crystal structures and our MD simulations, a mechanism for redox control of human Ngb (hNgb) activity via the influence of the CD loop on the active site is proposed. We provide evidence that the CD loop undergoes a strand-to-helix transition when the external environment becomes sufficiently oxidizing, and that this CD loop conformational transition causes critical restructuring of the active site. We postulate that the strand-to-helix mechanics of the CD loop allows hNgb to utilize the lability of Cys46/Cys55 disulfide bonding and of the Tyr44/His64/heme propionate interaction network for redox-controlled functioning of hNgb.

Theoretical study of HOCl-catalyzed keto-enol tautomerization of β-cyclopentanedione in an explicit water environment.

The mechanism of acid-catalyzed keto-enol tautomerization of β-cyclopentanedione (CPD) in solution is studied computationally. Reaction profiles are first calculated for a limited solvation environment using ab initio and density functional methods. Barrier heights for systems including up to one hydration shell of explicit water molecules depend strongly on the number of waters involved in proton transfer and to a lesser but significant extent on the number of waters forming hydrogen bonds with waters in the proton-transfer chain (each such water reduces the barrier by 4.4 kcal/mol on average). Barriers of 8-13 kcal/mol were obtained when a full or nearly full hydration shell was present, consistent with calculations for nonacid-catalyzed keto-enol tautomerization of related molecules. The presence of HOCl reduced the barrier by 4.5 kcal/mol in relation to the gas phase, consistent with the well-known principle that keto-enol tautomerization can be acid- or base-catalyzed. The reaction was also modeled beginning with snapshots of reactant conformations taken from a 300 K molecular dynamics simulation of CPD, HOCl, and 324 explicit waters. Reaction profiles were calculated at a QM/MM level with waters in the first hydration shell either fixed or energy-minimized at each step along the reaction coordinate. A substantial variation in barrier height was observed in both cases, depending primarily on electrostatic interactions (hydrogen bonding) with first-hydration-shell waters and, to a lesser extent, on electrostatic interactions with more distant waters and geometric distortion effects. For the lowest barriers, the extent of barrier reduction by waters involved in proton transfer is consistent with the limited solvation results, but further barrier reduction due to hydrogen bonding to waters involved in proton transfer is not observed. It is postulated that this is because highly flexible structures such as extensive hydrogen bonding networks optimal for reaction are entropically disfavored and so may not contribute significantly to the observed reaction rate.

Chloroperoxidase-Catalyzed Epoxidation of Cis-β-Methylstyrene: NH–S Hydrogen Bonds and Proximal Helix Dipole Change the Catalytic Mechanism and Significantly Lower the Reaction Barrier

Proximal hydrogen bonding of the axial sulfur with the backbone amides (NH-S) is a conserved feature of heme-thiolate enzymes such as chloroperoxidase (CPO) and cytochrome P450 (P450). In CPO, the effect of NH-S bonds is amplified by the dipole moment of the proximal helix. Our gas-phase DFT studies show that the proximal pocket effect significantly enhances CPOs reactivity toward the epoxidation of olefinic substrates. Comparison of models with and without proximal pocket residues shows that with them, the barrier for Cβ-O bond formation is lowered by about ∼4.6 kcal/mol, while Cα-O-Cβ ring closure becomes barrierless. The dipole moment of the proximal helix was estimated to contribute 1/3 of the decrease, while the rest is attributed to the effect of NH-S bonds. The decrease of the reaction barrier correlates with increased electron density transfer to residues of the proximal pocket. The effect is most pronounced on the doublet spin surface and involves a change in the electron-transfer mechanism. A full enzyme QMMM study on the doublet spin surface gives about the same barrier as the gas-phase DFT study. The free-energy barrier was estimated to be in agreement with the experimental results for the CPO-catalyzed epoxidation of styrene.

Proximal Pocket Hydrogen Bonds Significantly Influence the Mechanism of Chloroperoxidase Compound I Formation.

The influence of backbone hydrogen bonds to the sulfur atom of the proximal thiolate (NH···S hydrogen bonds) on the formation of compound I in chloroperoxidase is investigated with DFT calculations. Reaction profiles for the transformation of the ferric resting state into compound I in the presence of a peroxide substrate are calculated for a model system incorporating the heme and key proximal and distal amino acid residues. We find that NH···S hydrogen bonds (1) reduce the barrier for the formation of compound 0 by 7.6 kcal/mol, (2) increase the stability of compound 0 by 5.2 kcal/mol, (3) reduce the stability of compound I relative to compound 0 by 6.2 kcal/mol, and (4) reduce the stability of protonated compound 0, favoring a hybrid homo-heterolytic relative to a classic heterolytic mechanism for O-O bond scission. In general, the influence of the NH···S hydrogen bonds can be traced to a reduction in the pKa of the heme-bound substrate. We find that the hydrogen bond networks on the proximal and distal sides of the heme function together to modulate the mechanism of reaction. These results confirm and extend long-standing theories that the NH···S hydrogen bonds in heme thiolate proteins influence reactivity by tuning the thiolate push effect.

