Photooxidation of cytochrome P450-BM3
Photooxidation of cytochrome P450-BM3
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DOI：
10.1073/pnas.1012381107
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发表时间：
2010-11-02
影响因子：
11.1
通讯作者：
Cheruzel, Lionel
中科院分区：
文献类型：
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作者：
Ener, Maraia E.;Lee, Young-Tae;Cheruzel, Lionel
高价铁氧物种被认为是加氧酶和过氧化物酶催化循环的中间体。一个有吸引力的路线，这些铁氧中间体涉及激光闪光淬火氧化亚铁血红素，证明了我们的工作的铁（化合物II）和铁卟啉自由基阳离子（化合物I）辣根过氧化物酶的中间体。将这项工作扩展到包括细胞色素P450-BM 3（CYP 102 A1）需要将Ru（II）光敏剂共价连接到血红素附近的非天然半胱氨酸（Ru（K97 C）（II）-Fe（P450）（III）），以促进电子从Fe（III）卟啉转移到光生Ru（III）。Ru（K97 C）（II）-Fe（P450）（III）缀合物通过X射线晶体学（2.4埃分辨率; Ru-Fe距离，24埃）进行结构表征。闪淬氧化的铁-水合血红素产生的Fe（IV）-氢氧化物物种（化合物II）在2毫秒内。三个单氧化P450-BM 3中间体的差异光谱从动力学建模的瞬态吸收数据结合广义奇异值分解分析和多指数拟合。
High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru(II) photosensitizer to a nonnative cysteine near the heme (Ru(K97C)(II)-Fe(P450)(III)), in order to promote electron transfer from the Fe(III) porphyrin to photogenerated Ru(III). The Ru(K97C)(II)-Fe(P450)(III) conjugate was structurally characterized by X-ray crystallography (2.4 angstrom resolution; Ru-Fe distance, 24 angstrom). Flash-quench oxidation of the ferric-aquo heme produces an Fe(IV)-hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.