title: An EPR spin label study of the quinol oxidase, E. coli cytochrome bo3: a search for redox induced conformational changes.
abstract: A search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome bo3, E. coli, has been carried out using site directed nitroxide spin labeling (SDSL) of cysteine residues. These were positioned at R134 and R309, on loops that link helices II and III and VI and VII at the entrances to the D and K proton channels, respectively. The motional characteristics of both labels have been determined using X- and W-band EPR spectroscopy at room temperature in selected redox levels in the reaction sequence of QO with oxygen, namely, the mixed valence carbon monoxide form (COMV), the oxidized (O) and super-oxidized (PM) states. The O to PM step is accompanied by the uptake of protons through the K pathway. We find no evidence for changes in the motional characteristics of either label that are expected to be associated with helical motions at the entrances to the channels. Because kinetic studies of mutants show that the redox gating of protons occurs deep within the D channel close to the heme-copper site, the present study implies that no motion is transmitted to the ends of the helices.
authors.affiliation: School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, UK.
authors.forename: Gaye F
authors.initials: GF
authors.lastname: White
authors.forename: Sarah
authors.initials: S
authors.lastname: Field
authors.forename: Sophie
authors.initials: S
authors.lastname: Marritt
authors.forename: Vasily S
authors.initials: VS
authors.lastname: Oganesyan
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Lai Lai
authors.initials: LL
authors.lastname: Yap
authors.forename: Andromachi
authors.initials: A
authors.lastname: Katsonouri
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thomson
mesh_terms: D018360:Crystallography, X-Ray; D003580:Cytochromes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D011487:Protein Conformation; D013113:Spin Labels
chemical_list: D003580:Cytochromes; D013113:Spin Labels; D010088:Oxidoreductases; C020094:duroquinol oxidase