The formation of ferric haem during low-temperature photolysis of horseradish peroxidase Compound I.
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ABSTRACT:
SUBMITTER: Foote N
PROVIDER: S-EPMC1148330 | biostudies-other | 1987 Sep
REPOSITORIES: biostudies-other
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Project description:The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has definitively been observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881-1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252-2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75%, and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate of kBA ? (3.6 ps)(-1) is the dominant process, some CN(-) exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN(-) association rate, we find that the CN(-) escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ? (1-2) × 10(7) s(-1). This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1 × 10(7) s(-1)) under the same conditions. The analysis leads to an escape probability kout/(kout + kBA) ~ 10(-4), which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN(-) bimolecular association rate (170 M(-1) s(-1)), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN(-) bimolecular association rate is larger by ~10(3), making the CN(-) photolysis more difficult to observe.
| S-EPMC3631277 | biostudies-literature
Project description:BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.
| S-EPMC3987668 | biostudies-literature
Project description:1. The kinetics of formation of horseradish peroxidase Compound I were studied by using peroxobenzoic acid and ten substituted peroxobenzoic acids as substrates. Kinetic data for the formation of Compound I with H2O2 and for the reaction of deuteroferrihaem with H2O2 and peroxobenzoic acids, to form a peroxidatically active intermediate, are included for comparison. 2. The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10, in contrast with pH-independence of the reaction with H2O2. The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules. 3. The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2. Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects. The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution, but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions. 4. Possible reaction mechanisms are considered by which the apparent conflict between the pH-effect and substituent-effect data may be resolved. A model in which it is postulated that a negatively charged 'electrostatic gate' controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.
| S-EPMC1163839 | biostudies-other
Project description:The role of haem propionates in oxidative and reductive reactions catalysed by horseradish peroxidase (HRP) was studied after successful reconstitution of ferric protoporphyrin IX dimethyl ester (PPDME) into the apoperoxidase. The reconstituted enzyme oxidizes neither guaiacol (aromatic electron donor) nor iodide or thiocyanate (inorganic donor). Although the reconstituted enzyme binds guaiacol with a similar Kd (13 mM) to that of the native enzyme (10 mM), the Kd for SCN- binding (5 mM) is decreased 20-fold compared with that of the native enzyme (100 mM). This indicates that haem propionates hinder the entry or binding of inorganic anion to the active site of the native HRP. However, the reconstituted enzyme is catalytically inactive as it does not form spectroscopically detectable compound II with H2O2. CD measurements indicate a significant loss of haem CD spectrum of the reconstituted enzyme at 409 nm, suggesting a loss of asymmetry of the haem-protein interaction. Thus the inability of the reconstituted enzyme to form catalytic intermediates results from the change in orientation of the haem due to loss of interactions via the haem propionates. HRP also catalyses reductive reactions such as reduction of iodine (I+) in the presence of EDTA and H2O2. The reconstituted enzyme cannot catalyse I+ reduction because of the loss of I+ binding to the haem propionate. Since I+ reduction requires formation of the catalytically active enzyme-I+-EDTA ternary complex, the loss of reductive activity is primarily due to the loss of active enzyme formation. Haem propionates thus play a vital role in the oxidative and reductive reactions of HRP by favouring the formation of catalytic intermediates with H2O2 by maintaining the correct orientation of the haem with respect to the surrounding residues.
| S-EPMC1219660 | biostudies-other
Project description:Strong electron-paramagnetic-resonance signals in the g = 2.00 region were detected after irradiation of horseradish peroxidase Compound I at temperatures of 10 and 100 K. These signals establish the presence of new free-radical species in the peroxidase system. The new species are interpreted in terms of a haem-photosensitized oxidation of the protein's peptide groups close to the Compound I radical site. On warming to room temperature, the radicals decayed irreversibly to a species having a weak asymmetric electron-paramagnetic-resonance signal at 100 K, which could still be observed after incubation at room temperature for more than 1 h.
| S-EPMC1183618 | biostudies-other
Project description:We have investigated the effects of mutations at residues His-42, Arg-38 and Phe-41 in the distal haem pocket of horseradish peroxidase on the changes in protonation state that accompany redox- and ligand-linked changes to the haem group. The mutations H42L and R38L result in the loss of a characteristic pH dependency in the visible spectrum of the ferrous form and a diminished dependency of the midpoint redox potential of the haem group on pH. The results support the view that His-42, with its pK probably modulated by Arg-38, provides the protonation site on the reduced enzyme that is responsible for these pH dependencies. The mutations H42L and R38L also have major effects on the binding of cyanide to the haem. We have already reported that binding of cyanide to the ferrous forms of these mutants becomes too weak to be measurable [Meunier, Rodriguez-Lopez, Smith, Thorneley and Rich (1995) Biochemistry 34, 14687-14692]. The pH dependency of the rate constants for binding of cyanide to the oxidized form of H42L suggests that CN- is the kinetically active species, in contrast with wild-type horseradish peroxidase, where HCN is the binding form. For the R38L variant, the pH dependency of cyanide binding suggests that the pK of His-42 in the absence of Arg-38 is raised to 7.5-8, in the oxidized form. In contrast with these changes, the mutant F41A exhibits cyanide-binding behaviour that is similar to that of the wild type, both in its oxidized and reduced forms. However, the rate constant for carbon monoxide recombination increases substantially, suggesting that the access route for carbon monoxide, but not for cyanide, is perturbed by this amino acid substitution.
