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R Wever

Publications and source records attributed to R Wever.

At least 73 records · Page 4Linked to original sources
PubMed1988

Photodissociation of cytochrome c oxidase-nitric oxide complexes.

The dissociation of cytochrome c oxidase-nitric oxide complexes was studied by optical spectroscopy at cryogenic temperatures (15 degrees K). With the reduced cytochrome c oxidase-nitric oxide complex, the observations that were reported by Yoshida et al. were confirmed. Photodissociation of the oxidized cytochrome c oxidase-nitric oxide complex did not induce any significant absorbance changes between 350 and 875 nm. With the azide-nitrosyl-cytochrome c oxidase complex, the illumination caused the dissociation of the a2+(3).NO complex to the unligated state a2+(3). Increasing the temperature to 77 degrees K led to the formation of a new complex, probably a3+(3).N3-. The N3(-)-NO-cytochrome c oxidase complex was the only compound for which appreciable photodissociation was achieved by continuous illumination at room temperature (20 degrees C). The effect of illumination was biphasic. In the first phase the a2+(3).NO complex is dissociated and cytochrome a3 oxidized by an electron transfer to CuB. In the second phase nitric oxide, which is still bound to CuB after the first phase, is expelled from the complex by azide, with a concomitant electron transfer from CuB to cytochrome a.

Carbon Monoxide↗
PubMed1988

Structure and function of vanadium-containing bromoperoxidases.

The properties of the vanadium-containing bromoperoxidases from the seaweeds Ascophyllum nodosum, Laminaria saccharina and the lichen Xanthoria parietina were studied. Upon reduction with sodium dithionite, these bromoperoxidases show EPR spectra which are typical of a vanadyl cation (VO2+). From the spectral parameters and a comparison with inorganic vanadyl complexes, we conclude that the ligand environment largely consists of oxygen donors. The data also show that the structure of the active sites in these enzymes is very similar. Since EPR spectra of vanadium(IV) bromoperoxidase are only obtained after reduction, the metal ion is present in the native enzymes in the 5+ oxidation state. All these enzymes loose their enzymic activity upon dialysis against citrate-phosphate (PO4(3-)) buffer at pH 3.8, containing EDTA. The brominating activity could be reconstituted by the addition of vanadate (VO4(3-)). The experiments suggest that vanadate is incorporated into these enzymes. In line with the EPR data, we propose a structure of the active site in which at least 4 oxygen atoms are present as donors for the central vanadium(V) ion. Since several inorganic peroxovanadium(V) complexes have been described, we suggest that the vanadium ion in bromoperoxidases serves as a binding site for H2O2. Upon subsequent binding of bromide this ion is oxidized by the peroxo-intermediate to form hypobromite. This model does not require valence state changes of the metal ion itself and indeed no changes in the EPR spectrum of reduced bromoperoxidase are observed upon addition of H2O2 or Br-. Further, bromoperoxidase reduced with a small excess of sodium dithionite is not active in the bromination reaction. The bromoperoxidases from the various sources show similarity in the amino-acid composition with a predominance of acidic amino acids. Distinct pH optima are observed in the bromination reaction catalysed by the bromoperoxidases. Despite the presence of the same prosthetic group in these enzymes with comparable vanadium ligand-field environment, the enzymic properties are very different. The specific activity as well as the Km for bromide differ greatly. Unlike the enzymes from the seaweeds A. nodosum and L. saccharina the bromoperoxidase from the lichen X. parietina is inhibited by low concentrations (1-5 mM) of nitrate. These bromoperoxidases have a remarkable resistance towards organic solvents such as methanol, ethanol and propanol.

Amino Acids↗
PubMed1987

The bromoperoxidase from the lichen Xanthoria parietina is a novel vanadium enzyme.

A novel bromoperoxidase was isolated from the lichen Xanthoria parietina. The enzyme contained vanadium, which is essential for enzymic activity. Under denaturating conditions the preparation showed a single protein band with an Mr of 65,000. Thermal-denaturation studies showed that this bromoperoxidase could tolerate high temperatures. The affinity of the enzyme for its substrate bromide is high; the Km for bromide was 29 microM. Excess halides (50 mM) inhibited enzymic activity considerably.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

The reaction of nitric oxide with copper proteins and the photodissociation of copper-NO complexes.

The reactivity with nitric oxide was investigated for a number of type-1, type-2 and type-3 copper proteins azurin from Pseudomonas aeruginosa (type-1 copper); bovine superoxide dismutase, diamine oxidase from pig kidney and galactose oxidase from Dactylium dendroides (type-2 copper); haemocyanin from Helix pomatia (type-3 copper); the blue oxidases ceruloplasmin from pig serum, and ascorbate oxidase from Cucurbita pepo medullosa. Type-1 copper formed complexes with NO in the oxidised state, which complexes were only fully formed at low temperatures and could be photodissociated at 77K. Complex formation led to the disappearance of the EPR signal of type-1 copper and of the optical absorbance band in the 600 nm region. In azurin, photodissociation caused the reappearance of the original 625 nm absorbance band, but in the blue oxidases, a new band with lower intensity was found at 595 nm instead of the original absorbance band at 610 nm. In all cases, the EPR signal of type-1 copper did not return. These results are best explained by the formation of a photolabile type-1 Cu1+-NO+ complex. They also indicate that in the complex formed, the type-1 copper structure is probably not disrupted, and that after illumination, the nitric oxide molecule is still in the near vicinity of the copper atom. Type-2 copper did not react at all with nitric oxide, and type-3 copper formed complexes with nitric oxide in both the oxidised and the reduced state, but photodissociation of these complexes could not be demonstrated.

Amine Oxidase (Copper-Containing)↗
PubMed1987

Spectral properties of myeloperoxidase compounds II and III.

In order to resolve the confusion about the spectral properties of myeloperoxidase Compound II and Compound III (myeloperoxidase is donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), the absorbance spectra in the visible and ultraviolet regions were measured under conditions where either Compound II or Compound III was present. Peak positions, isosbestic points and absorption coefficients are presented. We conclude that in most studies on Compound II or Compound III, mixtures of these compounds had been present. Our data indicate that the relative contributions of Compound II and Compound III in a sample can be determined from the absorbance ratio A625nm/A456nm. The optical absorbance spectrum of myeloperoxidase compound III was not affected by pH (pH 3-8). The absorbance spectrum of Compound II, however, was dependent on pH. The absorbance spectrum of Compound II at high pH is described.

Humans↗
PubMed1987

The effect of D-penicillamine on myeloperoxidase: formation of compound III and inhibition of the chlorinating activity.

The inhibitory effect of the anti-arthritic drug D-penicillamine on the formation of hypochlorite (HOCl) by myeloperoxidase from H2O2 and Cl- was investigated. When D-penicillamine was added to myeloperoxidase under turnover conditions, Compound III was formed, the superoxide derivative of the enzyme. Compound III was not formed when D-penicillamine was added in the presence of EDTA or in the absence of oxygen. However, when H2O2 was added to myeloperoxidase, D-penicillamine and EDTA, Compound III was formed. Therefore it is concluded that formation of Compound III is initiated by metal-catalysed oxidation of the thiol group of this anti-arthritic drug, resulting in formation of superoxide anions. Once Compound III is formed, a chain reaction is started via which the thiol groups of other D-penicillamine molecules are oxidized to disulphides. Concomitantly, Compound I of myeloperoxidase would be reduced to Compound II and superoxide anions would be generated from oxygen. This conclusion is supported by experiments which showed that formation of Compound III of myeloperoxidase by D-penicillamine depended on the chloride concentration. Thus, an enzyme intermediate which is active in chlorination (i.e. Compound I) participated in the generation of superoxide anions from the anti-arthritic drug. From the results described in this paper it is proposed that D-penicillamine may exert its therapeutic effect in the treatment of rheumatoid arthritis by scavenging HOCl and by converting myeloperoxidase to Compound III, which is inactive in the formation of HOCl.

Chemical Phenomena↗
PubMed1986

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide.

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.

Animals↗
PubMed1986

The superoxide dismutase activity of myeloperoxidase; formation of compound III.

The reaction of superoxide anions with myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which results in the formation of Compound III of myeloperoxidase, was investigated. It is shown that myeloperoxidase has a high affinity for superoxide anions because formation of Compound III was only partially inhibited by high concentrations of superoxide dismutase. Furthermore, when superoxide anions were generated in a mixture of both cytochrome c and myeloperoxidase in the absence of Cl-, only Compound III was formed and reduction of cytochrome c was not observed. In the presence of Cl-, Compound III was also formed and reduction of cytochrome c was inhibited. From the results described in this paper we conclude that Compound III is able to react with superoxide anions, probably resulting in formation of an intermediate (Compound I) which is catalytically active in the oxidation of Cl- to yield hypochlorous acid (HOCl). Because Compound III of myeloperoxidase is formed in phagocytosing neutrophils (Winterbourn, C.C., Garcia, R.C. and Segal, A.W. (1985) Biochem. J. 228, 583-592) we propose that, in vivo, myeloperoxidase also acts as a superoxide dismutase, and via formation of Compound I uses superoxide anions in the formation of HOCl.

