Compound I formation is a partially rate-limiting process in chloroperoxidase-catalyzed bromination reactions

The kinetics of chloroperoxidase-catalyzed bromination and chlorination reactions were studied at various halide and hydrogen peroxide concentrations. At very high concentrations, both chloride (KI = 370 mM) and bromide (KI = 150 mM) are competitive substrate inhibitors versus hydrogen peroxide. Results at subinhibitory halide concentrations for bromination reactions (kcat = 4 ms-1, kcat/KPeroxide = 1.6 microM-1 x s-1 and kcat/KBr = 4.0 microM-1 x s-1) and chlorination reactions (kcat = 1.5 ms-1, kcat/Kperoxide = 2.3 microM-1 x s-1, and kcat/KBr = 0.32 microM-1 x s-1) indicate that halide oxidation is rate-limiting in chlorination reactions. However, in bromination reactions, both compound I formation and bromide oxidation are partially rate-limiting. This is the first documented case where compound I formation participates in determining the overall rate of a peroxidase reaction.     

The susceptibility of strains of Mycobacterium tuberculosis to catalase-mediated peroxidative killing

At low pH and with continuous low concentrations of hydrogen peroxide generated in situ, catalase was able to replace peroxidase in the peroxidase/hydrogen peroxide/iodide microbicidal system. The system was effective against Escherichia coli and Mycobacterium tuberculosis. Iodide could not be replaced by chloride. The system was effective in lactate buffer, but not in citrate/phosphate buffer. Strains of M. tuberculosis with high and low virulence were equally susceptible. The observations are discussed in the context of an involvement of host-cell catalase in a possible intracellular killing mechanism against M. tuberculosis.     

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.     

Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 A. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.     

Novel Haloperoxidase Reaction: Synthesis of Dihalogenated Products

The enzymatic synthesis of vicinal, dihalogenated products from alkenes and alkynes is described. The enzymatic reaction required an alkene or alkyne, dilute hydrogen peroxide, a haloperoxidase, and molar amounts of halide ions. Vicinal dichloro, dibromo, and diiodo products could be formed. A hydroxyl group on the carbon adjacent to the carbon-carbon double or triple bond lowered the halide ion concentration needed to produce the dihalo product. This reaction offers one explanation for the origin of natural, vicinal, dihalogenated products, such as those found frequently in marine microogranisms.     

Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase

A new approach, the immuno-spin trapping assay, used a novel rabbit polyclonal anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antiserum to detect protein radical-derived DMPO nitrone adducts in the hemoprotein Mycobacterium tuberculosis catalase-peroxidase (KatG). This work demonstrates that the formation of protein nitrone adducts is dependent on the concentrations of tert-BuOOH and DMPO as shown by Western blotting and an enzyme-linked immunosorbent assay (ELISA). We have also detected protein-protein cross-links formed during turnover of Mtb KatG driven by tert-butyl peroxide ( tert-BuOOH) or enzymatic generation of hydrogen peroxide. DMPO inhibits this dimerization due to its ability to trap the amino acid radicals responsible for the cross-linkage. Chemical modifications by tyrosine and tryptophan blockage suggest that tyrosine residues are one site of formation of the dimers. The presence of the tuberculosis drug isoniazid (INH) also prevented cross-linking as a result of its competition for the protein radical. Protein-DMPO nitrone adducts were also generated by a continuous flux of hydrogen peroxide. These findings demonstrated that the protein-based radicals were formed not only during Mtb KatG turnover with alkyl peroxides but also in the presence of hydrogen peroxide. Furthermore, the formation of protein-DMPO nitrone adducts was accelerated in the presence of isoniazid.     

Exploring the Chemistry and Biology of Vanadium-dependent Haloperoxidases

Nature has developed an exquisite array of methods to introduce halogen atoms into organic compounds. Most of these enzymes are oxidative and require either hydrogen peroxide or molecular oxygen as a cosubstrate to generate a reactive halogen atom for catalysis. Vanadium-dependent haloperoxidases contain a vanadate prosthetic group and utilize hydrogen peroxide to oxidize a halide ion into a reactive electrophilic intermediate. These metalloenzymes have a large distribution in nature, where they are present in macroalgae, fungi, and bacteria, but have been exclusively characterized in eukaryotes. In this minireview, we highlight the chemistry and biology of vanadium-dependent haloperoxidases from fungi and marine algae and the emergence of new bacterial members that extend the biological function of these poorly understood halogenating enzymes.     

Chloroperoxidase-Catalysed Halogenation of Apolar Compounds Using Reversed Micelles

The chloroperoxidase from the mold Caldariomyces fumago was entrapped into reversed micelles composed of aqueous buffer, cetyltrimethylammonium chloride or bromide, pentanol and octane. The surfactant serves a dual function: i) it stabilizes the reversed micelle, and ii) the halide counter ion is used as a substrate for the enzyme. 2-Monochlorodimedon and 1,3-dihydroxybenzene were halogenated with this system, giving their 2-halo and 4-halo derivatives, respectively. The reaction rates were about twice as high as in aqueous media. Enzyme activity was maximal at high water content of the micelles and at relatively low pentanol concentration. The enzyme was inactivated by high concentrations of hydrogen peroxide.     

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.     

Combined Effects of Radiation and Inorganic Reagents during Irradiation on Radiosensitivity of Bacterial Cells

The combined effects of inorganic reagents and radiation on the inactivation of E. coli in the resting state were studied. Among these reagents halides such as NaCI, KCI, KBr and KI were found to have a considerable synergistic action to radiation. Temperature effect on the halide action during irradiation was not observed, but removal of oxygen from halide solutions increased the radiosensitivity of cells. Combined effects of radiation and some other inorganic reagents were also investigated. Heavy metal salts and hydrogen peroxide were synergistic, nitrates and sulfates having no influence or a slightly protective action. Barium chloride and calcium chloride were protective in lower concentrations and synergistic in higher concentrations. These synergistic actions of inorganic reagents except ferric salts were observed during irradiation, but not after the irradiation.     

Haloperoxidases: Enzymatic Synthesis of alpha,beta-Halohydrins from Gaseous Alkenes

The enzymatic synthesis of alpha,beta-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from alpha,beta-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide.     

Decontamination with vaporized hydrogen peroxide is effective against Mycobacterium tuberculosis

AIMS: To determine the efficacy of room fumigation with vaporized hydrogen peroxide (VHP) in decontamination of viable Mycobacterium tuberculosis. METHODS AND RESULTS: About 8 x 10(4)-2.3 x 10(6) CFU of M. tuberculosis H37Rv and M. tuberculosis Beijing were dried in 10-microl drops in tissue culture plates, placed in steam-permeable Tyvek pouches and distributed on laboratory surfaces. The room was exposed to VHP delivered by air conditioning. Different exposure conditions were tested. Exposure to VHP resulted in sterilization of the bacterial samples in three different test runs. CONCLUSIONS: VHP treatment is an effective means of reducing and eliminating room contaminations of M. tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Fumigation with VHP represents an alternative to formaldehyde fumigation, particularly for decontamination of animal rooms in tuberculosis research laboratories.     

Effects of peroxidase and hydrogen peroxide on the dityrosine formation and the mixing characteristics of wheat-flour dough

The effects of adding hydrogen peroxide and peroxidase to wheat-flour dough on dityrosine formation and mixing characteristics were investigated. Dityrosine in wheat-flour dough was identified by HPLC with a fluorescence detector and by LC/MS/MS. Formation of dityrosine increased with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase, to wheat-flour dough, while the addition of peroxidase had no effect on the amount of dityrosine formed. The mixing curve obtained by a doughgraph changed with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase; the peak time was significantly delayed and the dough development time was extended. We found that dityrosine cross-links in wheat-flour dough increased with the addition of peroxidase plus hydrogen peroxide. It is thought that these cross-links can lead to polymerization of the proteins in wheat-flour dough.     

Effects of Peroxidase and Hydrogen Peroxide on the Dityrosine Formation and the Mixing Characteristics of Wheat-Flour Dough

The effects of adding hydrogen peroxide and peroxidase to wheat-flour dough on dityrosine formation and mixing characteristics were investigated. Dityrosine in wheat-flour dough was identified by HPLC with a fluorescence detector and by LC/MS/MS. Formation of dityrosine increased with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase, to wheat-flour dough, while the addition of peroxidase had no effect on the amount of dityrosine formed. The mixing curve obtained by a doughgraph changed with the addition of hydrogen peroxide, and hydrogen peroxide plus peroxidase; the peak time was significantly delayed and the dough development time was extended. We found that dityrosine cross-links in wheat-flour dough increased with the addition of peroxidase plus hydrogen peroxide. It is thought that these cross-links can lead to polymerization of the proteins in wheat-flour dough.     

Resistance of Serratia marcescens to Hydrogen Peroxide

Irradiation with ultraviolet (u.v.) light (71 J/m²) reduced the viable count of suspenrsions of Serratia marcescens , grown in a glycerol-salts defined medium, to five in 10⁴ cells. Subsequent incubation of irradiated cells in hydrogen peroxide failed to decrease the survivors, but u.v. irradiation in the presence of hydrogen peroxide reduced the viable count to fewer than two in 10⁶ cells. Cells grown in defined medium with added iron had more measurable catalase activity and were more resistant to hydrogen peroxide alone and to simultaneous treatment with u.v. irradiation and hydrogen peroxide. Cells grown in a non-defined medium contained little iron and measurable catalase activity but were more resistant to hydrogen peroxide. Treatment with toluene, heat killing or sonication increased the catalase activity detected in all cell suspensions and showed that resistance to hydrogen peroxide and to u.v. irradiation in hydrogen peroxide was related to the total catalase activity within cells.     

Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

黄腐酸引发软骨细胞产生过氧化氢及硒的抑制作用

为探讨黄腐酸（FA）能否直接刺激软骨细胞产生活性氧以及硒能否抑制由此产生的活性氧,采用二氯荧光素双酯（DCFH－DA）作为软骨细胞产生过氧化氢的探针,用流式细胞技术定量地检测了在FA作用下软骨细胞产生的过氧化氢,并同时检测了硒存在时的过氧化氢含量．发现FA不但能够刺激软骨细胞产生过氧化氢（P＜0．05）,并与FA浓度相关,随FA浓度增大,产生过氧化氢增多,当FA浓度为100mg／L时软骨细胞产生过氧化氢达到最大,之后再增大FA浓度,过氧化氢的产生量减少;硒的存在对FA刺激软骨细胞产生过氧化氢有抑制作用．     

The Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Non-sporing Bacteria

A kill of 99.99% was obtained in cell suspensions of Escherichia coli and Streptococcus faecalis by incubation with hydrogen peroxide 1.0% (w/v) for 75 and 180 min respectively. The same kill was produced by 30 s irradiation with ultraviolet (u.v.) light in the presence of hydrogen peroxide 1.0% (w/v). This simultaneous treatment with u.v. and hydrogen peroxide produced a synergistic kill at least 30-fold greater than that produced by irradiation of cell suspensions of Esch. coli with or without subsequent incubation with hydrogen peroxide.     

Effects of rifampicin and doxycycline on the production of hydrogen peroxide by macrophages]

The influence of rifampicin and doxycycline on oxidative metabolism of macrophages was estimated in vitro by production of hydrogen peroxide. It was shown that low concentrations of rifampicin and doxycycline stimulated production of hydrogen peroxide by macrophages of guinea pigs. In concentrations of 1 to 10 micrograms/ml corresponding to the mean therapeutic ones doxycycline increased both the spontaneous and zymosan-induced production of hydrogen peroxide by the macrophages. The potentiating activity of doxycycline on the cells activated by opsonized zymosan was higher. The maximum increase in the induced production of hydrogen peroxide (by 40 per cent) was observed when the antibiotic concentration was 1 microgram/ml. Rifampicin in concentrations of 0.1 to 1 microgram/ml corresponding to the mean therapeutic ones stimulated the zymosan-induced production of hydrogen peroxide by the macrophages. The maximum increase in the production of hydrogen peroxide (by 22 per cent) was noted at the rifampicin concentration of 1 microgram/ml.