Mechanism of and exquisite selectivity for O-O bond formation by the heme-dependent chlorite dismutase

Abstract

Chlorite dismutase (Cld) is a heme b-dependent, O-O bond forming enzyme that transforms toxic chlorite (ClO(2)(-)) into innocuous chloride and molecular oxygen. The mechanism and specificity of the reaction with chlorite and alternate oxidants were investigated. Chlorite is the sole source of dioxygen as determined by oxygen-18 labeling studies. Based on ion chromatography and mass spectrometry results, Cld is highly specific for the dismutation of chlorite to chloride and dioxygen with no other side products. Cld does not use chlorite as an oxidant for oxygen atom transfer and halogenation reactions (using cosubstrates guaiacol, thioanisole, and monochlorodimedone, respectively). When peracetic acid or H(2)O(2) was used as an alternative oxidant, oxidation and oxygen atom transfer but not halogenation reactions occurred. Monitoring the reaction of Cld with peracetic acid by rapid-mixing UV-visible spectroscopy, the formation of the high valent compound I intermediate, [(Por(*+))Fe(IV) = O], was observed [k(1) = (1.28 +/- 0.04) x 10(6) M(-1) s(-1)]. Compound I readily decayed to form compound II in a manner that is independent of peracetic acid concentration (k(2) = 170 +/- 20 s(-1)). Both compound I and a compound II-associated tryptophanyl radical that resembles cytochrome c peroxidase (Ccp) compound I were observed by EPR under freeze-quench conditions. The data collectively suggest an O-O bond-forming mechanism involving generation of a compound I intermediate via oxygen atom transfer from chlorite, and subsequent recombination of the resulting hypochlorite and compound I.

Fig. 1.

Mass spectrum of gaseous products from the reaction of 85 nM Cld with 50 mM chlorite in 0.1 M sodium phosphate buffer, pH 7, made in 95% oxygen-18 enriched water (A) and by using enriched 18-O chlorite in ¹⁶OH₂ (B). Signal assignments (m/z): H₂ (2), N (14), H₂O (18), N₂ (28), ¹⁶O₂ (32), ¹⁸O₂ (36).

Fig. 2.

Spectral changes within the first 20 ms after 10 μM Cld was mixed with 20 eq of PAA. Dashed line, ferric enzyme; dotted lines, compound I; solid lines, compound II. (A) Complete spectrum. (B) Visible region on an expanded scale.

Scheme 1.

Fig. 3.

EPR spectra of Cld in ferric form (A), during turnover with chlorite (B), same conditions as B but after warming to near the melting temperature (C), difference spectrum (B − C) (D), simulation of the axial S = 3/2 signal with g_⊥ = 4.5, and g_‖ = 1.99 (E). Concentration of enzyme and chlorite are 6.4 × 10⁻⁶ M and 1.0 M, respectively, in 0.010 M sodium phosphate buffer, pH 7.0. EPR spectra were recorded at 5 K.