Sites of Covalent Attachment of CYP4 Enzymes to Heme: Evidence for Microheterogeneity of P450 Heme Orientation

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• 作者：Brian R. Baer ; Jason T. Schuman ; A. Patricia Campbell ; Matthew J. Cheesman ; Mariko Nakano ; Nicole Moguilevsky ; Kent L. Kunze ; and Allan E. Rettie
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：October 25, 2005
• 年：2005
• 卷：44
• 期：42
• 页码：13914 - 13920
• 全文大小：144K
• 年卷期：v.44,no.42(October 25, 2005)
• ISSN：1520-4995

文摘

Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligandbound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobicinteractions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds hemethrough a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, massspectrometry, and NMR have now been utilized to identify the site of attachment on the heme. NativeCYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structuralheterogeneity may be common among recombinant CYP4 proteins because CYP4A3 exhibited this duality.Attempts to evaluate functional heterogeneity were complicated by the complexity of the system. Thephenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneityin heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems.