title: Hemozymes peroxidase activity of artificial hemoproteins constructed from the Streptomyces lividans xylanase A and iron(III)-carboxy-substituted porphyrins.
abstract: To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-alpha4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 microM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV-visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.
authors.affiliation: Institut de Chimie Moléculaire et des Matériaux d'Orsay, UMR 8182 CNRS, Laboratoire de Chimie Bioorganique et Bioinorganique, Bât. 420, Université Paris XI, 91405 Orsay Cedex, France.
authors.forename: Rémy
authors.initials: R
authors.lastname: Ricoux
authors.forename: Roger
authors.initials: R
authors.lastname: Dubuc
authors.forename: Claude
authors.initials: C
authors.lastname: Dupont
authors.forename: Jean-Didier
authors.initials: JD
authors.lastname: Marechal
authors.forename: Aurore
authors.initials: A
authors.lastname: Martin
authors.forename: Marion
authors.initials: M
authors.lastname: Sellier
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Mahy
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003962:Dianisidine / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000378:metabolism*; D005290:Ferric Compounds / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D011166:Porphyrins / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D048170:Streptomyces lividans / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D005290:Ferric Compounds; D006420:Hemeproteins; D007093:Imidazoles; D011166:Porphyrins; C029899:imidazole; D006861:Hydrogen Peroxide; D010544:Peroxidases; D043364:Endo-1,4-beta Xylanases; D003962:Dianisidine