Unique Biradical Intermediate in the Mechanism of the Heme Enzyme Chlorite Dismutase

Abstract

The heme enzyme chlorite dismutase (Cld) catalyzes O–O bond formation as part of the conversion of the toxic chlorite (ClO 2 – ) to chloride (Cl – ) and molecular oxygen (O 2 ). Enzymatic O–O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae ( Ao Cld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. Ao Cld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, Ao Cld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at k obs = 2–5 × 10 4 s –1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of Ao Cld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety’s reorganization and thereby maximize the enzyme’s catalytic efficiency.

Roles of High-Valent Hemes and pH Dependence in Halite Decomposition Catalyzed by Chlorite Dismutase fromDechloromonas aromatica

“…Thus, the possibility of a one-electron pathway cannot be discounted on the basis of this EPR alone. A recent hyperquench EPR study provided no evidence of an S = 1 Fe IV O heme exchanged coupled to a S = OCl• at pH 7.0 and 20 K …”

“…No spectroscopic evidence of a Da Cld–OBrO – complex was observed in this work. However, the association of resting Ao Cld with ClO 2 – was recently reported to form a 6cHS Fe­(III) complex within the dead time of a microsecond stopped-flow experiment and decay on the pre-steady-state time scale . The most widely accepted hypothesis is that the halite coordinates through one of its terminal oxo groups to the coordinately unsaturated heme of the resting enzyme.…”

“…Based on experimental and quantum computational studies, both homolytic and heterolytic O–ClO – (hereinafter O–Cl) bond cleavage mechanisms have been proposed for catalytic ClO 2 – decomposition by Clds (Scheme ). ,,,− In the homolytic pathway, accumulation of Cpd II and the one-electron reduction product of ClO 2 – , OCl•, are expected. Currently, there is no experimental evidence for the accumulation of OCl•.…”

“…The three mechanistic pathways proposed to date for catalytic ClO 2 – decomposition by Cld are illustrated in Scheme . Two of the three involve heterolytic cleavage of the O–ClO – bond following formation of the enzyme substrate complex. ,, The earliest suggested pathway leads to formation of Cpd I and hypochlorite (OCl – ) within the catalytic heme pocket. This geminate pair then recombines to yield product through catalytic O–O bond formation as a prelude to O 2 evolution.…”

“…This geminate pair then recombines to yield product through catalytic O–O bond formation as a prelude to O 2 evolution. In a recently proposed mechanism, reaction with the substrate introduces two holes into the protein as an exchange coupled biradical dication wherein the heme iron remains in its ferric oxidation state . This pathway also involves a rebound of the OCl – fragment to form the O–O bond.…”