Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.     

Resonance Raman spectroscopy shows different temperature-dependent coordination equilibria for native horseradish and cytochrome c peroxidase

Resonance Raman spectra are reported for native horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP) at 290, 77 and 9 K, using 406.7 nm excitation, in resonance with the Soret electronic transition. The spectra reveal temperature-dependent equilibria involving changes in coordination or spin state. At 290 K and pH 6.5, CCP contains a mixture of 5- and 6-coordinate high-spin Fe^III heme while at 9 K the equilibrium is shifted entirely to the 6-coordinate species. The spectra indicate weak binding of H₂O to the heme Pe, consistent with the long distance, 2.4 Å, seen in the crystal structure. At 290 K HRP also contains a mixture of high-spin Fe^III hemes with the 5-coordinate form predominant. At low temperature, a small 6-coordinate high-spin component remains but the 5-coordinate high-spin spectrum is replaced by another which is characteristic either of 6-coordinate low-spin or 5-coordinate intermediate spin heme. The latter species is definitely indicated by previous EPR studies at low temperature. This behavior implies that, in contrast to CCP, the distal coordination site of HRP is only partially occupied by H₂O at any temperature and that lowering the temperature significantly weakens the Fe-proximal imidazole bond. Consistent with this inference, the 77 K spectrum of reduced HRP shows an appreciable fraction of molecules having an Fe-imidazole stretching frequency of 222 cm⁻¹, a value indicating weakened H-bonding of the proximal imidazole.Resonance Roman spectroscopyHorseradish peroxidaseCytochrome c peroxidaseCoordination equilibrium     

Determination of isoelectric points in thin-layer isoelectric focusing: The importance of attaining the steady state and the role of CO2 interference

Isoelectric points differing by 1 to 2 pH units are measured for horseradish peroxidase and lactoperoxidase depending upon the technique of isoelectric focusing, namely, the density gradient technique or systems stabilized by either granulated (Sephadex, Bio-Gel) or compact polyacrylamide gels. Conditions standardized for the determination of pI values of selected pH marker proteins proved inadequate for the predominant isoenzyme of horseradish peroxidase which requires an excessively long focusing time to attain the steady state. Carbon dioxide interferes with the determination of pI values >8.2 to 8.3. Thin-layer isoelectric focusing in a CO₂-free atmosphere followed by pH measurements also in a CO₂-free atmosphere, yields for alkaline marker proteins and the predominant peroxidase isoenzyme, pI values in excellent agreement with these found by the density gradient technique. The isoionic point of the predominant peroxidase isoenzyme determined by ion exchange desalting is identical with the isoelectric point found by density gradient and thin-layer isoelectric focusing in a CO₂-free atmosphere.     

A biophysical characterization of the iron coordination environment in wheat germ peroxidase

 The heme protein wheat germ peroxidase (isoenzyme C₂) and its cyanide-inhibited form have been investigated by means of electronic, CD and paramagnetic NMR spectroscopy. The data indicate a protein environment of the active site distinct from that of horseradish peroxidase (HRP), with a larger solvent accessibility. The iron is pentacoordinated at neutral and low pH, whereas a hydroxyl anion may be bound at alkaline pH. The fifth axial ligand is a His residue with a partial anionic character, as found in other peroxidases. A spin equilibrium is observed at high enzyme concentrations. Received: 17 September 1996 / Accepted: 10 January 1997     

Spin-state equilibria and axial ligand bonding in FixL hydroxide: a resonance raman study

 Vibrational assignments for the Fe-OH unit of ferric alkaline forms of two deletion derivatives of Rhizobium meliloti FixL, FixL*, a functional O₂-sensing heme kinase, and FixLN, which contains only the heme domain, are made. Appearance of ²H- and ¹⁸O-sensitive Raman bands indicates that the heme group of FixL binds hydroxide as a distal ligand to form a six-coordinate complex. The alkaline FixLs are distributed between high- and low-spin states. The high- and low-spin bands corresponding to the ν (Fe-OH) modes occur at 479 and 539 cm^–1, respectively. Low temperature favors formation of the low-spin complex, indicative of a thermal spin-state equilibrium. The ν (Fe-OH) frequencies of FixLN and FixL* are 11 to 18 cm^–1 lower than those observed for the respective vibrations in alkaline myoglobin and hemoglobin. The weaker Fe-OH bond in the FixLs is attributed to a lack of hydrogen bonding on the distal side of the heme pocket. Received: 20 November 1997 / Accepted: 2 March 1998     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Proton nuclear magnetic resonance spectra of compounds I and II of horseradish peroxidase

Enzymatic reaction intermediates of horseradish peroxidase, compounds I and II, were studied by high-resolution nuclear magnetic resonance spectroscopy at 220 MHz. The heme peripheral proton peaks were successfully obtained in the downfield region of 50 to 80 ppm from 4,4-dimethyl-4-silapentane-5-sulfonate for compound I and of 10 to 20 ppm for compound II at pH 9.2. This indicates that no isoporphyrin appears in the catalytic cycle of the enzyme. Temperature dependences of the spectra also were determined for these compounds between 7 and 32 degrees C. With increasing temperature, all the peaks in the downfield region for compound I shifted upfield, obeying the Curie law. These results suggest that the Fe atoms in compounds I and II are in ferryl high- and low-spin states, respectively. The spectrum was also observed in solutions of horse metmyoglobin to which hydrogen peroxide (H2O2) was added. The electron formulations of the hemes in their spectra. Evidence was found against a pi-cation radical on the heme ring as a source of the oxidizing equivalent in compound I.     

Phenothiazines are slowly oxidizable substrates of horseradish peroxidase

Reactions of peroxidase oxidation of triftazine and thioproperazine have been investigated in the presence of horseradish peroxidase using steady state kinetic methods. It has been shown that phenothiazines are slowly oxidizable substrates for horseradish peroxidase. k _cat and K _m values have been determined in the range of pH from 4.5 to 7.5. The study of co-oxidation of phenothiazines and o-dianisidine (ODN) revealed that in the presence of aminazine and ODN in the reaction medium both substances follow sequential oxidation. ODN oxidation was not observed until full conversion of aminazine. At pH 4.5–5.5 thioproperazine bound to the enzyme-substrate complex and caused anticompetitive inhibition of peroxidase. At pH > 5.5 sequential substrate oxidation with preferential thioproperazine conversion occurred. In the range of pH from 4.5 to 7.5 triftazine did not influence ODN oxidation.     

Computer calculation-based quantitative structure-activity relationships for the oxidation of phenol derivatives horseradish peroxidase compound II

 The second-order rate constants for the oxidation of a series of phenol derivatives by horseradish peroxidase compound II were compared to computer-calculated chemical parameters characteristic for this reaction step. The phenol derivatives studied were phenol, 4-chlorophenol, 3-hydroxyphenol, 3-methylphenol, 4-methylphenol, 4-hydroxybenzoate, 4-methoxyphenol and 4-hydroxybenzaldehyde. Assuming a reaction of the phenolic substrates in their non-dissociated, uncharged forms, clear correlations (r = 0.977 and r = 0.905) were obtained between the natural logarithm of the second-order rate constants (ln k _app and ln k ₂ respectively) for their oxidation by compound II and their calculated ionisation potential, i.e. minus the energy of their highest occupied molecular orbital [E(HOMO)]. In addition to this first approach in which the quantitative structure-activity relationship (QSAR) was based on a calculated frontier orbital parameter of the substrate, in a second and third approach the relative heat of formation (ΔΔHF) calculated for the process of one-electron abstraction and H^• abstraction from the phenol derivatives was used as a parameter. Plots of the natural logarithms of the second-order rate constants (k _app and k ₂) for the reaction and the calculated ΔΔHF values for the process of one-electron abstraction also provide clear QSARs with correlation coefficients of –0.968 and –0.926 respectively. Plots of the natural logarithms of the second-order rate constants (k _app and k ₂) for the reaction and the calculated ΔΔHF values for the process of H^• abstraction provide QSARs with correlation coefficients of –0.989 and –0.922 respectively. Since both mechanisms considered, i.e. initial electron abstraction versus initial H^• abstraction, provided clear QSARs, the results could not be used to discriminate between these two possible mechanisms for phenol oxidation by horseradish peroxidase compound II. The computer calculation-based QSARs thus obtained for the oxidation of the various phenol derivatives by compound II from horseradish peroxidase indicate the validity of the approaches investigated, i.e. both the frontier orbital approach and the approach in which the process is described by calculated relative heats of formation. The results also indicate that outcomes from computer calculations on relatively unrelated phenol derivatives can be reliably compared to one another. Furthermore, as the actual oxidation of peroxidase substrates by compound II is known to be the rate-limiting step in the overall catalysis by horseradish peroxidase, the QSARs of the present study may have implications for the differences in the overall rate of substrate oxidation of the phenol derivatives by horseradish peroxidase. Received: 29 March 1996 / Accepted: 17 July 1996     