Catalase↗
PubMed1985

The oxidation of cytochrome c oxidase by hydrogen peroxide.

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.

Animals↗
PubMed1985

Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent.

Soret excitation resonance Raman spectroscopy has been used to characterize dimeric human leukocyte myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) and monomeric bovine spleen green haemoprotein. The spectra of the two proteins, under the same conditions of iron valence and ligation, are essentially identical. Owing to strong symmetry reduction effects, the spectra are more complex than usually observed for haemoproteins. It is possible, however, to assign the high-frequency vibrations and, from these assignments, to determine structural features of the iron chromophores. In the resting protein, the iron adopts a six-coordinate high-spin configuration in both proteins; cyanide addition produces six-coordinate low-spin species, and in the ferrous enzymes the iron appears to be five-coordinate and high-spin. The proteins are stable to laser excitation and do not photoreduce under illumination. No evidence is found for unusual peripheral substituents, such as formyl or protonated Schiff's base group, in conjugation with the main chromophore in the native protein. The vibrational data are consistent with an iron chlorin chromophore, although other electronic effects, in addition to those produced by porphyrin ring reduction, are necessary to account for the optical properties of the proteins. The similarity in Raman spectra for myeloperoxidase and green haemoprotein indicates that the two iron sites in myeloperoxidase are equivalent.

Animals↗
PubMed1985

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.

Amino Acids↗
PubMed1985

Antiarthritic drugs containing thiol groups scavenge hypochlorite and inhibit its formation by myeloperoxidase from human leukocytes. A therapeutic mechanism of these drugs in rheumatoid arthritis?

We investigated the effect of antiarthritic drugs containing thiol groups, such as D-penicillamine, tiopronin (N-[2-mercaptopropionyl]glycine), sodium aurothiomalate, and aurothioglucose, on the chlorinating activity of myeloperoxidase purified from human leukocytes. Hypochlorite, the reactive product of the reaction catalyzed by myeloperoxidase, was effectively scavenged by these antiarthritic drugs, and in addition, D-penicillamine and tiopronin inhibited myeloperoxidase itself. The above-mentioned effects of these drugs were observed at concentrations that occur in the serum of rheumatoid arthritis patients treated with these agents. We suggest that the therapeutic effect of these antiarthritic drugs may be due to the protection of tissues against the reactive HOCI released by activated granulocytes at inflamed sites.

Amino Acids, Sulfur↗
PubMed1984

The nitrosyl compounds of ferrous animal haloperoxidases.

Human myeloperoxidase, human eosinophil peroxidase and bovine lactoperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) reduced with ascorbic acid form nitrosyl compounds which show rhombic EPR signals centered at g = 2. Using 14NO (IN = 1), the central resonance signal exhibited a hyperfine structure of nine lines originating from a triplet with a small hyperfine splitting (AII(zeta) = 0.69 mT for myeloperoxidase and 0.73 mT for eosinophil peroxidase and lactoperoxidase) superimposed upon a triplet with a larger hyperfine splitting (AI(zeta) = 2.34, 2.32 and 2.09 mT for myeloperoxidase, eosinophil peroxidase and lactoperoxidase, respectively). Using 15NO (IN = 1/2), the nitrosyl compound of ferrous myeloperoxidase and ferrous lactoperoxidase showed a doublet of triplets superimposed upon the central resonance signal. These results demonstrate that a nitrogen nucleus is present at the fifth ligand position of the haem iron in these peroxidases.

Electron Spin Resonance Spectroscopy↗
PubMed1984

A kinetic study of the reaction between human myeloperoxidase, hydroperoxides and cyanide. Inhibition by chloride and thiocyanate.

The reaction between myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), hydrogen peroxide and ethyl hydroperoxide was investigated using the stopped-flow technique. Like other peroxidases, myeloperoxidase forms two sequential peroxide compounds. The pH-dependence of the apparent second-order rate constant of compound I formation shows that there is an acid/base group on the enzyme with a pKa of 4.30 +/- 0.15, which - when protonated - prevents the reaction of the enzyme with peroxides. The rate constants for the formation of compound I by hydrogen peroxide and ethyl hydroperoxide are (2.3 +/- 0.1) X 10(7) M-1 X s-1 and (2.8 +/- 0.3) X 10(5) M-1 X s-1, respectively. The binding of cyanide to myeloperoxidase (k1 = (1.30 +/- 0.05) X 10(6) M-1 X s-1) is also regulated by an acid/base group with a pKa of 4.00 +/- 0.05 as is the case with hydrogen peroxide; also, only the protonated uncharged form of cyanide reacts with the enzyme. From their effects on the binding of cyanide to the enzyme it is concluded that chloride and thiocyanate bind to myeloperoxidase only when the acid/base group is protonated. The pH-dependence of the dissociation constant of the myeloperoxidase-chloride complex obtained from the spectral changes induced by chloride is the same as observed in the inhibition by chloride of the binding of cyanide. It is concluded that hydrogen peroxide, cyanide, chloride and thiocyanate bind at the same site on the enzyme.

Chlorides↗
PubMed1984

The cytochrome c oxidase-azide-nitric oxide complex as a model for the oxygen-binding site.

The complex of cytochrome c oxidase with NO and azide has been studied by EPR at 9.2 and 35 GHz. This complex which shows delta ms = 2 EPR triplet and strong anisotropic signals, due to the interaction of cytochrome a2+3 X NO (S = 1/2) and Cu2+B (S = 1/2), is photodissociable . Its action spectrum is similar to that of cytochrome a2+3 X NO with bands at 430, 560 and 595 nm, but shows an additional band in the near ultraviolet region. The quantum yield of the photodissociation process of cytochrome a2+3 X NO in the metal pair appears to depend on the redox state of CuB. When the photolysed sample was warmed to 77 K, a complex was observed with the EPR parameters of cytochrome a3+3 - N-3 - Cu1 +B (S = 1/2). This process of electron and ligand transfer can be reversed by heating the sample to 220 K. It is suggested that in the triplet species azide is bound to Cu2+B whereas NO is bridged between Cu2+B and the haem iron of the cytochrome a2+3. The complex has a triplet ground state and a singlet excited state with an exchange interaction J = -7.1 cm-1 between both spins. The anisotropy in the EPR spectra is mainly due to a magnetic dipole-dipole interaction between cytochrome a2+3 X NO and Cu2+B. From simulations of the triplet EPR spectra obtained at 9 and 35 GHz, a value for the distance between the nitroxide radical and Cu2+B of 0.33 nm was found. A model of the NO binding in the cytochrome a3-Cu pair shows a distance between the haem iron of cytochrome a3 and CuB of 0.45 nm. It is concluded that the cytochrome a3-CuB pair forms a cage in which the dioxygen molecule is bidentate coordinated to the two metals during the catalytic reaction.

Azides↗
PubMed1984

Some properties of human eosinophil peroxidase, a comparison with other peroxidases.

Eosinophil peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) was isolated from outdated human white blood cells. The purified enzyme has a molecular weight of 71000 +/- 1000. The enzyme is composed of two subunits, of Mr 58000 and 14000, in a 1:1 stoichiometry. Amino-acid analyses showed that eosinophil peroxidase has a high content of the amino acids arginine, leucine and aspartic acid. The millimolar absorbance coefficient of the Soret band at 412 nm of eosinophil peroxidase was determined. Three independent methods yield a value for epsilon 412nm of 110 +/- 4 mm-1 X cm-1. Purified eosinophil peroxidase showed a homogeneous high-spin EPR signal with rhombic symmetry (gx = 6.50; gy = 5.40; gz = 1.982) for the haem group. EPR spectroscopy of low-spin cyanide and azide derivatives of eosinophil peroxidase, lactoperoxidase, myeloperoxidase and catalase revealed that the haem-ligand structure of eosinophil peroxidase is closely related to lactoperoxidase, whereas that of myeloperoxidase shows great resemblance to catalase.

Amino Acids↗
PubMed1984

Vitamin C stimulates the chlorinating activity of human myeloperoxidase.

Ascorbic acid (vitamin C) was found to stimulate the chlorinating activity of human myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) 3-fold in vitro and to shift the pH optimum of the reaction to higher pH values. These effects are due to the conversion by ascorbic acid of inactive compound II formed during turnover into native enzyme.

Ascorbic Acid↗