Hemozymes peroxidase activity of artificial hemoproteins constructed from the Streptomyces lividans xylanase A and iron(III)-carboxy-substituted porphyrins

Rémy Ricoux, Roger Dubuc, Claude Dupont, Jean Didier Marechal, Aurore Martin, Marion Sellier, Jean Pierre Mahy

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39 Citations (Scopus)

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-α4-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 μM. 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 H 2O2. 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. © 2008 American Chemical Society.
Original languageEnglish
Pages (from-to)899-910
JournalBioconjugate Chemistry
Volume19
Issue number4
DOIs
Publication statusPublished - 1 Jan 2008

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