Pirom ChenprakhonDuangthip TrisriviratKittisak ThotsapornJeerus SucharitakulPimchai ChaiyenMahidol UniversityChulalongkorn University2018-11-092018-11-092014-07-01Biochemistry. Vol.53, No.25 (2014), 4084-408615204995000629602-s2.0-84903740934https://repository.li.mahidol.ac.th/handle/20.500.14594/33247The 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. © 2014 American Chemical Society.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1021/bi500480n