A theoretical three-dimensional model for lactoperoxidase and eosinophil peroxidase, built on the scaffold of the myeloperoxidase X-ray structure

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).

Section: Discussionmentioning

confidence: 99%

“…The amino acid sequences of MPO, EPO, and LPO show a remarkable degree of similarity that ranges from 50 to 70% residue identity (37). This led us to investigate whether H 2 O 2 also causes covalent oligomerization of MPO and EPO.…”

Section: Spin Trapping Of Lpo Protein-derived Radicals By Db-nbs-mentioning

confidence: 99%

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

Lardinois

Medzihradszky

Montellano

1999

“…The properties of the heme in MPO, EPO, and LPO have been characterized by a wide variety of spectroscopic techniques, including optical absorption, stopped-flow, electron paramagnetic resonance, and resonance Raman spectroscopy (26 -37). These techniques, with a combination of structure analysis (16, 38 -39) and advanced computer modeling (40), have shown that the heme pockets of the mammalian peroxidases are envisioned to form the catalytic site where the stepwise reduction of H 2 O 2 takes place. The simplified mechanism that governs the catalytic activity of mammalian peroxidase superfamily can be represented by the classic peroxidases catalytic cycle.…”

Section: And Scnmentioning

confidence: 99%

Thiocyanate Modulates the Catalytic Activity of Mammalian Peroxidases

Tahboub¹,

Galijašević²,

Diamond³

et al. 2005

We investigated the potential role of the co-substrate, thiocyanate (SCN ؊ ), in modulating the catalytic activity of myeloperoxidase (MPO) and other members of the mammalian peroxidase superfamily (lactoperoxidase (LPO) and eosinophil peroxidase (EPO) ؊ concentration, depending on the experimental conditions. Collectively, these results illustrate for the first time the potential mechanistic differences of these three enzymes. A modified kinetic model, which incorporates our current findings with the mammalian peroxidases classic cycle, is presented. Myeloperoxidase (MPO)1 and other members of the mammalian peroxidase superfamily (eosinophil peroxidase (EPO) and lactoperoxidase (LPO)) display a crucial difference (within a wide range of biological processes) in their unique ability in catalyzing the H 2 O 2 -dependent peroxidation of halides and pseudohalides to produce antimicrobial agents and hypohalous acids (1-7). These heme-containing enzymes share 50 -70%

“…Furthermore, the highly conserved sequences of mammalian peroxidase enzymes in the region of covalent linkage for LPO and MPO suggest similar ester bonds between heme and protein throughout this family of enzymes. Hence, after decades of effort, the direct elucidation of heme l (17,18) and heme m (19,20) structures as well as indirect spectral (7,21) and modeling (36) correlations have resolved the controversy of a novel heme prosthetic group in mammalian peroxidases.…”

Section: Isolation Of Lpo Heme Species-mentioning

confidence: 99%

The Heme Prosthetic Group of Lactoperoxidase

Rae

Goff

1998

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme l, is isolated through an approach that combines proteolytic hydrolysis and reverse-phase high performance liquid chromatographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotrypsin) or a peptide-free heme (with proteinase K). Both heme l and heme l-peptide species were investigated by paramagnetic