Biochemical Evidence for Heme Linkage through Esters with Asp-93 and Glu-241 in Human Eosinophil Peroxidase

2002

103

The prosthetic heme group in the CYP4A family of cytochrome P450 enzymes is covalently attached to an I-helix glutamic acid residue. This glutamic acid is conserved in the CYP4 family but is absent in other P450 families. As shown here, the glutamic acid is linked, presumably via an ester bond, to a hydroxyl group on the heme 5-methyl group. Mutation of the glutamic acid to an alanine in CYP4A1, CYP4A3, and CYP4A11 suppresses covalent heme binding. In wild-type CYP4A3 68% of the heme is covalently bound to the heterologously expressed protein, but in the CYP4A3/E318D mutant, 47% of the heme is unchanged, 47% is present as noncovalently bound 5-hydroxymethylheme, and only 6% is covalently bound to the protein. In the CYP4A3/ E318Q mutant, the majority of the heme is unaltered, and <2% is covalently linked. The proportion of covalently bound heme in the recombinant CYP4A proteins increases with time under turnover conditions. The catalytic activity is sensitive in some, but not all, CYP4A enzymes to the extent of covalent heme binding. Mutations of Glu318 in CYP4A3 decrease the apparent k cat values for lauric acid hydroxylation. The key conclusions are that (a) covalent heme binding occurs via an ester bond to the heme 5-methyl group, (b) covalent binding of the heme is mediated by an autocatalytic process, and (c) fatty acid oxidation is sensitive in some CYP4A enzymes to the presence or absence of the heme covalent link.

mentioning

confidence: 87%

Autocatalytic Mechanism and Consequences of Covalent Heme Attachment in the Cytochrome P4504A Family

LeBrun

Hoch

2002

103

“…Spectra were externally calibrated using four identified molecular ions of a standard peptide mixture, yielding an accuracy of 0.01-0.05%. Non-heme peptides were observed as MH ϩ species, but heme peptides were observed as M ϩ species as described previously for lactoperoxidase and eosinophil peroxidase heme peptides (32). The intact heme peptides were observed along with the free peptides resulting from cleavage of the peptide-heme linkage in the instrument.…”

Section: Isolation and Characterization Of Heme-containingmentioning

confidence: 98%

“…The heme group has been shown to be cross-linked to the protein in lactoperoxidase (27,28), myeloperoxidase (29 -31), eosinophil peroxidase (32), and thyroid peroxidase (33). The heme prosthetic group in lactoperoxidase, the only one that has been fully characterized, is iron 1,5-bis(hydroxymethyl)-3,8-dimethyl-2,4-divinylporphyrin-6,7-dipropionic acid.…”

Section: Fig 5 Comparison Of Lc Analysis Of Cyp4a1 Before (A) and Amentioning

confidence: 99%

Covalently Linked Heme in Cytochrome P4504A Fatty Acid Hydroxylases

Hoch

2001

Three independent experimental methods, liquid chromatography, denaturing gel electrophoresis with heme staining, and mass spectrometry, establish that the CYP4A class of enzymes has a covalently bound heme group even though the heme is not cross-linked to the protein in other P450 enzymes. Covalent binding has been demonstrated for CYP4A1, -4A2, -4A3, -4A8, and -4A11 heterologously expressed in Escherichia coli. However, the covalent link is also present in CYP4A1 isolated from rat liver and is not an artifact of heterologous expression. The extent of heme covalent binding in the proteins as isolated varies and is substoichiometric. In CYP4A3, the heme is attached to the protein via an ester link to glutamic acid residue 318, which is on the I-helix, and is predicted to be within the active site. This is the first demonstration that a class of cytochrome P450 enzymes covalently binds their prosthetic heme group. Heme1 is the essential prosthetic group of many important proteins, including the hemoglobins, catalases, peroxidases, and cytochromes. In cytochrome P450 enzymes, the heme sits deep inside the protein with its iron ligated to a cysteine thiolate group and the sixth iron coordination site occupied by a water molecule in the substrate-free state (1). This coordination arrangement gives cytochrome P450 enzymes their unique spectral and catalytic properties. These enzymes are involved in many vital processes, including the biosynthesis of steroids and other lipophilic physiological effectors, the metabolism of drugs, and the degradation of xenobiotics. A unique feature of the cytochrome P450 enzymes is their ability to catalyze the hydroxylation of unactivated hydrocarbons under physiological conditions rather than the extreme conditions required for the reaction to occur under uncatalyzed conditions. Sequence homologies within the heme-binding region of the protein suggest that, despite the existence of numerous P450 forms with different substrate specificities, individual P450 enzymes share a common catalytic mechanism of oxygen activation.Work in this laboratory has focused on the P450 enzymes of family 4 (CYP4), especially the four known rat isoforms (CYP4A1, -4A2, -4A3, and -4A8) and the single characterized human isoform (CYP4A11) (2-5). The first three rat enzymes are present in liver and are induced by clofibrate, whereas all four enzymes are constitutively expressed in the rat kidney. The enzymes of the CYP4 family are distinguished by their unique ability to hydroxylate preferentially the thermodynamically disfavored terminal methyl group of medium and long chain fatty acids, prostaglandins, and other eicosanoids. Although other P450 enzymes hydroxylate fatty acids at internal positions of the hydrocarbon chain (6 -8), only the CYP4 enzymes preferentially hydroxylate the energetically disfavored terminal methyl group. Site-directed mutagenesis studies have begun to shed some light on the mechanism by which these enzymes achieve this specificity (4, 5, 9), but the detailed mechanism remains obscur...

“…We established earlier that the prosthetic group is bound to the protein in LPO via a self-activating process in which noncovalently bound heme becomes covalently bound on exposure of the heme-protein complex to H 2 O 2 (4). Recent evidence indicates that a similar mechanism is involved in covalent attachment of the heme to the protein in EPO and thyroid peroxidase (12,13).…”

Section: Lpomentioning

confidence: 99%

Spin Trapping and Protein Cross-linking of the Lactoperoxidase Protein Radical

Lardinois

Medzihradszky

1999

Lactoperoxidase (LPO) reacts with H 2 O 2 to sequentially give two Compound I intermediates: the first with a ferryl (Fe IV ‫؍‬O) species and a porphyrin radical cation, and the second with the same ferryl species and a presumed protein radical. However, little actual evidence is available for the protein radical. We report here that LPO reacts with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid to give a 1:1 protein-bound radical adduct. Furthermore, LPO undergoes the H 2 O 2 -dependent formation of dimeric and trimeric products. Proteolytic digestion and mass spectrometric analysis indicates that the dimer is held together by a dityrosine link between Tyr-289 in each of two LPO molecules. The dimer retains full catalytic activity and reacts to the same extent with the spin trap, indicating that the spin trap reacts with a radical center other than Tyr-289. The monomeric protein recovered from incubations of LPO with H 2 O 2 is fully active but no longer forms dimers when incubated with H 2 O 2 , clear evidence that it has also been structurally modified. Myeloperoxidase, a naturally dimeric protein, and eosinophil peroxidase do not undergo H 2 O 2 -dependent oligomerization. Analysis of the interface in the LPO dimers indicates that the same protein surface is involved in LPO dimerization as in the normal formation of myeloperoxidase dimers. Oligomerization of LPO alters its physical properties and may alter its ability to interact with macromolecular substrates. LPO1 (EC 1.11.1.7; donor-H 2 O 2 oxidoreductase), which oxidizes thiocyanate (HSCN) to hypothiocyanate (HOSCN) and more highly oxidized species, is a component of the mammalian antimicrobial defense system (1). It is closely related by sequence and function to MPO and EPO and also by sequence to thyroid peroxidase (2, 3). LPO is not only of intrinsic interest but is also a useful model for the study of MPO and EPO, neither of which has been successfully expressed in other than mammalian cells (4).Bovine LPO is a protein of 612 amino acids with a molecular mass of approximately 78,000 Da, approximately 10% of which is carbohydrate (2, 5). The prosthetic group of the enzyme is a heme in which the 1-and 5-methyl groups bear hydroxyl groups esterified with the side chains of respectively (4,[6][7][8]. The prosthetic heme in myeloperoxidase is bound via two similar ester bonds to the protein (9) but in addition is linked to the protein by a bond between the 2-vinyl group and a methionine residue (10, 11). We established earlier that the prosthetic group is bound to the protein in LPO via a self-activating process in which noncovalently bound heme becomes covalently bound on exposure of the heme-protein complex to H 2 O 2 (4). Recent evidence indicates that a similar mechanism is involved in covalent attachment of the heme to the protein in EPO and thyroid peroxidase (12, 13).The peroxidative mechanism for LPO, as is true for all hemoprotein peroxidases, involves the following three-step sequence, where AH is a substrate (14).