Open Access
Mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase and 2,4‐diacetyl‐[ 2 H]heme‐substituted horse‐radish peroxidase
Author(s) -
SANDRO Virginie,
DUPUY Corinne,
KANIEWSKI Jacques,
OHAYON Renée,
DÈME Danielle,
VIRION Alain,
POMMIER Jacques
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb16310.x
Subject(s) - chemistry , peroxidase , superoxide dismutase , catalysis , heme , stereochemistry , diacetyl , superoxide , enzyme , medicinal chemistry , photochemistry , biochemistry
The mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase (HRP) and 2,4‐diacetyl‐[ 2 H]heme‐substituted horse‐radish peroxidase (DHRP) was studied. The roles of the different H 2 O 2 /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 H 2 O 2 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 O 2 producing O 2 . Consequently H 2 O 2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence. In the absence of a mediating molecule, the H 2 O 2 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 H 2 O 2 was produced slowly by the glucose/ glucose‐oxidase system, compound II was never formed and a pure O 2 adduct of DHRP (compound III) accumulated.