Roles of High-valent Hemes and pH Dependence in Halite Decomposition Catalyzed by Chlorite Dismutase from...

The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO₂ ^-) to yield Cl^- and O₂. The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO₂ ^-) with the evolution of O₂ (k _cat = (2.0±0.2)×10² s^-1; k _cat/K _M = (1.2±0.2)×10⁵ M^-1 s^-1 at pH 5.2). Stopped-flow studies of this BrO₂ ^- decomposition as a function of pH show that 1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O₂ evolution in acidic solution, 2) Cpd I and its one-electron reduction product, compound II (Cpd II) are present in varying ratios at intermediate pHs, and 3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a pK of 6.7±0.1 in good agreement with the pK of DaCld activity with ClO₂ ^-. The observation of a protein-based amino acid radical (AA•) whose appearance coincides with that of Cpd II supports the hypothesis that conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having pK 6.7. Examination of reaction mixtures comprising DaCld and ClO₂ ^- by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO₂, which forms in preference to the analogous to AA• in the BrO₂ ^- reaction. Addition of ClO^- to Cpd II did not yield O₂. Together these results are consistent with heterolytic cleavage of the O-BrO^- and O-ClO^- bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O₂ production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.