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Detection and Characterization of Heme-Thiolate Compound II from AaeAPO PeroxygenaseOpen image in new window

  • Xiaoshi WangEmail author
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Part of the Springer Theses book series (Springer Theses)

Abstract

A kinetic and spectroscopic characterization of the unusual, high-valent Cys–FeIV–OH intermediate (compound II) from AaeAPO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. AaeAPO-II was generated by the reaction of ferric enzyme with mCPBA in the presence of nitroxides and detected with the use of fast-mixing UV-vis stopped-flow spectroscopy. The nitroxides served as a selective reductant of AaeAPO-I, reacting only slowly with AaeAPO-II. AaeAPO-II displayed a characteristic split Soret UV-vis spectrum (370 and 428 nm). Rapid-mixing, pH-jump spectrophotometry revealed a basic pK a of 10 for the FeIV–O–H of AaeAPO-II, indicating that AaeAPO-II is protonated under typical turnover conditions. Kinetic characterization showed that AaeAPO-II is unusually reactive toward a panel of benzylic C–H and phenolic substrates, with second-order rate constants in the range of 10–107 M−1 s−1. Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the Fe-oxo, AaeAPO compound I, thus providing driving force of C–H bonds oxygenation.

Keywords

Phenol Oxidation Kinetic Characterization Nitrosyl Complex Hydrogen Atom Abstraction Cyclohexane Carboxylic 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.PhiladelphiaUSA

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