title: Role of C-terminal acidic cluster in stabilization of heme spin state of ascorbate peroxidase from Leishmania major.
abstract: Architecture of hemoprotein is solely responsible for different nature of heme coordination. Here we report that substitution of the acidic surface residue Glu226 to Ala in ascorbate peroxidase from Leishmania major alters the 5 coordinate high spin (5cHS) to a 6 coordinate low spin (6cLS) form at pH 7.5. Using UV-visible spectrophotometry, we show that the sixth ligand of heme in Glu226Ala at pH 7.5 is hydroxo. When the pH is decreased to 5.5, a new species of Glu226Ala appeared that had a spectrum characteristic of a 6cHS derivative. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I was not seen in the Glu226Ala in presence of H(2)O(2). Similarly guaiacol, ascorbate and ferrocytochrome c oxidation rate was 10(3) orders less for the Glu226Ala mutants compared to the wild type. These data suggested that surface acidic residue Glu226 might play role in proper maintenance of active site conformation.
authors.affiliation: Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India.
authors.forename: Rajesh K
authors.initials: RK
authors.lastname: Yadav
authors.forename: Subhankar
authors.initials: S
authors.lastname: Dolai
authors.forename: Swati
authors.initials: S
authors.lastname: Pal
authors.forename: Subrata
authors.initials: S
authors.lastname: Adak
mesh_terms: D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D020134:Catalytic Domain; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D040681:Structural Homology, Protein
chemical_list: D006418:Heme; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases