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

Abstract

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 enzyme’s versatility: catalytic reactivity is tuned by flexibility of the distal pocket.

Nuclear Magnetic Resonance Spectroscopic and Computational Investigations of Chloroperoxidase Catalyzed Regio- and Enantio-Selective Transformations

“…contribute -6.6 to -9.4 kJ/mol per residue to the formation of CPO-indole complex (see Table 2.4). This agrees with reports that these hydrophobic residues interact significantly with hydrophobic substrates such as cis-β-methylstyrene [107,108] and anthracene [176].…”

“…Although the use of computational method for the study of CPO is limited at status quo, this computational method is promising because of the rapid-growing computer technology. Recently, Morozov et al proposed that the simple steric considerations from the hydrophobic core (residues Phe 103, Ile 179, Val 182, and Phe 186) around the oxyferryl heme center govern the stereoselectivity of CPO [107,108], based on the molecular model of cis-β-methylstyrene, a substrate can be oxidized enantioselectively by CPO and H 2 O 2 .…”

“…In 1998, Sundaramoorthy, et al first used computer simulations to address which factors give rise to the pronounced enantioselectivity in epoxidation reactions [27] and suggested that the high enantioselective product is related to favorable interactions between the substrates and Glu 183. Futhermore, according to molecular dynamic simulations of CPO-cis-β-methylstyrene complex, Morozov, et al proposed that enantiomer is likely due to a subtle balance of steric interactions caused by residues Phe 103, Ile 179, Val 182, and Phe 186 around the oxyferyl heme center [107,108].…”

The Influence of the Proximal Amide Hydrogen Bonds and the Proximal Helix Dipole on the Catalytic Activity of Chloroperoxidase

“…Of particular interest to our group is the epoxidation of olefinic substrates. The Chatfield research group has focused on the epoxidation of cis-β-methylstyrene (CBMS), 13,42 as CPO is capable of catalyzing this substrate to an enantiomeric excess of 96%, making CBMS an excellent substrate for determining the structural features responsible for enantioselectivity. 39 Additionally, CBMS has been used in prior experimental and computational studies.…”

“…The imidazole side-chain nitrogens of His105 form hydrogen bonds with the carboxylate side chain of Glu183 and the backbone carbonyl oxygen of Asp106. This set of hydrogen bonds influences the effective pKa of the Glu183 carboxylate side chain, which participates directly in the formation of Cpd I. Additionally, His105 participates in a proton-shuttle during the acidbase catalysis of Cpd I. Molecular dynamics (MD) simulations 13 have shown that the distal pocket changes character from polar (peroxidase-like) to hydrophobic (P450-like)…”