title: Covalent linkage of prosthetic heme to CYP4 family P450 enzymes.
abstract: An extensive body of research on the structural properties of cytochrome P450 enzymes has established that these proteins possess a b-type heme prosthetic group which is noncovalently bound at the active site. Coordinate, electrostatic, and hydrogen bond interactions between the protein backbone and heme functional groups are readily overcome upon mild acid treatment of the enzyme, which releases free heme from the protein. In the present study, we have used a combination of HPLC, LC/ESI-MS, and SDS-PAGE techniques to demonstrate that members of the mammalian CYP4B, CYP4F, and CYP4A subfamilies bind their heme in an unusually tight manner. HPLC chromatography of CYP4B1 on a POROS R2 column under mild acidic conditions caused dissociation of less than one-third of the heme from the protein. Moreover, heme was not substantially removed from CYP4B1 under electrospray or electrophoresis conditions that readily release the prosthetic group from other non-CYP4 P450 isoforms. This was evidenced by an intact protein mass value of 59,217 +/- 3 amu for CYP4B1 (i.e., apoprotein plus heme) and extensive staining of this approximately 60 kDa protein with tetramethylbenzidine/H(2)O(2) following SDS-PAGE. In addition, treatment of CYP4B1, CYP4F3, and CYP4A5/7 with strong base generated a new, chromatographically distinct, polar heme species with a mass of 632.3 amu rather than 616.2 amu. This mass shift is indicative of the incorporation of an oxygen atom into the heme nucleus and is consistent with the presence of a novel covalent ester linkage between the protein backbone of the CYP4 family of mammalian P450s and their heme catalytic center.
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington 98195, USA.
authors.forename: K R
authors.initials: KR
authors.lastname: Henne
authors.forename: K L
authors.initials: KL
authors.lastname: Kunze
authors.forename: Y M
authors.initials: YM
authors.lastname: Zheng
authors.forename: P
authors.initials: P
authors.lastname: Christmas
authors.forename: R J
authors.initials: RJ
authors.lastname: Soberman
authors.forename: A E
authors.initials: AE
authors.lastname: Rettie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000378:metabolism; D001560:Benzidines / Q000494:pharmacology; D001665:Binding Sites; D020134:Catalytic Domain; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D000072470:Cytochrome P450 Family 4; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014466:Ultraviolet Rays
chemical_list: D001560:Benzidines; D020033:Protein Isoforms; C021758:3,3',5,5'-tetramethylbenzidine; D006418:Heme; D006639:Histidine; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D000072470:Cytochrome P450 Family 4; C095029:cytochrome P-450 CYP4B1; C040780:leukotriene B4 20-hydroxylase; D042926:Cytochrome P-450 CYP4A