P450の、APO-II (Fe(IV)-OH：たぶんCompound IIと呼んでも差し支えない)の活性に関する論文です。Fe(IV)–OHのpKaは10だそうです。ヒドロキソにも関わらず、この錯体はC–HやO–HからのH•引き抜きを行います。
Compound Iとの活性比較も行っており、Compound Iのほうが活性は高いのですが、Compound Iによるアルカンの酸化より、Compound IIによるアルカンの酸化の速度の方が、より大きな BDE依存性が出ているところが興味深いです。なんでなんでしょう？
- Contributed by John T. Groves, February 19, 2015 (sent for review January 6, 2015; reviewed by John H. Dawson
Significance
The heme-thiolate peroxygenase of Agrocybe aegerita is a remarkably capable biocatalyst and a mechanistic analog of cytochrome P450. The stability of this fungal protein has provided a rare opportunity to study P450-like C−H hydroxylation in a novel and unrelated enzyme. Both APO-I and APO-II have been generated, and their redox potentials have been determined. The ferryl species Cys−S−FeIV−OH (APO-II) has been generated cleanly via reduction of the corresponding APO-I and a basic pKa revealed for the Cys−S−FeIV−OH ⇄ Cys−S−FeIV=O equilibrium. Most significantly, APO-II displays surprisingly high reactivity toward benzylic C−H (bond-dissociation energy 80−86 kcal/mol) and phenolic substrates with rate constants orders of magnitude larger than those of typical peroxidases or model compounds due to the basic ferryl.
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 FeIV−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 10–107 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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