Abstract
© 2021 The Authors. Published by American Chemical Society.Flavin as a cofactor is an extremely versatile molecule that participates in a wide range of biochemical reactions. A special characteristic of the flavin cofactor, unique for a metal free cofactor, is its ability to react in its reduced form with molecular oxygen. This is exploited in flavoprotein oxidases and monooxygenases. The flavin-mediated reduction of dioxygen can lead to various reactive oxygen species (ROS), primarily hydrogen peroxide and superoxide. No systematic analysis of the formation of such reduced oxygen species produced by flavoprotein oxidases and monooxygenases had been performed before. In this work, we investigated which ROS are formed by several prototype flavoprotein oxidases and monooxygenases: Phenylacetone monooxygenase (TfPAMO), an engineered TfPAMO variant that acts as an NADPH oxidase (TfPAMO C65D), eugenol oxidase (RjEUGO), and 5-hydroxymethyl furfural oxidase (MHMFO). The formed amounts of superoxide and hydrogen peroxide were measured under various conditions (different substrate concentrations, pH values and cosolvents). Interestingly, all flavoenzymes were found to produce, except for hydrogen peroxide, significant amounts of superoxide. Moreover, increased superoxide levels were measured at higher pH, which could be indicative for a pH-sensitive caged radical pair dissociation. To probe the effect of ROS formation on biocatalytic performance, conversions catalyzed by TfPAMO or RjEUGO, with or without catalase, were monitored. This revealed that catalase has a beneficial effect. No detrimental effect of the accumulation of superoxide on biocatalysis could be demonstrated. The results reveal that formation of ROS by flavoenzymes is highly dependent on the experimental conditions used. The results provide a better insight into the mechanism by which ROS is formed in flavoenzymes and may help studies or applications in which ROS formation should be promoted or minimized for industrial biocatalysis.
Original language | English |
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Pages (from-to) | 4948-4959 |
Journal | ACS Sustainable Chemistry and Engineering |
Volume | 11 |
Issue number | 13 |
DOIs | |
Publication status | Published - 2021 |
Externally published | Yes |
Funding
A Ph.D. fellowship from ANID (ex-CONICYT, Comision Nacional de Investigacion Cientifica y Tecnologica de Chile) and a scholarship from Pontificia Universidad Católica de Chile and from the Faculty of engineering of Pontificia Universidad Católica de Chile to A.G.-S. is acknowledged. This work was supported by the Institute for Biological and Medical Engineering from Pontificia Universidad Catolica de Chile and CORFO 14ENI2–26862. A Ph.D. fellowship from CONICYT and a scholarship from Pontificia Universidad Catolica de Chile to A.G-.S. is also acknowledged.
Funders | Funder number |
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ANID | |
Institute for Biological and Medical Engineering | |
Corporación de Fomento de la Producción | 14ENI2–26862 |
Comisión Nacional de Investigación Científica y Tecnológica | |
Pontificia Universidad Católica de Chile | |
Agencia Nacional de Investigación y Desarrollo |