title: Mechanism of Oxygen Activation in a Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer.
abstract: Understanding how flavin-dependent enzymes activate oxygen for their oxidation and oxygenation reactions is one of the most challenging issues in flavoenzymology. Density functional calculations and transient kinetics were performed to investigate the mechanism of oxygen activation in the oxygenase component (C2) of p-hydroxyphenylacetate 3-hydroxylase (HPAH). We found that the protonation of dioxygen by His396 via a proton-coupled electron transfer mechanism is the key step in the formation of the triplet diradical complex of flavin semiquinone and (•)OOH. This complex undergoes intersystem crossing to form the open-shell singlet diradical complex before it forms the closed-shell singlet C4a-hydroperoxyflavin intermediate (C4aOOH). Notably, density functional calculations indicated that the formation of C4aOOH is nearly barrierless, possibly facilitated by the active site arrangement in which His396 positions the proximal oxygen of the (•)OOH in an optimum position to directly attack the C4a atom of the isoalloxazine ring. The nearly barrierless formation of C4aOOH agrees well with the experimental results; based on transient kinetics and Eyring plot analyses, the enthalpy of activation for the formation of C4aOOH is only 1.4 kcal/mol and the formation of C4aOOH by C2 is fast (∼10(6) M(-1) s(-1) at 4 °C). The calculations identified Ser171 as the key residue that stabilizes C4aOOH by accepting a hydrogen bond from the H(N5) of the isoalloxazine ring. Both Ser171 and Trp112 facilitate H2O2 elimination by donating hydrogen bonds to the proximal oxygen of the OOH moiety during the proton transfer. According to our combined theoretical and experimental studies, the existence of a positively charged general acid at the position optimized for facilitating the proton-coupled electron transfer has emerged as an important catalytic feature for the oxygen activation process in flavin-dependent enzymes.
authors.affiliation: §Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, †Institute for Innovative Learning, and ∥Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Surawit
authors.initials: S
authors.lastname: Visitsatthawong
authors.affiliation: §Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, †Institute for Innovative Learning, and ∥Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Pirom
authors.initials: P
authors.lastname: Chenprakhon
authors.affiliation: §Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, †Institute for Innovative Learning, and ∥Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Pimchai
authors.initials: P
authors.lastname: Chaiyen
authors.affiliation: §Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, †Institute for Innovative Learning, and ∥Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Panida
authors.initials: P
authors.lastname: Surawatanawong
mesh_terms: D020134:Catalytic Domain; D004579:Electron Transport; D004789:Enzyme Activation; D005415:Flavins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism*; D011522:Protons; D011789:Quantum Theory; D013696:Temperature; D013816:Thermodynamics
chemical_list: C531259:C4a-hydroperoxyflavin; D005415:Flavins; D011522:Protons; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D010100:Oxygen