Hypochlorous Acid-Induced Heme Degradation from Lactoperoxidase as a Novel Mechanism of Free Iron Release and Tissue Injury in Inflammatory Diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H2O2) in the airways through its ability to oxidize thiocyanate (SCN−) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 µM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O2, Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN−. On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function

Galactose and its Metabolites Deteriorate Metaphase II Mouse Oocyte Quality and Subsequent Embryo Development by Disrupting the Spindle Structure

Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare spontaneous pregnancies have been reported. Here, we evaluate the effect of D-galactose and its metabolites, galactitol and galactose 1-phosphate, on oocyte quality as well as embryo development to elucidate the mechanism through which these compounds mediate oocyte deterioration. Metaphase II mouse oocytes (n=240), with and without cumulus cells (CCs), were exposed for 4hours to D-galactose (2μM), galactitol (11μM) and galactose 1-phosphate (0.1mM), (corresponding to plasma concentrations in patients on galactoserestricted diet) and compared to controls. The treated oocytes showed decreased quality as a function of significant enhancement in production of reactive oxygen species (ROS) when compared to controls. The presence of CCs offered no protection, as elevated ROS was accompanied by increased apoptosis of CCs. Our results suggested that D-galactose and its metabolites disturbed the spindle structure and chromosomal alignment, which was associated with significant decline in oocyte cleavage and blastocyst development after in-vitro fertilization. The results provide insight into prevention and treatment strategies that may be used to extend the window of fertility in these patients

Kinetic Evidence Supports the Existence of Two Halide Binding Sites that Have a Distinct Impact on the Heme Iron Microenvironment in Myeloperoxidase †

ABSTRACT: Myeloperoxidase (MPO) structural analysis has suggested that halides and pseudohalides bind to the distal binding site and serve as substrates or inhibitors, while others have concluded that there are two separate sites. Here, evidence for two distinct binding sites for halides comes from the bell-shaped effects observed when the second-order rate constant of nitric oxide (NO) binding to MPO was plotted versus Cl -concentration. The chloride level used in the X-ray structure that produced Cl -binding to the amino terminus of the helix halide binding site was insufficient to populate either of the two sites that appear to be responsible for the two phases. Biphasic effects were also observed when the I -, Br -, and SCN -concentrations were plotted against the NO combination rate constants. Interestingly, the trough concentrations obtained from the bell-shaped curves are comparable to normal plasma levels of halides and pseudohalides, suggesting the potential relevance of these molecules in modulating MPO function. The second-order rate constant of NO binding in the presence of plasma levels of I -, Br -, and SCN -is 1-2-fold lower compared to that obtained in the absence of these molecules and remains unaltered through the Cl -plasma level. When Cl -exceeded the plasma level, the NO combination rate becomes indistinguishable from the second phase of the bell-shaped curve that was obtained in the absence of halides. Our results are consistent with two halide binding sites that could be populated by two halides in which both display distinct effects on the MPO heme iron microenvironment. Myeloperoxidase (MPO) 1 is an abundant heme-containing protein found in neutrophil granules, monocytes, and selected tissue macrophages (1-3). MPO plays an important role in generating an array of toxic oxidants important to host defense (1-3). The molecular mass of the enzyme is 150-165 kDa and the enzyme is comprised of two identical subunits joined by a single disulfide bridge (2). Each subunit consists of a light chain and a heavy chain derived from a single gene product (4). The heavy chains contain an iron bound to a novel protoporphyrin IX derivative that is covalently attached to the heavy chain polypeptide (5, 6). The heme prosthetic groups are approximately 50 Å apart, and a variety of observations suggest that both are functionally identical (7-10). They presumably operate independently in the oxidation of Cl -and in the bactericidal activity of the enzyme (7). Structural studies of both canine and human MPO demonstrate that the heme of MPO is positioned at the base of a deep and narrow cleft and is axially coordinated to the protein through His933 (7-10). The imidazole ring of His95 is located 5.7 Å from the heme iron, while the guanidinium group of Arg239 and the side chain of Gln91 are close to the heme surface and have minimum interatomic distances from the iron atom of 7.0 and 4.5 Å, respectively (7-10). The location of these residues above the heme iron is consistent with the heme iron being the site where hydrogen peroxide •-, nitric oxide (NO), and ascorbic aci