title: Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive.
abstract: 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 rapid-mixing 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 pKa 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.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544; and.
authors.forename: Xiaoshi
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Bio- and Environmental Sciences, International Institute Zittau, Dresden University of Technology, 02763 Zittau, Germany.
authors.forename: René
authors.initials: R
authors.lastname: Ullrich
authors.affiliation: Department of Bio- and Environmental Sciences, International Institute Zittau, Dresden University of Technology, 02763 Zittau, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Hofrichter
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544; and jtgroves@princeton.edu.
authors.forename: John T
authors.initials: JT
authors.lastname: Groves
mesh_terms: D055362:Agrocybe / Q000201:enzymology*; D002244:Carbon / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D019800:Phenol / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013696:Temperature
chemical_list: D013438:Sulfhydryl Compounds; D019800:Phenol; D006418:Heme; D002244:Carbon; D006859:Hydrogen; D006899:Mixed Function Oxygenases; C022333:peroxygenase; D009584:Nitrogen; D010100:Oxygen
keywords: APO; HAT; UPO; compound II; ferryl