Crystal Structures of Chloroperoxidase with Its Bound Substrates and Complexed with Formate, Acetate, and Nitrate

Abstract

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 Å . This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me 2 SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.

Visible light-driven and chloroperoxidase-catalyzed oxygenation reactions

“…Similar effects have been reported recently for the CPO-catalyzed epoxidation of limonene 4d and can be rationalized by assuming binding of formate to the heme-iron as suggested by crystallographic data. 19 Thus, presence of formate might impair precise positioning of thioanisole within the CPO active site.…”

Characterization of Recombinant Chloroperoxidase, and F103A and C29H/C79H/C87H Mutants

“…Initially, deprotonated Glu183 removes a proton from the incoming HOOH (Kuhnel, Blankenfeldt et al 2006). -anion then binds at the heme producing a ferric-hydroperoxo species, also called compound 0 (Cpd 0) (Denisov, Dawson et al 2007).…”

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

“…The deprotonated Glu183 abstracts a proton from the substrate hydrogen peroxide, and then the formed hydroperoxo-anion binds to the heme iron, yielding a ferric-hydroperoxo species known as Compound 0. Glu183 then protonates the distal oxygen of Cpd 0 and the peroxide O-O is cleaved heterolytically to generate Cpd I and release a water molecule, similar to other peroxidase mechanisms (107,108). CPO is adept in catalyzing a number of chiral oxidations with high yields and high enantioselectivity, in addition to alkyne hydroxylation and heteroatom dealkylation (109)(110)(111)(112)(113)(114).…”