| S-EPMC1219140 | biostudies-other
Project description:Background and aimsPlants have evolved a number of inducible defence mechanisms against pathogen attack, including synthesis of pathogenesis-related proteins. The aim of the study was to purify and characterize antifungal protein from leaves of Acorus calamus.MethodsLeaf proteins from A. calamus were fractionated by cation exchange chromatography and gel filtration and the fraction inhibiting the hyphal extension of phytopathogens was characterized. The temperature stability and pH optima of the protein were determined and its presence was localized in the leaf tissues.Key resultsThe purified protein was identified as a class III haem peroxidase with a molecular weight of approx. 32 kDa and pI of 7.93. The temperature stability of the enzyme was observed from 5 degrees C to 60 degrees C with a temperature optimum of 36 degrees C. Maximum enzyme activity was registered at pH 5.5. The pH and temperature optima were corroborated with the antifungal activity of the enzyme. The enzyme was localized in the leaf epidermal cells and lumen tissues of xylem, characteristic of class III peroxidases. The toxic nature of the enzyme which inhibited hyphal growth was demonstrated against phytopathogens such as Macrophomina phaseolina, Fusarium moniliforme and Trichosporium vesiculosum. Microscopic observations revealed distortion in the hyphal structure with stunted growth, increased volume and extensive hyphal branching.ConclusionsThis study indicates that peroxidases may have a role to play in host defence by inhibiting the hyphal extension of invading pathogens.
| S-EPMC2803583 | biostudies-literature
Project description:The formation of a reversible adsorption complex between a dimer of N-acetyl-L-tyrosine [di-(N-acetyl-L-tyrosine), (NAT)2] and horseradish peroxidase (HRP) compound II (CII) was demonstrated using a kinetic approach. A specific KIIm value (0.58 mM) was deduced for this step from stopped-flow measurements. The dimerization of the dipeptide Gly-Tyr was analysed at the steady state and compared with (NAT)2 dimerization [(NAT)2-->(NAT)4]. A saturation of the enzyme was observed for both substrates within their range of solubility. In each case the rate of dimerization reflected the rate-limiting step of compound II reduction to the native HRP (E) (kappcat/Kappm approximately kII-->E). The kappcat values for (Gly-Tyr)2 and (NAT)4 formation were 254 s-1 and 3.6 s-1 respectively. The KappM value of Gly-Tyr was 24 mM. It was observed that the value (0.7 mM) for (NAT)2 was close both to its specific KIIm value for the second step of reduction (CII-->E) and to its thermodynamic dissociation constant (Kd=0.7 mM) with the resting form of the enzyme. As (NAT)2 was a tighter ligand but a poorer substrate than Gly-Tyr, a steady-state kinetic study was performed in the presence of both substrates. A kinetic model which includes an enzyme-substrate adsorption prior to each of the two steps of reduction was derived. This one agreed reasonably well with the experimental data.
| S-EPMC1220252 | biostudies-other
Project description:The present study explains computational methods to design thermostable horseradish peroxidase enzyme using the crystal structure available from Protein Data Bank (PDB ID: 6ATJ). Multiple mutations were introduced to the original enzyme and developed a model by using Modeler9.14. After designing the model functional effect was confirmed in terms of protein ligand binding by molecular docking using Autodock 4.2. The implementation of modeling steps is demonstrated in the context of performing mutations for particular amino acid residue on the ligand pocket of the horseradish peroxidase, to derive the desired ligand binding properties. The docking investigation of modelled HRP with Quercetindihydroxide using Autodock 4.2 software that six amino acid residues, P139, H42, A31, L174, A38, and G169 are involved in hydrogen bonding. More importantly, it provides insight into understanding and properly interpreting the data produced by these methods. The 3D model was docked with Quercetindihydroxide (a known horseradish modulator) to understand molecular interactions at the active site region.
| S-EPMC5322315 | biostudies-literature
Project description:Hydrogels provide promising applications in tissue engineering and regenerative medicine, with silk fibroin (SF) offering biocompatibility, biodegradability and tunable mechanical properties. The molecular weight (MW) distribution of SF chains varies from ∼80 to 400 kDa depending on the extraction and purification process utilized to prepare the protein polymer. Here, we report a fundamental study on the effect of different silk degumming (extraction) time (DT) on biomaterial properties of enzymatically crosslinked hydrogels, including secondary structure, mechanical stiffness, in vitro degradation, swelling/contraction, optical transparency and cell behaviour. The results indicate that DT plays a crucial role in determining material properties of the hydrogel; decrease in DT increases β-sheet (crystal) formation and mechanical stiffness while decreasing degradation rate and optical transparency. The findings on the relationships between properties of silk hydrogels and DT should facilitate the more rational design of silk-based hydrogel biomaterials to match properties needed for diverse purpose in biomedical engineering.
| S-EPMC7390697 | biostudies-literature