Probing the catalytic cycle of cytochrome P450 for reaction intermediates
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- Tez No: 403395
- Danışmanlar: Prof. Dr. WILLEM H. KOPPENOL
- Tez Türü: Doktora
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2014
- Dil: İngilizce
- Üniversite: Eidgenössische Technische Hochschule Zürich (ETH)
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 123
Özet
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Özet (Çeviri)
Cytochrome P450 (CYP) enzymes are haem-containing mono-oxygenases that catalyze the hydroxylation of non-activated C–H bonds stereo- and regioselectively. The crucial step in substrate oxidation by cytochrome P450 is the insertion of one atom from molecular dioxygen, which is activated by a reduced haem iron, to the substrate. Different high-valent iron intermediates have been proposed as primary oxidant in this crucial step. The candidates are compound 0 (Cpd 0, [FeOOH]2+) and compound I (Cpd I, Fe(IV)=O por+). Due to extremely short half-lives, characterization of those oxidants is not possible under real-time catalytic turnover with standard spectroscopic techniques. Cpd I of CYP119, a thermophilic isozyme of cytochrome P450, has been detected by exploring the peroxide shunt reaction. However, this intermediate has never been convincingly detected in the otherwise well-studied catalytic cycle of the camphor-hydroxylating soluble cytochrome P450 isozyme from the soil bacterium Pseudomonas putida (P450cam, CYP101). In order to investigate the catalytic cycle of CYP101 under conditions as closely as possible to the natural reaction sequence of the enzyme, when a typical substrate is present in the reaction medium, expression of highly pure CYP101 was an essential step in this thesis. Because of the short half-lives of crucial intermediates, we needed to follow an approach that relies on ultra-fast reduction of the [FeO2]2+ intermediate in the catalytic cycle. We used pulse radiolysis at 4° C to deliver an electron rapidly to the CYP101[FeO2]2+ intermediate in the presence of substrate (camphor), thereby circumventing the slow enzymatic reduction. An important achievement of this thesis is the imitation of the catalytic cycle of CYP101 under quantitative control with pulse radiolysis, without damaging the enzyme. Moreover, we have clearly shown that the Cpd 0 signature at 440 nm builds up within 5 μs, and that the typical product (5-hydroxycamphor) is formed after pulse irradiation. In Chapter 3, the analysis of the CYP101 catalytic cycle by pulse radiolysis, product quantification, and kinetic isotope effect studies to resolve intermediates of the catalytic mechanism, are reported in detail. Chapter 4 focuses on spectroscopic studies of the CYP119 catalytic cycle with the same approach described in Chapter 3 and used on CYP101, i.e. reduction of [FeO2]2+ intermediate by pulse-irradiation at 4° C, and by exploring the shunt reaction with m-chloroperoxybenzoic acid as oxygen source. Although we observed an increase in absorbance expected for Cpd I formation after the shunt reaction, carried out by the stopped-flow technique, we did not detect any spectral fingerprint of Cpd I in the pulse radiolysis study. Electron paramagnetic resonance (EPR) is a useful tool to characterize Cpd I intermediates in haem enzymes and corresponding model compounds. Cpd I, Fe(IV)=O por+ has been described as a spin system combination with S=1 from oxido-iron(IV) and with S'=1/2 from the porphyrin radical cation, both weakly coupled. In Chapter 5, we show the preparation of a model Cpd I from an iron porphyrin with 2,6-di-tert-butylphenol substituents at the meso-aryl positions of the porphyrin ring (FeR4P). We characterized its Cpd I by applying continuous wave (CW) EPR, pulse-EPR, and two-dimensional hyperfine sublevel correlation (HYSCORE) methods. When we prepared the Cpd I model in acetonitrile:2-methylbutane solvent mixture, we could not observe a paramagnetic resonance signal arising from a radical. A comprehensive result of this work is that the pulse radiolysis approach to identify crucial reactive intermediates in cytochrome P450 catalytic cycles can be combined with EPR to confirm and extend the spectral evidence obtained after pulse irradiation.
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