| 1. |
- Kvist, Malin, et al.
(author)
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An investigation of the peroxidase activity of Vitreoscilla hemoglobin
- 2007
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In: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 1432-1327 .- 0949-8257. ; 12:3, s. 324-334
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Journal article (peer-reviewed)abstract
- In order to investigate the ability of the Vitreoscilla hemoglobin (VHb) to act as a peroxidase, the protein was overexpressed in Escerichia coli and purified using a 6xHis-tag. The peroxidase activity of VHb was studied using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferrocene carboxylic acid (FcCOOH) dopamine and L-dopa as substrates. The effects of external agents such as pH, salt concentration/ionic strength, and the thermal stability of VHb on the catalytic activity were assessed. The optimum pH for VHb using ABTS as a substrate was estimated to be 6-7. The VHb protein proved to be stable up to 80 degrees C, as judged by its peroxidase activity. Furthermore, NaCl concentrations up to 100 mM did not exert any significant effect on the activity. The catalytic activity against ABTS and FcCOOH was similar to that measured for horseradish peroxidase, whereas in the case of the phenolic substrates dopamine and L-dopa the activity was several orders of magnitude lower. The Michaelis constants, K-m(H2O2), were in good agreement with the data for human and bovine hemoglobin. No activity could be detected for the negative controls lacking VHb. These results demonstrate that VHb exhibits peroxidase activity, a finding in line with the hypothesis that VHb has cellular functions beyond the role as an oxygen carrier.
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| 2. |
- Ryabova, Ekaterina, et al.
(author)
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A comparative reactivity study of microperoxidases based on hemin, mesohemin and deuterohemin
- 2005
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In: Journal of Inorganic Biochemistry. - : Elsevier BV. - 1873-3344 .- 0162-0134. ; 99:3, s. 852-863
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Journal article (peer-reviewed)abstract
- Three microperoxidases-hemin-6(7)-gly-gly-his methyl ester (HGGH), mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH)-have been prepared as models for heme-containing peroxidases by condensation Of glycyl-glycyl-L-histidine methyl ester with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. The three microperoxidases differ in two substituents, R, of the protoporphyrin IX framework (HGGH: R = vinyl, MGGH: R = ethyl, DGGH: R = H). X-band and high field EPR spectra show that the microperoxidases exhibit spectroscopic properties similar to those of metmyoglobin, i.e. a high spin ferric S = 5/2 signal at g(perpendicular to) = 6 and g(parallel to) = 2 and an estimated D value of 7.5 +/- 1 cm(-1). The catalytic activities of the microperoxidases towards K-4[Fe(CN)(6)], L-tyrosine methyl ester and 2,2'-azino(bis(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS) have been investigated. It was found that all three microperoxidases exhibit peroxidase activity and that the reactions follow the generally accepted peroxidase reaction scheme [Biochem. J. 145 (1975) 93-103] with the exception that the initial formation of a Compound I analogue is the rate-limiting step for the whole process. The general activity trend was found to be MGGH approximate to DGGH > HGGH. For each microperoxidase, DFT calculations (B3LYP) were made on the reactions of compounds 0, I and II with H+, e(-) and H+ + e(-), respectively, in order to probe the possible relationship between the nature of the 2- and 4-substituents of the hemin and the observed reactivity. The computational modeling indicates that the relative energy differences are very small; solvation and electrostatic effects may be factors that decide the relative activities of the microperoxidases. (C) 2005 Elsevier Inc. All rights reserved.
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| 3. |
- Ryabova, Ekaterina
(author)
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An Investigation of Peroxidase Activity in Biomimetic and Biological Systems
- 2004
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Doctoral thesis (other academic/artistic)abstract
- In order to create a heme environment which permits biomimicry of heme-containing plant peroxidases and investigate the role played by the vinyl side chains of the porphyrin ring on the stability and, possibly, catalytic activity of model compounds, a number of new synthetic microperoxidases -– hemin-6(7)-gly-his methyl ester (HGH) and hemin-6(7)-gly-gly-his methyl ester (HGGH) and hemin-6,7-bis(gly-gly-his methyl ester) (H2GGH) as well as mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH) have been prepared by condensation of a peptide residue with the propionic side chains of hemin, mesohemin and deuterohemin, respectively. Reactivity studies towards different organic and inorganic substrates showed that not only five- but also six-coordinate hemin peptide complexes catalyze the oxidation of substrates by H2O2. The HGGH complex showed higher catalytic activity than HGH due to a less strained structure provided by the longer peptide arm. Microperoxidases based on meso- and deuterohemin (MGGH and DGGH, respectively) exhibited at least one order of magnitude higher reactivity relative to their hemin-based analogues. However, computational modelling showed that the relative energy differences between the different hemins are small, suggesting that more subtle factors (e.g. properties of reaction media) rather that only electronic properties of substituents of the porphyrin ring determine the reactivity order of the hemin peptide complexes studied. In general, the hemin-peptide complexes showed relatively low peroxidase reactivity. On the contrary, manganese(III) microperoxidase (MnGGH) showed relatively high reactivity in peroxidase-type reactions. Rate constants for Compound I formation obtained by fast kinetics studies as well as steady-state rate constants for the reaction of MnGGH with the substrates were comparable to these observed for manganese microperoxidase-8 (MnMP-8). The higher peroxidase reactivity of MnGGH as compared to its iron(III) analogue, HGGH, may be attributed to the higher operational stability of the manganese complexes as well as higher stability of Mn(III) oxo intermediates. An investigation of the peroxidase reactivity of bacterial hemoglobin from Vitreoscilla stercoraria (VHb) showed for the first time that VHb does exhibit peroxidase catalytic activity which for certain substrates is comparable with the activity of horseradish peroxidase. VHb also showed unusual substrate specificity (for peroxidases); i.e. good activity was observed only for substrates with more than one hydrogen bond donor/acceptor. In order to create a heme environment which permits biomimicry of heme-containing plant peroxidases and investigate the role played by the vinyl side chains of the porphyrin ring on the stability and catalytic activity of model compounds, a number of new synthetic microperoxidases – hemin-6(7)-gly-his methyl ester (HGH), hemin-6(7)-gly-gly-his methyl ester (HGGH) and hemin-6,7-bis(gly-gly-his methyl ester) (H2GGH) as well as mesohemin-6(7)-gly-gly-his methyl ester (MGGH) and deuterohemin-6(7)-gly-gly-his methyl ester (DGGH) have been prepared by condensation of a peptide residue with the propionic acid side chains of hemin, mesohemin and deuterohemin, respectively. Reactivity studies towards different organic and inorganic substrates showed that not only five- but also six-coordinate hemin peptide complexes catalyze the oxidation of substrates by H2O2. The HGGH complex showed higher catalytic activity than HGH, possibly due to a less strained structure provided by the longer peptide arm. Microperoxidases based on meso- and deuterohemin (MGGH and DGGH, respectively) exhibited at least one order of magnitude higher reactivity relative to their hemin-based analogues. However, computational modelling showed that the relative energy differences between the different hemins are small, suggesting that more subtle factors (e.g. properties of reaction media) rather that only electronic properties of the substituents of the porphyrin ring determine the reactivity order of the hemin peptide complexes studied. Manganese(III) microperoxidase (MnGGH) showed relatively high reactivity in peroxidase-type reactions. Rate constants for Compound I formation obtained by fast kinetics studies as well as steady-state rate constants for the reaction of MnGGH with substrates were comparable to these observed for manganese microperoxidase-8. The higher peroxidase reactivity of MnGGH as compared to its iron(III) analogue, HGGH, may be attributed to the higher operational stability of the manganese complex as well as higher stability of Mn(III) oxo intermediates. An investigation of the peroxidase reactivity of the bacterial hemoglobin from Vitreoscilla stercoraria (VHb) showed for the first time that VHb does exhibit peroxidase catalytic activity which for certain substrates is comparable with the activity of horseradish peroxidase. VHb also showed unusual substrate specificity (for peroxidases); i.e. good activity was observed only for substrates with more than one hydrogen bond donor/acceptor.
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| 4. |
- Ryabova, Ekaterina, et al.
(author)
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Preparation and reactivity studies of synthetic microperoxidases containing b-type heme
- 2004
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In: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 1432-1327 .- 0949-8257. ; 9:4, s. 385-395
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Journal article (peer-reviewed)abstract
- In order to create a heme environment that permits biomimicry of heme-containing peroxidases, a number of new hemin–peptide complexes—hemin-2(18)-glycyl-l-histidine methyl ester (HGH), hemin-2(18)-glycyl-glycyl-l-histidine methyl ester (HGGH), and hemin-2,18-bis(glycyl-glycyl-l-histidine methyl ester) (H2GGH)—have been prepared by condensation of glycyl-l-histidine methyl ester or glycyl-glycyl-l-histidine methyl ester with the propionic side chains of hemin. Characterization by means of UV/vis- and 1H NMR spectroscopy as well as cyclic- and differential pulse voltammetry indicates the formation of five-coordinate complexes in the case of HGH and HGGH, with histidine as an axial ligand. In the case of H2GGH, a six-coordinate complex with both imidazoles coordinated to the iron center appears to be formed. However, 1H NMR of H2GGH reveals the existence of an equilibrium between low-spin six-coordinate and high-spin five-coordinate species in solution. The catalytic activity of the hemin–peptide complexes towards several organic substrates, such as p-cresol, l-tyrosine methyl ester, and ABTS, has been investigated. It was found that not only the five-coordinate HGH and HGGH complexes, but also the six-coordinate H2GGH, catalyze the oxidation of substrates by H2O2. The longer and less strained peptide arm provides the HGGH complex with a slightly higher catalytic efficiency, as compared with HGH, due to formation of more stable intermediate complexes.
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| 5. |
- Ryabova, Ekaterina, et al.
(author)
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Synthesis and reactivity studies of a manganese microperoxidase containing b-type heme
- 2005
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In: Dalton Transactions. - : Royal Society of Chemistry (RSC). - 1477-9234. ; :7, s. 1228-1233
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Journal article (peer-reviewed)abstract
- Mn(III) protoporphyrin IX-6(7)-gly-gly-his methyl ester (MnGGH) has been prepared by condensation of glycyl-glycyl-L-histidine methyl ester with the propionic side chains of Mn(III) protoporphyrin IX. It was characterised by mass spectrometry and UV/VIS spectroscopy. Stopped-flow spectrophotometry was used to study the reaction of the Mn microperoxidase with hydrogen peroxide. The formation of active intermediates analogous to previously described metal-hydroperoxo (compound 0) and metal oxo (compound I) intermediates of the natural Fe(III) microperoxidase-8 and Mn(III) microperoxidase-8 was observed. The rate of formation of the MnGGH-based compound I analogue was found to increase dramatically with increasing pH. A steady-state kinetic analysis of the catalytic peroxidase activity of MnGGH towards K-4[Fe(CN)(6)], L-tyrosine methyl ester, o-dianisidine, o-methoxyphenol and ascorbic acid showed that the peroxidase reaction proceeds via the formation of a microperoxidase substrate complex followed by electron transfer from the substrate to the metal. The reactivity of MnGGH depends on the size and hydrophobicity of the substrate, and these properties appear to influence the rate of the electron transfer, which is the rate-limiting step for the whole process. MnGGH showed higher reactivity towards reducing substrates than its Fe(III) analogue.
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