Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5

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• 作者：Chunsheng Zhao ; Qiuxia Gao ; Arthur G. Roberts ; Scott A. Shaffer ; Catalin E. Doneanu ; Song Xue ; David R. Goodlett ; Sidney D. Nelson ; William M. Atkins
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：November 27, 2012
• 年：2012
• 卷：51
• 期：47
• 页码：9488-9500
• 全文大小：646K
• 年卷期：v.51,no.47(November 27, 2012)
• ISSN：1520-4995

文摘

Cytochrome b₅ (cyt b₅) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b₅ modulation of CYP catalysis. Both cyt b₅ and apo b₅ are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b₅ with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b₅ with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b₅ and apo b₅ were predicted to bind to the same groove on CYP3A4 with close contacts to the B鈥揃鈥?loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b₅ binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b₅ and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b₅ can dock with CYP3A4 in a manner analogous to that of holo b₅, so electron transfer from cyt b₅ is not required for its effects.