期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 112, 期 12, 页码 3686-3691出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1503340112
关键词
APO; UPO; compound II; ferryl; HAT
资金
- National Institutes of Health [2R37 GM036298]
- European Union integrated project, Peroxicats and Indox [KBBE-2010-4-265397, KBBE-2013-7-613549]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R37GM036298] Funding Source: NIH RePORTER
A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapidmixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pK(a) of 10.0 for the Fe-IV-O-H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C-H and phenolic substrates, with second-order rate constants for C-H and O-H bond scission in the range of 10-10(7) M-1.s(-1). Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C-H and O-H bond scission.
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