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

Karin Kühnel,Wulf Blankenfeldt,James Terner,Ilme Schlichting

doi:10.1074/jbc.m603166200

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 A. 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 (Me2SO) 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.

Highlights

Reactions can be performed in a stereoselective manner by CPO [7]
Of particular interest for commercial applications is the enantioselective formation of epoxides from alkenes [9, 10], because CPO utilizes H2O2, whereas P450 enzymes depend on both molecular oxygen and electron transfer proteins
CPO is reactive at low pH with an optimum at pH 2.8 [1], which is likely due to the high redox potential required to oxidize a chloride anion, and a glutamic acid residue instead of a histidine is used as a catalytic acid base

Summary

EXPERIMENTAL PROCEDURES

Co-crystallization with Halides—CPO was expressed and purified as described previously [25]. Crystals grown in mother liquor containing KBr were transferred into a soaking solution containing 200 mM CPD, 10% Me2SO, 50 mM KBr, 22% PEG 3000, and 0.1 M citrate at pH 3.6 and were incubated for 15 min at 20 °C. Crystals were subsequently moved into a cryoprotectant containing 15% ethylene glycol, 50 mM KCN, 200 mM CPD, 10% Me2SO, 50 mM KBr, 22% PEG 3000, and 0.1 M sodium citrate at pH 6 and cryogenically cooled. The acetate complex was prepared by soaking CPO crystals grown in the presence of 50 mM KBr in a solution containing 300 mM acetic acid, 50 mM KBr, 22% PEG 3000, and 0.1 M citrate at pH 2 for 10 min at 20 °C. Crystals were transferred into a cryoprotectant consisting of mother liquor supplemented with either 15% ethylene glycol, or 10% (w/v) xylitol and 10% (w/v) sucrose, and cryo-cooled in liquid nitrogen. Figures were prepared with Molscript [32], Bobscript [33], and Pymol.

RESULTS

Root mean square deviations

Ethylene glycol

DISCUSSION