title: An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.
abstract: The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.
authors.affiliation: Department of Molecular Biology, University of California, Irvine 92717-3900, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Bonagura
authors.forename: M
authors.initials: M
authors.lastname: Sundaramoorthy
authors.forename: H S
authors.initials: HS
authors.lastname: Pappa
authors.forename: W R
authors.initials: WR
authors.lastname: Patterson
authors.forename: T L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D000818:Animals; D001483:Base Sequence; D002412:Cations; D018360:Crystallography, X-Ray; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006736:Horses; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D002412:Cations; D014364:Tryptophan; D003578:Cytochrome-c Peroxidase