title: Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species.
abstract: The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.
authors.affiliation: Department of Biotechnology, Section Enzymology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Angela
authors.initials: A
authors.lastname: Paulus
authors.forename: Sebastiaan Gijsbertus Hendrik
authors.initials: SG
authors.lastname: Rossius
authors.forename: Madelon
authors.initials: M
authors.lastname: Dijk
authors.forename: Simon
authors.initials: S
authors.lastname: de Vries
mesh_terms: D002384:Catalysis; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011166:Porphyrins / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D011166:Porphyrins; D017382:Reactive Oxygen Species; D006418:Heme; C080647:heme d; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli