Potential role of tryptophan and chloride in the inhibition of human myeloperoxidase

Myeloperoxidase (MPO) binds H2O2 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 by-products may have a wide application in biological systems. Using direct rapid kinetic measurements and H2O2-selective electrodes, we show that tryptophan (Trp), an essential amino acid, is linked kinetically to the inhibition of MPO catalysis under physiological conditions. Trp inactivated MPO in the absence and presence of plasma levels of Cl(-), to various degrees, through binding to MPO, forming the inactive complexes Trp-MPO and Trp-MPO-Cl, and accelerating formation of MPO Compound II, 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 toward Cl(-) versus Trp, and (3) the slow conversion of MPO Compound II to MPO-Fe(III). Importantly, Trp-dependent inhibition of MPO occurred at a wide range of concentrations that span various physiological and supplemental ranges.     

Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidase (EPO), and lactoperoxidase (LPO), homologous members of the mammalian peroxidase superfamily, can all serve as catalysts for generating nitric oxide- (nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prosthetic groups that are ligated through a histidine nitrogen and use H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic and/or conformational changes in their heme pockets using a combination of rapid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe(III) state] causes immediate buildup of the corresponding Fe(II) complexes. Spectral changes indicate that two LPO-Fe(II) species are present in solution at equilibrium. Analyses of stopped-flow traces collected when EPO, MPO, or LPO solutions rapidly mixed with NO were accurately fit by single-exponential functions. Plots of the apparent rate constants as a function of NO concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed significantly lower affinity toward NO compared to Fe(III) forms, suggesting that heme reduction causes a dramatic change in the heme pocket electronic environment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-spin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward binding of diatomic ligands. Our results suggest that, upon reduction, the heme pocket of MPO collapses, LPO adopts two spectroscopically and kinetically distinguishable forms (one partially open and the other relatively closed), and EPO remains open.     

A novel multistep mechanism for oxygen binding to ferrous hemoproteins: rapid kinetic analysis of ferrous-dioxy myeloperoxidase (compound III) formation

Myeloperoxidase (MPO), a hemoprotein that uses H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions, is implicated as a participant in inflammatory injury and cardiovascular diseases. Mechanisms for turning off this enzyme once released, preventing unwanted tissue injury, are poorly understood. We recently demonstrated that MPO heme reduction causes collapse of the heme pocket, as monitored by significant reductions in the rates of diatomic ligand binding to the heme iron. Using spectral and rapid kinetic measurements, we now demonstrate that molecular oxygen (O(2)) binds to ferrous MPO (MPO-Fe(II)) in a distinct and novel mechanism. Rather than occurring through a simple, reversible, one-step mechanism, as is typical for O(2) binding to other ferrous hemoproteins, the reaction involves several kinetically and spectroscopically distinguishable intermediates. Diode array spectrophotometric and stopped-flow studies reveal that the formation of the MPO-Fe(II)-O(2) complex consists of at least three elementary steps and includes at least two sequential transient intermediates. The first step involves reversible formation of a transient intermediate via an O(2)-dependent mechanism, followed by two sequential O(2)-independent steps that appear to be conformational in origin. Insights into mechanisms for inactivating MPO and the novel mode of O(2) binding to the hemoprotein may provide important clues toward understanding the catalytic action of MPO.     

Thiocyanate modulates the catalytic activity of mammalian peroxidases

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.     

Nitric oxide is a physiological substrate for mammalian peroxidases

We now show that NO serves as a substrate for multiple members of the mammalian peroxidase superfamily under physiological conditions. Myeloperoxidase (MPO), eosinophil peroxidase, and lactoperoxidase all catalytically consumed NO in the presence of the co-substrate hydrogen peroxide (H(2)O(2)). Near identical rates of NO consumption by the peroxidases were observed in the presence versus absence of plasma levels of Cl(-). Although rates of NO consumption in buffer were accelerated in the presence of a superoxide-generating system, subsequent addition of catalytic levels of a model peroxidase, MPO, to NO-containing solutions resulted in the rapid acceleration of NO consumption. The interaction between NO and compounds I and II of MPO were further investigated during steady-state catalysis by stopped-flow kinetics. NO dramatically influenced the build-up, duration, and decay of steady-state levels of compound II, the rate-limiting intermediate in the classic peroxidase cycle, in both the presence and absence of Cl(-). Collectively, these results suggest that peroxidases may function as a catalytic sink for NO at sites of inflammation, influencing its bioavailability. They also support the potential existence of a complex and interdependent relationship between NO levels and the modulation of steady-state catalysis by peroxidases in vivo.     

Melatonin is a potent inhibitor for myeloperoxidase

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.     

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 (H(2)O(2)) 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)-O(2), 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.     

Crystal structures of the ferric,ferrous, and ferrous-NO forms of the Asp140Ala mutant of human heme oxygenase-1: catalytic implications

Site-directed mutagenesis studies have shown that Asp140 in both human and rat heme oxygenase-1 is critical for enzyme activity. Here, we report the D140A mutant crystal structure in the Fe(III) and Fe(II) redox states as well as the Fe(II)-NO complex as a model for the Fe(II)-oxy complex. These structures are compared to the corresponding wild-type structures. The mutant and wild-type structures are very similar, except for the distal heme pocket solvent structure. In the Fe(III) D140A mutant one water molecule takes the place of the missing Asp140 carboxylate side-chain and a second water molecule, novel to the mutant, binds in the distal pocket. Upon reduction to the Fe(II) state, the distal helix running along one face of the heme moves closer to the heme in both the wild-type and mutant structures thus tightening the active site. NO binds to both the wild-type and mutant in a bent conformation that orients the NO O atom toward the alpha-meso heme carbon atom. A network of water molecules provides a H-bonded network to the NO ligand, suggesting a possible proton shuttle pathway required to activate dioxygen for catalysis. In the wild-type structure, Asp140 exhibits two conformations, suggesting a dynamic role for Asp140 in shuttling protons from bulk solvent via the water network to the iron-linked oxy complex. On the basis of these structures, we consider why the D140A mutant is inactive as a heme oxygenase but active as a peroxidase.     

Nitric oxide dioxygenation reaction in DevS and the initial response to nitric oxide in Mycobacterium tuberculosis

DevS and DosT from Mycobacterium tuberculosis (MTB) are paralogous heme-based sensor kinases that respond to hypoxia and to low concentrations of nitric oxide (NO). Both proteins work with the response regulator DevR as a two-component regulatory system to induce the dormancy regulon in MTB. While DevS and DosT are inactive when dioxygen is bound to the heme Fe(II) at their sensor domain, autokinase activity is observed in their heme Fe(II)-NO counterparts. To date, the conversion between active and inactive states and the reactivity of the heme-oxy complex toward NO have not been investigated. Here, we use stopped-flow UV-vis spectroscopy and rapid freeze quench resonance Raman spectroscopy to probe these reactions in DevS. Our data reveal that the heme-O(2) complex of DevS reacts efficiently with NO to produce nitrate and the oxidized Fe(III) heme through an NO dioxygenation reaction that parallels the catalytic reactions of bacterial flavohemoglobin and truncated hemoglobins. Autophosphorylation activity assays show that the Fe(III) heme state of DevS remains inactive but exhibits a high affinity for NO and forms an Fe(III)-NO complex that is readily reduced by ascorbate, a mild reducing agent. On the basis of these results, we conclude that upon exposure to low NO concentrations, the inactive oxy-heme complex of DevS is rapidly converted to the Fe(II)-NO complex in the reducing environment of living cells and triggers the initiation of dormancy.     

Activation of heme-regulated eukaryotic initiation factor 2alpha kinase by nitric oxide is induced by the formation of a five-coordinate NO-heme complex: optical absorption, electron spin resonance, and resonance raman spectral studies

Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.     

Active site structure and catalytic mechanisms of human peroxidases

Myeloperoxidase (MPO), eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase are heme-containing oxidoreductases (EC 1.7.1.11), which bind ligands and/or undergo a series of redox reactions. Though sharing functional and structural homology, reflecting their phylogenetic origin, differences are observed regarding their spectral features, substrate specificities, redox properties, and kinetics of interconversion of the relevant redox intermediates ferric and ferrous peroxidase, compound I, compound II, and compound III. Depending on substrate availability, these heme enzymes path through the halogenation cycle and/or the peroxidase cycle and/or act as poor (pseudo-)catalases. Based on the published crystal structures of free MPO and its complexes with cyanide, bromide and thiocyanate as well as on sequence analysis and modeling, we critically discuss structure-function relationships. This analysis highlights similarities and distinguishing features within the mammalian peroxidases and intents to provide the molecular and enzymatic basis to understand the prominent role of these heme enzymes in host defense against infection, hormone biosynthesis, and pathogenesis.     

Nitrosyl-heme structures of Bacillus subtilis nitric oxide synthase have implications for understanding substrate oxidation

The crystal structures of nitrosyl-heme complexes of a prokaryotic nitric oxide synthase (NOS) from Bacillus subtilis (bsNOS) reveal changes in active-site hydrogen bonding in the presence of the intermediate N(omega)-hydroxy-l-arginine (NOHA) compared to the substrate l-arginine (l-Arg). Correlating with a Val-to-Ile residue substitution in the bsNOS heme pocket, the Fe(II)-NO complex with both l-Arg and NOHA is more bent than the Fe(II)-NO, l-Arg complex of mammalian eNOS [Li, H., Raman, C. S., Martasek, P., Masters, B. S. S., and Poulos, T. L. (2001) Biochemistry 40, 5399-5406]. Structures of the Fe(III)-NO complex with NOHA show a nearly linear nitrosyl group, and in one subunit, partial nitrosation of bound NOHA. In the Fe(II)-NO complexes, the protonated NOHA N(omega) atom forms a short hydrogen bond with the heme-coordinated NO nitrogen, but active-site water molecules are out of hydrogen bonding range with the distal NO oxygen. In contrast, the l-Arg guanidinium interacts more weakly and equally with both NO atoms, and an active-site water molecule hydrogen bonds to the distal NO oxygen. This difference in hydrogen bonding to the nitrosyl group by the two substrates indicates that interactions provided by NOHA may preferentially stabilize an electrophilic peroxo-heme intermediate in the second step of NOS catalysis.     

Myeloperoxidase metabolizes thiocyanate in a reaction driven by nitric oxide

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.     

Influence of the covalent heme-protein bonds on the redox thermodynamics of human myeloperoxidase

Myeloperoxidase (MPO) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride to generate the potent bleaching hypochlorous acid, thus contributing to pathogen killing as well as inflammatory diseases. Its catalytic properties are closely related with unique posttranslational modifications of its prosthetic group. In MPO, modified heme b is covalently bound to the protein via two ester linkages and one sulfonium ion linkage with a strong impact on its (electronic) structure and biophysical and chemical properties. Here, the thermodynamics of the one-electron reduction of the ferric heme in wild-type recombinant MPO and variants with disrupted heme-protein bonds (M243V, E242Q, and D94V) have been investigated by thin-layer spectroelectrochemistry. It turns out that neither the oligomeric structure nor the N-terminal extension in recombinant MPO modifies the peculiar positive reduction potential (E°' = 0.001 V at 25 °C and pH 7.0) or the enthalpy or entropy of the Fe(III) to Fe(II) reduction. By contrast, upon disruption of the MPO-typical sulfonium ion linkage, the reduction potential is significantly lower (-0.182 V). The M243V mutant has an enthalpically stabilized ferric state, whereas its ferrous form is entropically favored because of the loss of rigidity of the distal H-bonding network. Exchange of an adjacent ester bond (E242Q) induced similar but less pronounced effects (E°' = -0.094 V), whereas in the D94V variant (E°' = -0.060 V), formation of the ferrous state is entropically disfavored. These findings are discussed with respect to the chlorination and bromination activity of the wild-type protein and the mutants.     

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.     

Electronic and stereochemical characterizations of the photoinduced intermediates of nitrosyl complexes of metal (S = 5/2)-substituted hemoproteins trapped at low temperature

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric myoglobin (NO-Fe(III)Mb) and manganese(II)-porphyrin-substituted myoglobin (NO-Mn(II)Mb) was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the electronic and structural natures of the photoinduced intermediates of these hemoprotein-ligand complexes trapped at low temperature. The photoproduct of NO-Fe(III)Mb at 5 K exhibited entirely new X-band EPR absorptions in the magnetic field strength from 0 to 0.4 tesla. The widespread absorption together with distinct, sharp zero-field absorption was consistently observed in the photoproduct of the isoelectronic NO-Mn(II)Mb. These novel ERP signals indicate a spin-coupled pair with an effective spin of S = 2 between the high spin metal center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. On the other hand, the photolyzed form of nitrosyl complexes of Fe(III)- and Mn(II)-Glycera hemoglobins, in which the distal histidine of Mb is replaced by a leucyl residue, exhibited somewhat broader EPR absorptions similar to those of the corresponding native Fe(III)- or unliganded Mn(II)-Glycera hemoglobins, respectively, indicating that the photodissociated NO molecule moved farther away from the metal center in the heme pocket. These observations show the importance of the interaction of the distal residue with the ligand in determining the nature of the photolyzed states.     

