Journal
BIOCHEMISTRY
Volume 53, Issue 25, Pages 4084-4086Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi500480n
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Funding
- Thailand Research Fund [RTA5680001, TRG5780122, MRG5580151, RSA5580050]
- Mahidol University
- Development and Promotion of Science and Technology Talent Project
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The protonation status of the peroxide moiety in C4a-(hydro)peroxyflavin of p-hydroxyphenylacetate-3-hydroxylase can be directly monitored using transient kinetics. The pK(a) 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.
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