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.

Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase

“…A view has therefore emerged that any peroxidase, or perhaps any haem protein, that can react with H 2 O 2 and that has a suitable amino acid side chain poised in the right place will, under the right conditions, form a covalent link to the haem. This view is reinforced by other protein engineering work on the CYP4 family of cytochrome P450s, which also contain a covalent link between a carboxy residue and a haem methyl group [2][3][4]. However, from the present study, we can conclude that cysteine can be inert to formation of a bond under oxidative conditions.…”

“…Other examples of unexpected haem-protein links have also come to light. These include the CYP4 family of cytochrome P450s, which contain ester links between the 5-methyl group and a glutamate residue [2][3][4], cyanobacterial haemoglobin, which contains a covalent link between the 2-vinyl group and a histidine residue [5], and the haem chaperone CcmE, which uses a different vinyl-histidine covalent link [6,7].…”

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

“…Heme detection by SDS-PAGE was previously reported [23]. Briefly, SDS-gel electrophoresis was carried out for purified CYP52A21 and CYP4A1 (as a positive control).…”

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

“…One interesting feature of the CYP4 family is that the P450s belonging to this family covalently bind their heme groups via an ester link between heme and a carboxylic acid (Henne et al, 2001b;Ortiz De Montellano, 2008). In CYP4B1, this ester link is formed via the conserved I-helix residue Glu310 .…”