title: Control of C4a-hydroperoxyflavin protonation in the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase.
abstract: The protonation status of the peroxide moiety in C4a-(hydro)peroxyflavin of p-hydroxyphenylacetate-3-hydroxylase can be directly monitored using transient kinetics. The pKa for the wild-type (WT) enzyme is 9.8 ± 0.2, while the values for the H396N, H396V, and H396A variants are 9.3 ± 0.1, 7.3 ± 0.2, and 7.1 ± 0.2, respectively. The hydroxylation efficiency of these mutants is lower than that of the WT enzyme. Solvent kinetic isotope effect studies indicate that proton transfer is not the rate-limiting step in the formation of C4a-OOH. All data suggest that His396 may act as an instantaneous proton provider for the proton-coupled electron transfer that occurs before the transition state of C4a-OOH formation.
authors.affiliation: 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.forename: Duangthip
authors.initials: D
authors.lastname: Trisrivirat
authors.forename: Kittisak
authors.initials: K
authors.lastname: Thotsaporn
authors.forename: Jeerus
authors.initials: J
authors.lastname: Sucharitakul
authors.forename: Pimchai
authors.initials: P
authors.lastname: Chaiyen
mesh_terms: D005415:Flavins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011522:Protons
chemical_list: C531259:C4a-hydroperoxyflavin; D005415:Flavins; D011522:Protons; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase