Modulating the coupling efficiency of human cytochrome P450 CYP3A4 at electrode surfaces through protein engineering
Modulating the coupling efficiency of human cytochrome P450 CYP3A4 at electrode surfaces through protein engineering
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DOI：
10.1016/j.elecom.2008.09.007
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发表时间：
2008-11-01
影响因子：
5.4
通讯作者：
Gilardi, Gianfranco
中科院分区：
文献类型：
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作者：
Dodhia, Vikash R.;Sassone, Carlo;Gilardi, Gianfranco
本研究表明，利用人工氧化还原链调节人细胞色素P450-CYP3A4的电子流向血红素，可以显著提高其偶联效率和电极表面的催化活性。在基因水平上，将人的细胞色素P3A4基因与细胞色素P102A1的还原酶域(BMR)进行融合，构建了CYP3A4/BMR基因，并与普通黄色弧菌(FLD)进行了基因融合，构建了CYP3A4/FLD基因。在玻碳电极和金电极上对细胞色素P3A4、细胞色素P3A4/BMR和细胞色素P3A4/FLD的直接电化学研究表明，BMR和FLD黄素蛋白降低了向细胞色素P3A4血红素的电子转移速率。由于偶联效率的提高，电催化在两个表面上固定化的CYP3A4/BMR和CYP3A4/FLD的产物生成量显著增加。旋转圆盘电极的研究和过氧化氢的定量与所提出的在固定化的CYP3A4/BMR和CYP3A4/FLD中存在较长寿命的过氧铁物种的机理是一致的。这项研究中的方法有助于更好地了解细胞色素P450在电极表面的解偶联，并有助于构建改进的细胞色素P450生物传感器和生物电催化剂。(C)2008爱思唯尔B.V.保留所有权利。
This study shows that regulating the electron flow to the heme of human cytochrome P450 CYP3A4, using artificial redox chains, can significantly enhance its coupling efficiency and catalytic activity at electrode surfaces. The human CYP3A4 was fused at the genetic level either to the reductase domain of CYP102A1 (BMR) to create the CYP3A4/BMR or to Desulfovibrio vulgaris flavodoxin (FLD) to create the CYP3A4/FLD. Direct electrochemistry of the CYP3A4, CYP3A4/BMR and CYP3A4/FLD on glassy carbon and gold electrodes showed that the BMR and FLD flavo-proteins reduced the electron transfer rate to the CYP3A4 heme. Electrocatalysis resulted in appreciably higher product formation with the immobilized CYP3A4/BMR and CYP3A4/FLD on both surfaces due to an increased coupling efficiency. Rotating disk electrode studies and quantification of hydrogen peroxide were consistent with the proposed mechanism of a longer lived iron-peroxy species in the immobilized CYP3A4/BMR and CYP3A4/FLD. The approaches in this study provide a better understanding of cytochrome P450 uncoupling at electrode surfaces and aids in the construction of improved cytochrome P450 biosensors; and bioelectrocatalysts. (C) 2008 Elsevier B.V. All rights reserved.