Myeloperoxidase (MPO) catalyzes the formation of potent oxidants that have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Melatonin plays an important part in the regulation of various body functions including circadian sleep rhythms, blood pressure, oncogenesis, retinal function, seasonal reproduction, and immunity. Here, we demonstrate that melatonin serves as a potent inhibitor of MPO under physiological-like conditions. In the presence of chloride (Cl-), melatonin inactivated MPO at two points in the classic peroxidase cycle through binding to MPO to form an inactive complex, melatonin-MPO-Cl, and accelerating MPO compound II formation, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO compound II without any sign of compound I accumulation. This behavior indicates that melatonin binding modulates the formation of MPO intermediates and their decay rates. The Cl- presence enhanced the affinity of MPO toward melatonin, which switches the enzyme activity from peroxidation to catalase-like activity. In the absence of Cl-, melatonin served as a 1e- substrate for MPO compound I, but at higher concentration it limited the reaction by its dissociation from the corresponding complex. Importantly, melatonin-dependent inhibition of MPO occurred with a wide range of concentrations that span various physiological and supplemental ranges. Thus, the interplay between MPO and melatonin may have a broader implication in the function of several biological systems. This dual regulation by melatonin is unique and represents a new means through which melatonin can control MPO and its downstream inflammatory pathways.

Kinetic and structure analysis of inducible nitric oxide synthase (iNOS) revealed that, in addition to the increase of iNOS expression in inflamed areas, the major pathway causing overproduction of NO is destabilization of the iNOS-nitrosyl complex(es) that form during steady-state catalysis. Formation of such a complex allows iNOS to operate at only a fraction (20 -30%) of its maximum activity. Thus, bioavailability of NO scavengers at sites of inflammation may play an essential role in up-regulation of the catalytic activity of iNOS, by preventing the catalytic activity inhibition that is attributed to nitrosyl complex formation. Myeloperoxidase (MPO), a major NO scavenger, is a pivotal enzyme involved in leukocyte-mediated host defenses. It is thought to play a pathogenic role under circumstances such as acute inflammatory tissue injury and chronic inflammatory conditions. However, a detailed understanding of the interrelationship between iNOS and MPO at sites of inflammation is lacking. We used direct spectroscopic, HPLC, and selective NO-electrode measurements to determine the interdependent relationship that exists between iNOS and MPO and the role of the MPO͞H 2O2 system in up-regulating the catalytic activity of iNOS that occurs at sites of inflammation. Scavenging free NO from the iNOS milieu by the MPO͞H2O2 system subsequently restores the full capacity of iNOS to convert L-aginine to product (NO), as judged by the increase in the rates of citrulline and nitrite͞nitrate production. Studies of iNOS catalytic mechanisms and function are essential to a more fundamental understanding of these factors, which govern iNOS-dependent processes in human health and disease.inflammation ͉ peroxidase N itric oxide (NO) is a ubiquitous signaling molecule that plays essential bioregulatory roles in a wide range of processes, including vasodilation, cell proliferation, nerve transmission, tumor surveillance, antimicrobial defense, and regulation of inflammatory responses (1-4). NO is generated enzymatically by three distinct isoforms of NO synthase (NOS): neuronal, inducible (iNOS), and endothelial. All of these isoforms typically use L-arginine, O 2 , and NADPH to generate NO and citrulline (5). The biological effects of NO are governed in part, by its intrinsic instability, reactivity, lipophilicity, and affinity toward iron; these characteristics make it ideal for both signal transduction and defense (6). NO is freely diffusible; its effect in a given circumstance depends on its diffusion to reach the target cell, and the bioavailability of NO scavengers such as superoxide (O 2 ⅐Ϫ ) and oxyhemoglobin binding, which limit its ability to exert biologic effects (7). Although NO generated under normal conditions appear to serve a signaling function, under pathological conditions, such as during atherosclerosis, asthma, and other inflammatory processes, rates of NO production become excessive (8-15). Importantly, the cytokine-iNOS isoform that is present in many tissues, including lung, liver, kidney, heart, and smooth ...

