Understanding the Mechanism of Chlorite Dismutation by the Heme Dependent Enzyme Chlorite Dismutase

Chlorite Dismutase is a heme dependent enzyme that catalyses the decomposition of the substrate chlorite into ClÌ¢åÈ and OÌ¢'_'_. A basic characterization of the protein under steady-state conditions shows a robust enzyme capable of catalyzing the reaction at rapid rates. It was determined using EPR and stopped flow spectroscopy that the enzyme forms the highly oxidizing ferryl-oxo porphyrin ̏‰â cation radical intermediate (Compound 1) upon reaction with peracetic acid. Unlike other heme enzymes, however, when given chlorite as an oxidant, Cld seems specifically poised to carry out the dismutation exclusively. Measurement of the binding affinity for neutral ligands as a function of pH show an enzyme based pKa at 6.4. Resonance Raman spectroscopy at alkaline pH indicates a weak Fe-OH bond suggestive of a strong H-bond donating residue in the distal pocket. With UV/visible spectroscopy this HO- ligation to the heme Fe is observed with a pKa of 8.7. Resonance Ramen of CO bound Cld shows both a weak and a strong Fe-CO stretch at low pH with loss of the weak Fe-CO stretch at high pH. A crystal structure of Cld shows a hydrophobic distal pocket with only one residue, Arg-183, capable of carrying out an H-bond donating role. Together these data suggest that deprotonation of Arg-183 with a pKa = 6.4 is followed by a hydrolysis reaction of the active site at pH 8.9, yielding HOÌ¢åÈ binding to the Fe center and reprotonation of the Arg-183. The catalytic data as a function of pH show a pKa at pH 6.5, likely from the protonation of Arg-183 and suggest a positively charged Arg is needed for properly positioning the anionic ClOÌ¢åÈ within the hydrophobic distal pocket for reaction with Compound I, generating ClÌ¢åÈ and OÌ¢'_'_. The positively charged Arg-183, along with a hydrophobic distal pocket are likely major determinants for the unique reactivity of the enzyme. This research suggests that formation of an electrophilic high valent metal-oxo species in close vicinity to a nucleophilic O-atom containing anion may be a structural motif utilized by biology for the difficult reaction of forming an O-O.