title: Sites of covalent attachment of CYP4 enzymes to heme: evidence for microheterogeneity of P450 heme orientation.
abstract: Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, mass spectrometry, and NMR have now been utilized to identify the site of attachment on the heme. Native CYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1 from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structural heterogeneity 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. The phenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneity in heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Baer
authors.forename: Jason T
authors.initials: JT
authors.lastname: Schuman
authors.forename: A Patricia
authors.initials: AP
authors.lastname: Campbell
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cheesman
authors.forename: Mariko
authors.initials: M
authors.lastname: Nakano
authors.forename: Nicole
authors.initials: N
authors.lastname: Moguilevsky
authors.forename: Kent L
authors.initials: KL
authors.lastname: Kunze
authors.forename: Allan E
authors.initials: AE
authors.lastname: Rettie
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006418:Heme / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D006418:Heme; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1