title: A conserved glutamic acid in helix VI of cytochrome bo3 influences a key step in oxygen reduction.
abstract: We have compared the reactions with dioxygen of wild-type cytochrome bo3 and a mutant in which a conserved glutamic acid at position-286 of subunit I has been changed to an alanine. Flow-flash experiments reveal that oxygen binding and the rate of heme-heme electron transfer are unaffected by the mutation. Reaction of the fully (3-electron) reduced mutant cytochrome bo3 with dioxygen yields a binuclear center which is substantially in the P (peroxy) state, not the well-characterized F (oxyferryl) state which is the product of the reaction of the fully reduced wild-type enzyme with dioxygen [Puustinen, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1545-1548]. These results confirm that proton uptake is important in controlling the later stages of dioxygen reduction in heme-copper oxidases and show that E286 is an important component of the channel that delivers these protons to the active site.
authors.affiliation: Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norfolk, United Kingdom. n.watmough@uea.ac.uk
authors.forename: N J
authors.initials: NJ
authors.lastname: Watmough
authors.forename: A
authors.initials: A
authors.lastname: Katsonouri
authors.forename: R H
authors.initials: RH
authors.lastname: Little
authors.forename: J P
authors.initials: JP
authors.lastname: Osborne
authors.forename: E
authors.initials: E
authors.lastname: Furlong-Nickels
authors.forename: R B
authors.initials: RB
authors.lastname: Gennis
authors.forename: T
authors.initials: T
authors.lastname: Brittain
authors.forename: C
authors.initials: C
authors.lastname: Greenwood
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects; D017124:Conserved Sequence / Q000235:genetics; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013053:Spectrophotometry
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; D018698:Glutamic Acid