title: Direct detection of Fe(IV)[double bond]O intermediates in the cytochrome aa3 oxidase from Paracoccus denitrificans/H2O2 reaction.
abstract: We report the first evidence for the formation of the "607- and 580-nm forms" in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280. The pKa of the 607-580-nm transition is 7.5. The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280. Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin pi-cation radical. We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm-1. The former two are assigned to the Fe(IV)[double bond]O stretching mode of the 607- and 580-nm forms, respectively. The 14 cm-1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607-580-nm transition. We suggest that the 804-790 cm-1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site. The 358 cm-1 mode has been found for the first time to accumulate with the 804 cm-1 mode in the peroxide reaction. These results indicate that the mechanism of oxygen reduction must be reexamined.
authors.affiliation: Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece.
authors.forename: Eftychia
authors.initials: E
authors.lastname: Pinakoulaki
authors.forename: Ute
authors.initials: U
authors.lastname: Pfitzner
authors.forename: Bernd
authors.initials: B
authors.lastname: Ludwig
authors.forename: Constantinos
authors.initials: C
authors.lastname: Varotsis
mesh_terms: D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D010103:Oxygen Isotopes; D010231:Paracoccus denitrificans / Q000201:enzymology*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
chemical_list: D010103:Oxygen Isotopes; C066317:ferryl iron; D006861:Hydrogen Peroxide; D007501:Iron; D003576:Electron Transport Complex IV; D010100:Oxygen