Effect of metal ions on the kinetics of tyrosine oxidation catalysed by tyrosinase.

The conversion of tyrosine into dopa [3-(3,4-dihydroxyphenyl)alanine] is the rate limiting step in the biosynthesis of melanins catalysed by tyrosinase. This hydroxylation reaction is characterized by a lag period, the extent of which depends on various parameters, notably the presence of a suitable hydrogen donor such as dopa or tetrahydropterin. We have now found that catalytic amounts of Fe2+ ions have the same effect as dopa in stimulating the tyrosine hydroxylase activity of the enzyme. Kinetic experiments showed that the shortening of the induction time depends on the concentration of the added metal and the nature of the buffer system used and is not suppressed by superoxide dismutase, catalase, formate or mannitol. Notably, Fe3+ ions showed only a small delaying effect on tyrosinase activity. Among the other metals which were tested, Zn2+, Co2+, Cd2+ and Ni2+ had no detectable influence, whereas Cu2+ and Mn2+ exhibited a marked inhibitory effect on the kinetics of tyrosine oxidation. These findings are discussed in the light of the commonly accepted mechanism of action of tyrosinase.     

Heavier halides of transition metals by halide exchange

Examples are reported of heavier (bromides or iodides) metal halides of the d or f transition series being prepared through the halide exchange reaction from the lighter congeners (fluorides or chlorides, easily prepared by direct combination from the elements), by using gaseous hydrogen halides HX or alkyl halides RX in an anhydrous organic solvent at room temperature or even below. This represents a considerable improvement with respect to the traditional high-temperature experimental procedures from the elements. Thermodynamic data show that this synthetic route is of quite general validity.     

Superoxide-dependent oxidation of melatonin by myeloperoxidase

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.     

Formation of reactive halide species by myeloperoxidase and eosinophil peroxidase

The formation of chloro- and bromohydrins from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine following incubation with myeloperoxidase or eosinophil peroxidase in the presence of hydrogen peroxide, chloride and/or bromide was analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. These products were only formed below a certain pH threshold value, that increased with increasing halide concentration. Thermodynamic considerations on halide and pH dependencies of reduction potentials of all redox couples showed that the formation of a given reactive halide species in halide oxidation coupled with the reduction of compound I of heme peroxidases is only possible below a certain pH threshold that depends on halide concentration. The comparison of experimentally derived and calculated data revealed that Cl(2), Br(2), or BrCl will primarily be formed by the myeloperoxidase-H(2)O(2)-halide system. However, the eosinophil peroxidase-H(2)O(2)-halide system forms directly HOCl and HOBr.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Adenylyl cyclase activation by halide anions other than fluoride

Adenylyl cyclase of rat liver and fat cells is activated by chloride, bromide, and iodide in addition to fluoride, previously believed to be uniquely effective among the halide anions. Liver homogenates are activated approximately 6 fold by fluoride while chloride and bromide increase cyclase by 3 fold and iodide about 2 fold. Optimal concentrations of chloride, bromide and iodide are about 100 times higher than those required for activation by fluoride. The cyclase of fat cell ghosts is activated some 9 fold by fluoride, but the other halide anions produced effects very similar in magnitude to those seen with liver, although for fat the optimally effective concentrations were lower. These observations appear to relate adenylate cyclase to a number of other anion activated enzymes, some of which have already been studied in pure form by a number of physico-chemical techniques, and which may serve as models for understanding the action of fluoride and other anions on adenylyl cyclase.     

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.     

Enzymatic oxidation of manganese ions catalysed by laccase

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.     

Kinetics of oxidation of serotonin by myeloperoxidase compounds I and II.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.     

The oxidation of l-ascorbic acid catalysed by pear tyrosinase

The ability of partially purified pear tyrosinase (PPO) to catalyse the oxidation of l -ascorbic acid (AA) has been reported here for the first time. The ascorbate oxidase activity of PPO was studied by oxymetric assays. The activity was linearly related to the enzyme concentration with a Michaelis constant (K_m) for AA of 0.55±0.03 m M at pH 7. The stoichiometry was found to be 1:2 (O₂:AA). The action of the PPO inhibitors tropolone and sodium chloride was studied to exclude a possible interference of endogenous pear ascorbate oxidase in the oxidation of AA. A possible role of the 'AA/PPO' system in the browning of pears is proposed.     

Some observations on the NADP+-linked oxidation of methylglyoxal catalysed by 2-Oxoaldehyde dehydrogenase.

In the oxidation of methylglyoxal by 2-oxoaldehyde dehydrogenase, the apparent Km value for NADP+ was about 2.5 times lower than the corresponding Km for NAD+; the apparent Km values for methylglyoxal and for the amine activator L-2-aminopropan-1-ol, with NADP+ as cofactor, were also different from those obtained with NAD+. In the presence of NADP+, the enzyme was not activated by P1, in contrast with the activation of the enzyme when NAD+ was used. The significance of the results is discussed.     

Microperoxidase 8 catalysed nitrogen oxides formation from oxidation of N-hydroxyguanidines by hydrogen peroxide.

Nitric oxide (NO) is a potent intra- and intercellular messenger involved in the control of vascular tone, neuronal signalling and host response to infection. In mammals, NO is synthesized by oxidation of l-arginine catalysed by hemeproteins called NO-synthases with intermediate formation of Nomega-hydroxy-l-arginine (NOHA). NOHA and some hydroxyguanidines have been shown to be able to deliver nitrogen oxides including NO in the presence of various oxidative systems. In this study, NOHA and a model compound, N-(4-chlorophenyl)-N'-hydroxyguanidine, were tested for their ability to generate NO in the presence of a haemprotein model, microperoxidase 8 (MP8), and hydrogen peroxide. Nitrite and nitrate production along with selective formation of 4-chlorophenylcyanamide was observed from incubations of N-(4-chlorophenyl)-N'-hydroxyguanidine in the presence of MP8 and hydrogen peroxide. In the case of NOHA, the corresponding cyanamide, Ndelta-cyano-L-ornithine, was too unstable under the conditions used and l-citrulline was the only product identified. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl and formation of MP8-Fe-NO complexes were observed by EPR spectroscopy and low-temperature UV/visible spectroscopy, respectively. These results clearly demonstrate the formation of nitrogen oxides including NO from the oxidation of exogenous hydroxyguanidines by hydrogen peroxide in the presence of a minienzyme such as MP8. The importance of the bioactivation of endogenous (NOHA) or exogenous N-hydroxyguanidines by peroxidases of physiological interest remains to be established in vivo.     

The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions

The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyzes the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H2O2, whereas at higher pH, H2O2 inhibits the chlorination reaction. The dissociation constant (Kd) of the spectroscopically detectable complex and the Km for chloride are considerably smaller than the inhibition constant (Ki) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the Km for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/[H2O2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.     

Novel findings on the copper catalysed oxidation of cysteine

Summary The oxidation of cysteine (RSH) has been studied by using O₂, ferricytochrome c (Cyt c) and nitro blue tetrazolium (NBT) as electron acceptors. The addition of 200M Cu^II to a solution of 2mM cysteine, pH 7.4, produces an absorbance with a peak at 260 nm and a shoulder at 300 nm. Generation of a cuprous bis-cysteine complex (RS-Cu^I-SR) is responsible for this absorbance. In the absence of O₂ the absorbance is stable for long time while in the presence of air it vanishes slowly only when the cysteine excess is consumed. The neocuproine assay and the EPR analysis show that the metal remains reduced in the course of the oxidation of cysteine returning to the oxidised form at the end of reaction when all RSH has been oxidised to RSSR. Addition of Cu^II enhances the reduction rate of Cyt c and of NBT by cysteine also under anaerobiosis indicating the occurrence of a direct reduction of the acceptor by the complex. It is concluded that the cuprous bis-cysteine complex (RS-Cu^I-SR) is the catalytic species involved in the oxidation of cysteine. The novel finding of the stability of the complex together with the metal remaining in the reduced form during the oxidation suggest sulfur as the electron donor in the place of the metal ion.Abbreviations RSH cysteine - RS^– cysteine in the thiolate form - RS^· thiyl radical of cysteine - RSSR cystine - Cyt c cytochrome c - SOD superoxide dismutase - NBT nitro blue tetrazolium - NBF nitro blue formazan - DTNB 5,5-dithiobis-2-nitrobenzoic acid - DTPA diethylenetriaminepentaacetic acid Dedicated to prof. A. Ballio ob the occasion of his 75th birthday.     

Two-electron reduction and one-electron oxidation of organic hydroperoxides by human myeloperoxidase

The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t-butyl hydroperoxide, 3-chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi-mixing stopped-flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two-electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15 degrees C, the rate constant of the reaction between 3-chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8x10(7) M(-1) s(-1) and 1.4x10(7) M(-1) s(-1), respectively). With the exception of t-butyl hydroperoxide, the rates of compound I formation varied between 5.2x10(5) M(-1) s(-1) and 2.7x10(6) M(-1) s(-1). Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3-chloroperoxybenzoic acid this reaction is most effective (6. 6x10(4) M(-1) s(-1) at pH 7 and 15 degrees C). Both reactions are controlled by the same ionizable group (average pK(a) of about 4.0) which has to be in its conjugated base form for reaction.     

Selective oxidation in vitro by myeloperoxidase of the N-terminal amine in apolipoprotein B-100.

In contrast to the multiple low abundance 2,4-dinitrophenylhydrazine-reactive tryptic peptides formed by oxidation of LDL with reagent HOCl in vitro, myeloperoxidase-catalyzed oxidation produces a dominant product in considerably greater yield and selectivity. This modified peptide had a single amino-terminal sequence corresponding to amino acids 53-66 of apolipoprotein B-100 (apoB-100), but its mass spectra indicated a significantly higher mass than could be reconciled with simple modifications of this peptide. Subsequent studies indicate that this product appears to result from N-chlorination of the N-terminal amino group of apoB-100 and dehydrohalogenation to the corresponding imine, which may form the hydrazone derivative directly, or after hydrolysis to the ketone. The methionine residue is oxidized to the corresponding sulfoxide, and the primary sequence peptide (residues 1-14 of apoB-100) is linked by the intramolecular disulfide bond between C-12 and C-61 to the peptide composed of residues 53-66, as we have observed previously (Yang, C-Y., T. W. Kim, S. A. Weng, B. Lee, M. Yang, and A. M. Gotto, Jr. 1990. Proc. Natl. Acad. Sci. USA. 87: 5523-5527) in unmodified LDL. The selective oxidation by myeloperoxidase of the N-terminal amine suggests strong steric effects in the approach of substrate to the enzyme catalytic site, an effect that may apply to other macromolecules and to cell surface molecules.     

1,3-Butadiene oxidation by human myeloperoxidase. Role of chloride ion in catalysis of divergent pathways.

1,3-Butadiene was oxidized by human myeloperoxidase in the absence of KCl to yield butadiene monoxide (BM) and crotonaldehyde (CA), but at KCl concentrations higher than 50 mM, 1-chloro-2-hydroxy-3-butene (CHB) was the major metabolite detected; metabolite formation was dependent on incubation time, pH, KCl, 1,3-butadiene, and H2O2 concentrations. The data are best explained by 1,3-butadiene being oxidized by myeloperoxidase by two different mechanisms. First, oxygen transfer from the hemoprotein would occur to either C-1 or C-4 of 1,3-butadiene to form an intermediate which may cyclize to form BM or undergo a hydrogen shift to form 3-butenal, an unstable precursor of CA. Further evidence for this mechanism was provided by the inability to detect methyl vinyl ketone, a possible product of an oxygen transfer reaction to C-2 or C-3 of 1,3-butadiene, and by the finding that CA was not simply a decomposition product of BM under assay conditions. In the second mechanism, however, chloride ion is oxidized by myeloperoxidase to HOCl which reacts with 1,3-butadiene to yield CHB. Further evidence for this mechanism was provided by the finding that CHB was readily formed when 1,3-butadiene was added to the filtrate of a myeloperoxidase/H2O2/KCl incubation and when 1,3-butadiene was allowed to react with authentic HOCl. In addition, CHB was not detected when BM or CA was incubated with myeloperoxidase, H2O2, and KCl for up to 60 min, or when 1,3-butadiene and KCl were incubated with chloroperoxidase and H2O2 or with mouse liver microsomes and NADPH, enzyme systems which catalyze 1,3-butadiene oxidation to BM and CA, but unlike myeloperoxidase, do not catalyze chloride ion oxidation to HOCl. These results provide clear evidence for novel olefinic oxidation reactions by myeloperoxidase.