Uncoupled human flavin-containing monooxygenase 3 releases superoxide radical in addition to hydrogen peroxide

Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolizing enzyme capable of performing N- or S-oxidation using the C4a-hydroperoxy intermediate. In this work, we employ both wild type hFMO3 as well as an active site polymorphic variant (N61S) to unravel the uncoupling reactions in the c...

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Bibliographic Details
Published in:Free radical biology & medicine 2019-12, Vol.145, p.250-255
Main Authors: Catucci, Gianluca, Gao, Chongliang, Rampolla, Giulia, Gilardi, Gianfranco, Sadeghi, Sheila J.
Format: Article
Language:English
Subjects:
Flavin-containing monooxygenase
Hydrogen peroxide
N61S
Polymorphic variant
Superoxide radical
Uncoupling
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Summary:Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolizing enzyme capable of performing N- or S-oxidation using the C4a-hydroperoxy intermediate. In this work, we employ both wild type hFMO3 as well as an active site polymorphic variant (N61S) to unravel the uncoupling reactions in the catalytic cycle of this enzyme. We demonstrate that in addition to H2O2 this enzyme also produces superoxide anion radicals as its uncoupling products. The level of uncoupling was found to vary between 50 and 70% (WT) and 90–98% (N61S) for incubations with NADPH and benzydamine over a period of 5 or 20 min, respectively. For the first time, we were able to follow the production of the superoxide radical in hFMO3, which was found to account for 13–18% of the total uncoupling of this human enzyme. Moreover, measurements in the presence or absence of the substrate show that the substrate lowers the level of uncoupling only related to the H2O2 and not the superoxide radical. This is consistent with the entry point of the substrate in this enzyme's catalytic cycle. These findings highlight the importance of the involvement of hFMO3 in the production of radicals in the endoplasmic reticulum, as well as the relevance of single-nucleotide polymorphism leading to deleterious effects of oxidative stress. [Display omitted] •Purified human flavin-containing monooxygenase produces reactive oxygen species.•Both hydrogen peroxide and superoxide radical are detected.•N61S polymorphic variant is more uncoupled than the wild type enzyme.
ISSN:0891-5849
1873-4596
DOI:10.1016/j.freeradbiomed.2019.09.038