Covalent Linkage of Prosthetic Heme to CYP4 Family P450 Enzymes
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- 作者：Kirk R. Henne ; Kent L. Kunze ; Yi-Min Zheng ; Peter Christmas ; Roy J. Soberman ; and Allan E. Rettie
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：October 30, 2001
- 年：2001
- 卷：40
- 期：43
- 页码：12925 - 12931
- 全文大小：84K
- 年卷期：v.40,no.43(October 30, 2001)
- ISSN：1520-4995
文摘
An extensive body of research on the structural properties of cytochrome P450 enzymes hasestablished that these proteins possess a b-type heme prosthetic group which is noncovalently bound atthe active site. Coordinate, electrostatic, and hydrogen bond interactions between the protein backboneand heme functional groups are readily overcome upon mild acid treatment of the enzyme, which releasesfree heme from the protein. In the present study, we have used a combination of HPLC, LC/ESI-MS, andSDS-PAGE techniques to demonstrate that members of the mammalian CYP4B, CYP4F, and CYP4Asubfamilies bind their heme in an unusually tight manner. HPLC chromatography of CYP4B1 on a POROSR2 column under mild acidic conditions caused dissociation of less than one-third of the heme from theprotein. Moreover, heme was not substantially removed from CYP4B1 under electrospray or electrophoresisconditions that readily release the prosthetic group from other non-CYP4 P450 isoforms. This was evidencedby an intact protein mass value of 59 217 ± 3 amu for CYP4B1 (i.e., apoprotein plus heme) and extensivestaining of this ~60 kDa protein with tetramethylbenzidine/H2O2 following SDS-PAGE. In addition,treatment of CYP4B1, CYP4F3, and CYP4A5/7 with strong base generated a new, chromatographicallydistinct, polar heme species with a mass of 632.3 amu rather than 616.2 amu. This mass shift is indicativeof the incorporation of an oxygen atom into the heme nucleus and is consistent with the presence of anovel covalent ester linkage between the protein backbone of the CYP4 family of mammalian P450s andtheir heme catalytic center.