Remarkably selective NH4+ binding and fluorescence sensing by tripodal tris(pyrazolyl) receptors derived from 1,3,5-triethylbenzene: structural and theoretical insights on the role of ion pairing

A fluorescent sensor for NH4+ based on 1,3,5-triethylbenzene shows remarkable binding and sensing selectivity for NH4+vs. K+. Fluorescence and NMR titrations reveal surprising differences in the sensing properties and binding constants of tris-(3,5-dimethyl)pyrazole 1vs. tris(3,5-diphenyl)pyrazole 2. The roles of ion pairing and solvation are revealed by X-ray and DFT studies.

How the Proximal Pocket May Influence the Enantiospecificities of Chloroperoxidase-Catalyzed Epoxidations of Olefins.

Chloroperoxidase-catalyzed enantiospecific epoxidations of olefins are of significant biotechnological interest. Typical enantiomeric excesses are in the range of 66%–97% and translate into free energy differences on the order of 1 kcal/mol. These differences are generally attributed to the effect of the distal pocket. In this paper, we show that the influence of the proximal pocket on the electron transfer mechanism in the rate-limiting event may be just as significant for a quantitatively accurate account of the experimentally-measured enantiospecificities.

1,3,5-Tris-(4-(iso-propyl)-phenylsulfamoylmethyl)benzene as a potential Am(III) extractant: experimental and theoretical study of Sm(III) complexation and extraction and theoretical correlation with Am(III)

ABSTRACT The problem of legacy alkaline high-level waste (HLW) both in the US and Russia, as a result of weapons production, has prompted studies of ligands for extraction of actinides, that could be possibly used in the future together with Cs extractants for combined HLW extraction processes. The tripodal trisulphonamide ligand 1,3,5-tris-(4-(iso-propyl)-phenylsulfamoylmethyl)benzene (4-iPr-tsa), which has pre-organised functional groups for An(III) binding was synthesised and studied for potential Sm(III) and Am(III) binding and extraction by theoretical (DFT) and experimental (extraction) methods (for Sm(III) only). Both theory and experiments suggest that even though this family of ligands shows promise for Ln(III) and An(III) binding with minima for complex formation, complexation is competing with hydrolysis, and extraction is only feasible in alkaline solutions, in the presence of high concentrations of nitrate ions. Nevertheless, up to 51.8% of Sm(III) was removed under optimal conditions (NaOHu2009=u20092u2009×u200910−4u2009M, NaNO3u2009=u20090.1u2009M, [Sm]initu2009=u20095u2009×u200910−5u2009M). Quantum chemical calculations demonstrate that the extraction of Sm(III) and Am(III) from the aqueous phase in the form of [M·(H2O)4·(OH)2·(NO3)] to the organic phase in the form of [M·4-iPr-tsa·(H2O)3] is thermodynamically favourable. Theory also shows that Sm(III) is a reasonably good surrogate for Am(III), as the optimised structures of the Sm and Am complexes show remarkable similarities. Even though the ligand was designed with the goal of introducing favourable cation–arene interactions, along with the expected N-binding mode of the ligand in its deprotonated form, it was found that these cation–arene interactions are rather weak in this case, and coordination with O atoms of the sulphonamide, and external water molecules, is favoured instead. GRAPHICAL ABSTRACT

Proximal Pocket Controls Alkene Oxidation Selectivity of Cytochrome P450 and Chloroperoxidase toward Small, Nonpolar Substrates

This paper examines the influence of the proximal pockets of cytochrome P450CAM and chloroperoxidase (CPO) on the relative favorability of catalytic epoxidation and allylic hydroxylation of olefins, a type of alkene oxidation selectivity. The study employs quantum mechanical models of the active site to isolate the proximal pockets influence on the barrier for the selectivity-determining step for each reaction, using cyclohexene and cis-β-methylstyrene as substrates. The proximal pocket is found to preference epoxidation by 2-5 kcal/mol, the largest value being for CPO, converting the active heme-thiolate moiety from being intrinsically hydroxylation-selective to being intrinsically epoxidation-selective. This theoretical study, the first to correctly predict these enzymes preference for epoxidation of allylic substrates, strongly suggests that the proximal pocket is the key determinant of alkene oxidation selectivity. The selectivity for epoxidation can be rationalized in terms of the proximal pockets modulation of the thiolates electron push and consequent influence on the heme redox potential and the basicity of the trans ligand.