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%

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Myeloperoxidase (MPO) is a hemoprotein, involved in the leukocyte mediated defense mechanism, and uses hydrogen peroxide (H2O2) and chloride (Cl-) to produce hypochlorous acid. In human saliva and hypochloremic alkalosis syndrome occurring in breast fed infants, the MPO-H2O2 system functions in lower Cl- concentration (10-70 mM) compared to plasma levels (100 mM) as part of the antibacterial defense system. The impact of low Cl- concentration and exposure to high peroxynitrite (ONOO-) synthesized from cigarette smoke or oxidative stress on MPO function is still unexplored. Rapid mixing of ONOO- and MPO caused immediate formation of a transient intermediate MPO Compound II which then decayed to MPO-Fe (III). Double mixing of MPO with ONOO- followed by H2O2 caused immediate formation of Compound II followed by MPO heme depletion, a process that occured independent of ONOO- concentration. Peroxynitrite/H2O2-mediated MPO heme depletion was confirmed by HPLC analysis and in-gel heme staining showing 60-70% less heme content compared to the control. A non-reducing denaturing SDS PAGE showed no fragmentation or degradation of protein. Myeloperoxidase heme loss was completely prevented by pre-incubation of MPO with saturated amounts of Cl-. Chloride binding to the active site of MPO constrains ONOO- binding by filling the space directly above the heme moiety or by causing a protein conformational change that constricts the distal heme pocket, thus preventing ONOO- from binding to MPO heme iron. Peroxynitrite interaction with MPO may serve as a novel mechanism for modulating MPO catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Background and purpose: Eosinophil peroxidase (EPO) catalyses the formation of oxidants implicated in the pathogenesis of various respiratory diseases including allergy and asthma. Mechanisms for inhibiting EPO, once released, are poorly understood. The aim of this work is to determine the mechanisms by which melatonin, a hormone produced in the brain by the pineal gland, inhibits the catalytic activity of EPO. Experimental approach: We utilized H 2 O 2 -selective electrode and direct rapid kinetic measurements to determine the pathways by which melatonin inhibits human EPO. Key results: In the presence of plasma levels of bromide (Br À ), melatonin inactivates EPO at two different points in the classic peroxidase cycle. First, it binds to EPO and forms an inactive complex, melatonin-EPO-Br, which restricts access of H 2 O 2 to the catalytic site of the oxidation enzyme. Second, melatonin competes with Br À and switches the reaction from a two electron (2e À ) to a one electron (1e À ) pathway allowing the enzyme to function with catalase-like activity. Melatonin is a bulky molecule and binds to the entrance of the EPO haem pocket (regulatory sites). Furthermore, Br À seems to enhance the affinity of this binding. In the absence of Br À , melatonin accelerated formation of EPO Compound II and its decay by serving as a 1eÀ substrate for EPO Compounds I and II. Conclusions and implications:The interplay between EPO and melatonin may have a broader implication in the function of several biological systems. This dual regulation by melatonin is unique and represents a new mechanism for melatonin to control EPO and its downstream inflammatory pathways.

Myeloperoxidase (MPO) binds H 2 O 2 in the absence and presence of chloride (Cl − ) and catalyzes the formation of potent oxidants through 1e − and 2e − oxidation pathways. These potent oxidants have been implicated in the pathogenesis of various diseases including atherosclerosis, asthma, arthritis, and cancer. Thus, inhibition of MPO and its byproducts may have a much wide application in biological systems. Using direct rapid kinetic measurements and H 2 O 2 -selective electrodes, we showed that tryptophan (Trp), an essential amino acid, was linked kinetically to the inhibition of MPO catalysis under physiological-like conditions. Trp inactivated MPO in the absence and presence of plasma levels of chloride (Cl − ), in various degrees, through binding to MPO forming the inactive complexes, Trp-MPO and Trp-MPO-Cl, respectively; and accelerating MPO Compound II formation, an inactive form of MPO. Inactivation of MPO was mirrored by the direct conversion of MPO-Fe(III) to MPO Compound II without any sign of Compound I accumulation. This behavior indicates that Trp binding modulates the formation of MPO intermediates and their decay rates. Importantly, Trp is a poor substrate for MPO Compound II and has no role in destabilizing complex formation. Thus, the overall MPO catalytic activity will be limited by: 1) the dissociation of Trp from Trp-MPO and Trp-MPO-Cl complexes; 2) the affinity of MPO Compound I towards Cl − versus Trp; and 3) the slow conversion of MPO Compound II to MPO-Fe(III). Importantly, Trp dependentinhibition of MPO occurred with a wide range of concentrations that span various physiological and supplemental ranges.