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.

Eosinophil recruitment and enhanced nitric oxide (NO) production are characteristic features of asthma and other airway diseases. Eosinophil peroxidase (EPO), a highly cationic hemoprotein secreted by activation of eosinophils, is believed to play a central role in host defense against invading pathogens. The enzyme uses hydrogen peroxide (H2O2) and bromide (Br-), a preferred cosubstrate of EPO, to generate the cytotoxic oxidant hypobromous acid. The aim of this work was to determine whether NO can compete with plasma levels of Br- and steer the enzyme reaction from a 2e- oxidation to a 1e- oxidation pathway. Rapid kinetic measurements were utilized to measure the rate of EPO compounds I and II formation, duration, and decay at 412 and 432 nm, respectively, at 10 degrees C. An EPO-Fe(III) solution supplemented with increasing Br- concentrations was rapidly mixed with fixed amounts of H2O2 in the absence and in the presence of increasing NO concentrations. In the absence of NO, EPO-Fe(III) primarily converted to compound I and, upon H2O2 exhaustion, it decayed rapidly to the ferric form. NO caused a significant increase in the accumulation of EPO compound II, along with a proportional increase in its rate of formation and duration as determined by the time elapsed during catalysis. The time courses for these events have been incorporated into a comprehensive kinetic model. Computer simulations carried out supported the involvement of a conformational intermediate in the EPO compound II complex decay. Collectively, our results demonstrated that NO displays the potential capacity to promote substrate switching by modulating substrate selectivity of EPO.

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.

We examined the potential physiological relevance of myeloperoxidase (MPO)-nitric oxide (NO) interactions as they may relate to the cosubstrate, pseudo halide thiocyanate (SCN(-)), and substrate switching. Direct spectroscopic and rapid kinetics studies revealed that SCN(-) interaction with MPO facilitates formation of the MPO catalytic intermediate Compound II, limiting overall activity. However, a physiological NO concentration (2 microM or less) dramatically influences the build-up, duration, and decay of the steady-state level of MPO Compound II during the metabolism of SCN(-), allowing the enzyme to function at full capacity. At higher NO concentrations, we observed significant increases in the rate of MPO Compound II formation, along with proportional increases in its duration as determined by the time elapsed during catalysis. Surprisingly, the decay rate of MPO Compound II remained unaltered as NO concentrations were increased. Computer simulations were carried out to model the kinetics of MPO Compound II formation, duration, and decay during the metabolism of SCN(-) as a function of NO concentration. These simulation traces closely approximate what was observed experimentally and support the involvement of a conformational intermediate of MPO Compound II complex decay, altering the overall capacity of MPO to promote two electrons versus one-electron oxidation reactions during steady-state catalysis. Collectively, the present studies reveal that (patho)physiologically relevant levels of NO have significant effects on MPO Compound II accumulation. Thus, NO affects the overall rate of peroxidation of substrates and the overall ability of the peroxidase to execute one- versus two-electron oxidation reactions.

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)). Pre-incubation of SCN– with MPO generates a more complex biological setting, because SCN– serves as either a substrate or inhibitor, causing diverse impacts on the MPO heme iron microenvironment. Consistent with this hypothesis, the relationship between the association rate constant of nitric oxide binding to MPO-Fe(III) as a function of SCN– concentration is bell-shaped, with a trough comparable with normal SCN– plasma levels. Rapid kinetic measurements indicate that MPO, EPO, and LPO Compound I formation occur at rates slower than complex decay, and its formation serves to simultaneously catalyze SCN– via 1e– and 2e– oxidation pathways. For the three enzymes, Compound II formation is a fundamental feature of catalysis and allows the enzymes to operate at a fraction of their possible maximum activities. MPO and EPO Compound II is relatively stable and decays gradually within minutes to ground state upon H2O2 exhaustion. In contrast, LPO Compound II is unstable and decays within seconds to ground state, suggesting that SCN– may serve as a substrate for Compound II. Compound II formation can be partially or completely prevented by increasing SCN– 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.

Heme reduction of ferric lactoperoxidase (LPO) into its ferrous form initially leads to the accumulation of the unstable form of LPO-Fe(II), which spontaneously converts to a more stable species, the two of which can be identified by Soret peaks at 440 and 434 nm, respectively. Our data demonstrate that both LPO-Fe(II) species are capable of binding O2 at a similar rate to generate the ferrous-dioxy complex. Its formation with respect to O2 was first order and monophasic and with rate constants of kon = 3.8 × 104 m–1 s–1 and koff = 11.2 s–1. The dissociation rate constant for the formation of LPO-Fe(II)-O2 is relatively high, in contrast to hemoprotein model compounds. This high dissociation rate can be attributed to a combination of effects that include the positive trans effect of the proximal ligand, the heme pocket environment, and the geometry of the Fe-O2 linkage. Our results have also shown that the decay of the LPO-Fe(II)-O2 complex occurs by two sequential O2-independent steps. The first step involves formation of a short-lived intermediate that can be characterized by its Soret absorption peak at 416 nm and may be attributed to the weakening of the Fe(II)-O2 linkage with a rate constant of 0.5 s–1. The second step is spontaneous conversion of this intermediate to generate the native enzyme and presumably superoxide as end products with a rate constant of 0.03 s–1. A comprehensive kinetic model that links LPO-Fe(II)-O2 complex formation to the LPO catalase-like activity, combined with the classic catalytic cycle, is presented here.

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 MPO/H2O2 system in up-regulating the catalytic activity of iNOS that occurs at sites of inflammation. Scavenging free NO from the iNOS milieu by the MPO/H2O2 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 nitrite/nitrate 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.

The kinetic behavior of electron-transfer reactions involving several copper(II/I) complexes has previously been attributed to a dual-pathway “square scheme” mechanism in which changes in the coordination geometry occur sequentially, rather than concertedly, with the electron-transfer step. In the case of 14-membered macrocyclic quadridentate ligand complexes studied to date, the major geometric change appears to be the inversion of two coordinated donor atoms during the overall electron-transfer process. However, the relative importance of these two inversions has been a matter of speculation. In the current investigation, a comparison is made of Cu(II/I) systems involving two pairs of ligands with S4 and NS3 donor sets: 1,4,8,11-tetrathiacyclotetradecane ([14]aneS4-a); 1,4,7,11-tetrathiacyclotetradecane ([14]aneS4-b); 1,4,8-trithia-11-azacyclotetradecane ([14]aneNS3-a); and 1,7,11-trithia-4-azacyclotetradecane ([14]aneNS3-b). In each pair of ligands, isomer a has the common chelate ring size sequence 5,6,5,6 while isomer b has the sequence 5,5,6,6. A crystal structure for [CuII([14]aneNS3-b)(H2O)](ClO4)2 demonstrates that, when coordinated to Cu(II), the b isomers stabilize the relatively rare ligand conformation designated as conformer II in which one donor atom is oriented opposite to the other three relative to the plane of the macrocycle. This eliminates one of the donor atom inversion steps which normally occurs during Cu(II/I) electron transfer. The copper complexes formed with these a and b isomers are examined in terms of (i) their CuIIL and CuIL stability constants, (ii) their CuIIL formation and dissociation rate constants, (iii) their CuII/IL redox potentials and (iv) their apparent electron self-exchange rate constants. Of the two donor atom inversions which occur in the case of the a-isomer complexes, the specific donor atom inversion which is common to the b-isomer complexes is judged to exhibit the larger energy barrier. Thus, it is presumed to represent the rate-limiting process responsible for the onset of “gated” electron transfer in previous studies on a-isomer complexes.