Mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase and 2,4‐diacetyl‐[2H]heme‐substituted horse‐radish peroxidase

• Published in: European journal of biochemistry Vol. 201; no. 2; pp. 507 - 513
• Main Authors: SANDRO, Virginie, DUPUY, Corinne, KANIEWSKI, Jacques, OHAYON, Renée, DÈME, Danielle, VIRION, Alain, POMMIER, Jacques
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 15.10.1991; Blackwell
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catalysis; Deuteroporphyrins; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glucose - metabolism; Glucose Oxidase - metabolism; Horseradish Peroxidase - metabolism; Kinetics; NADP - metabolism; Oxidation-Reduction; Oxidoreductases; NADH; Enzyme; Nucleotide; Hydrogenperoxides; Enzymatic catalysis; Oxidation; Peroxidase; Kinetics; Mechanism of action
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1991.tb16310.x

The mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase (HRP) and 2,4‐diacetyl‐[2H]heme‐substituted horse‐radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used. In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP°, which in turn reacted with O2 producing O2. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence. In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single‐electron transfer, producing NADP°; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two‐electron transfer, producing NADP+. There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/ glucose‐oxidase system, compound II was never formed and a pure O2 adduct of DHRP (compound III) accumulated.