Oxides of Nitrogen (NO· and NO2 –) as Cofactors of the Myeloperoxidase System

Myeloperoxidase is the main peroxisomal protein of neutrophils, monocytes, and a subpopulation of tissue macrophages; it plays the key role in protective and inflammatory responses of the organism. This role is mediated by various diffusible radicals formed during oxidative reactions catalyzed by the enzyme heme. Myeloperoxidase and nitric oxide synthase are stored in peroxisomes. Nitric oxide reacts with the heme of myeloperoxidase. Low nitric oxide concentrations increase peroxidase activity through reduction of Compound II to native myeloperoxidase. Conversely, high nitric oxide concentrations inhibit the catalytic activity of myeloperoxidase through formation of inactive nitrosyl–heme complexes. Such effect of nitric oxide on catalytic activity of myeloperoxidase has various consequences for infectious and local inflammatory processes. Another oxide of nitrogen, nitrite, is a good substrate for myeloperoxidase Compound I but slowly reacts with Compound II. Nitrogen dioxide is formed after nitrite oxidation by myeloperoxidase. Formation of nitrogen dioxide is another protective mechanism and nitration of microbial proteins by myeloperoxidase can represent an additional protective response of peroxisomes.     

Myeloperoxidase binds to low-density lipoprotein: potential implications for atherosclerosis

Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can modify low-density lipoprotein (LDL) to a form that converts macrophages into lipid-laden or 'foam' cells, the hallmark of atherosclerotic lesions. Since MPO has been shown to bind to a number of different cell types, we investigated binding of MPO to LDL. Using the precipitation reagents phosphotungstate or isopropanol, MPO co-precipitated with LDL, retaining its catalytic activity. The association of MPO with LDL was confirmed using native gel electrophoresis. MPO was also found to co-precipitate with apolipoprotein B-100-containing lipoproteins in whole plasma. No precipitation of MPO was observed in lipoprotein-deficient plasma, and there was a dose-dependent increase in precipitation following addition of LDL to lipoprotein-deficient plasma. Binding of MPO to LDL could potentially enhance site-directed oxidation of the lipoprotein and limit scavenging of reactive oxygen species by antioxidants.     

Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis

Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from l-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O(2) to form a transient heme Fe(II)O(2) species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine. In the latter case, disappearance of the Fe(II)O(2) species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O(2) intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS.     

Iron, Nitric Oxide, and Myeloperoxidase in Asthmatic Patients

Plasma nitric oxide (NO), myeloperoxidase (MPO), and iron (Fe) levels were determined in bronchial asthma. The relations among these parameters in different stages of asthma were interpreted. Their association with airway inflammation observed in patients with bronchial asthma as well as the roles and the contributions to the pathological processes were evaluated. A total of 62 individuals, 32 asthmatics and 30 controls, were included into the scope of this study. Plasma nitric oxide metabolites (NOx) and MPO and Fe levels were determined by the Griess reaction, ELISA, and the automated TPTZ (2,4,6-tri[2-pyridyl]-5-triazine) method, respectively. In the asthmatic individuals, plasma NOx, MPO, and Fe concentrations were 133 +/- 13 microM, 95 +/- 20 ng/ml, and 159 +/- 20 microg/dl, respectively; in the control group these values were 82 +/- 11 microM, 62 +/- 11 ng/ml, and 96 +/- 9 microg/dl. Increased values were detected for plasma MPO (p > 0.05), NOx (p < 0.01), and Fe (p < 0.01) concentrations in asthmatic individuals. Considering the facts that NO modulates the catalytic activity of MPO and induces the expression of heme oxygenase as important contributors to the mechanisms causing free Fe release, it is concluded that elevated NOx, MPO, and Fe levels observed in the asthmatic group act in a concerted manner and appear to be involved in the pathogenesis of asthma.