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 P450 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 pocket's 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.

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.

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 CPO's reactivity toward the epoxidation of olefinic substrates.

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.

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.

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.

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.

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.

The conformation of end-groups is one determinant of carotenoid topology suitable for high fidelity molecular recognition: a study of beta- and epsilon-end-groups.

Conformation affects a carotenoid's ability to bind selectively to proteins. We calculated adiabatic energy profiles for rotating the ring end-groups around the C6C7 bond and for flexing of the ring with respect to the polyene chain. The choice of computational methods is important.

1,3-dipolar cycloadditions of trimethylsilyldiazomethane revisited: steric demand of the dipolarophile and the influence on product distribution.

The 1,3-dipolar cycloaddition of trimethylsilyldiazomethane with alpha,beta-unsaturated esters was examined. The resulting 1-pyrazolines isomerize to regioisomeric 2-pyrazolines (a or b) or undergo desilylation (c). Acrylates yield only b or c.

Correlation times and adiabatic barriers for methyl rotation in SNase.

The relation of rotational correlation times to adiabatic rotational barriers for alanine methyl groups in staphylococcal nuclease (SNase) is investigated. The hypothesis that methyl rotational barriers may be useful probes of local packing in proteins is supported by an analysis of ten X-ray crystal structures of SNase mutants. The barrier heights are consistent across a set of ten structures of a native SNase and mutants containing single-point mutations or single or double insertions, most in a ternary SNase complex.

Methyl dynamics in crystalline amino acids: MD and NMR.

Correlation times for rotation of deuterated methyls in crystalline leucine, valine, and cyclo-L-alanyl-L-alanine are calculated with molecular dynamics and compared with NMR data. The simulations distinguish between methyls having different steric environments in the crystal, yielding correlation times differing by a factor of up to 30 for methyls within a given crystal. MD and NMR correlation times agree to within a factor of 2.