Application of optimized chemiluminescence assay for determination of the antioxidant capacity of herbal extracts

A chemiluminescence (CL) assay for the determination of antioxidant capacity (AOC) has been optimized and applied to analyses of herbal extracts in the present study. The optimal concentrations of reagents (luminol, H₂O₂, horseradish peroxidase) have been determined, as well as the optimal reaction conditions (wavelength, pH, temperature, sample volume). All of the measurements were performed at the emission maximum of the oxidized form of luminol (425 nm). The optimal concentrations of the reagents were determined as follows: 1.6 mmol/L luminol, 7.5 mmol/L H₂O₂ and 0.14 U/mL horseradish peroxidase activity in the reaction mixture. Analyses were carried out in phosphate buffer, pH 7.4, at room temperature. With the optimized CL assay, the AOCs of various water and methanol herbal extracts were determined (dog rose hips, plantain leaves and coltsfoot and thyme flowers) and the results were compared to those obtained by other classical methods for the evaluation of antioxidants. Strong correlations (r > 0.9) with the Folin–Ciocalteau assay and the 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH)^● assay are confirmed, although there is no correlation between AOC and the concentration of ascorbic acid in the samples analysed. This optimized CL assay is simple, rapid and reliable, and it represents a good alternative to classical methods (Folin–Ciocalteau, DPPH^●) for the determination of AOC of herbal extracts and other food samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Polydisulfides of substituted phenols as effective protectors of peroxidase against inactivation by ultrasonic cavitation

Kinetics of inactivation of horseradish peroxidase (HP) induced by low-frequency ultrasonic (US) treatment (27 kHz) with the specific power of 60 W/cm² were studied in phosphate (pH 7.4) and acetate (pH 5.2) buffers within the temperature range of 36.0 to 50.0°C and characterized by effective first-order rate constants of US inactivation k _in (us) in min^–1. Values of k _in (us) depend on the specific ultrasonic power within the range of 20-60 W/cm², on the concentration of HP, and on pH and temperature of the solutions. The activation energy of US inactivation of HP is 9.4 kcal/mole. Scavengers of HO^· radicals, mannitol and dimethylformamide, significantly inhibit the US inactivation of HP at 36.0°C, whereas micromolar concentrations of polydisulfide of gallic acid (poly(DSG)) and of poly(2-aminodisulfide-4-nitrophenol) (poly(ADSNP)) virtually completely suppress the US inactivation of peroxidase at the ultrasonic power of 60 W/cm² on the sonication of the enzyme solutions for more than 1 h at pH 5.2. Various complexes of poly(DSG) with human serum albumin effectively protect HP against the US inactivation in phosphate buffer (pH 7.4). The findings unambiguously confirm a free radical mechanism of the US inactivation of HP in aqueous solutions. Polydisulfides of substituted phenols are very effective protectors of peroxidase against inactivation caused by US cavitation.     

Kinetic and redox properties of MnP II, a major manganese peroxidase isoenzyme from Panus tigrinus CBS 577.79

A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed “MnP II,” with a specific activity of 288 IU mg⁻¹ protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45 °C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 °C, with half-lives of 11 min and 105 s, respectively. K _m values for H₂O₂ and Mn²⁺ were 16 and 124 μM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn²⁺-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H₂O₂ and Mn²⁺; in addition, low turnover numbers and K _m values higher than 300 μM characterized the Mn²⁺-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus.     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (E _m7 = –254 mV) and one low-spin, high-potential (E _m7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c ₅₅₅ as the electron donor, the K _M and V _max for peroxide reduction were 510 ± 100 nM and 425 ± 22 mol ferrocytochrome c ₅₅₅ oxidized min^–1 (mole cytochrome c peroxidase)^–1, respectively. Received: 6 January 1997 / Accepted: 27 May 1997     

Kinetic regulations of dihydroquercetin oxidation with horseradish peroxide

The steady-state kinetics of horseradish peroxidase-catalyzed dihydroquercetin oxidation was studied. Dihydroquercetin was shown to be a slowly oxidized substrate of horseradish peroxidase. Two dihydroquercetin isoforms (cis and trans forms) that were selectively involved in peroxidase-induced oxidation were found in water-alcohol and buffer solutions. The k _cat and K _m were determined in the pH range of 4.5–8.0.     

Temperature and pH optima of extremely halophilic archaea: a mini-review

Archaeal microorganisms that grow optimally at Na⁺ concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review encompasses extremely halophilic archaea and their growth characteristics with respect to the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance. The focus is on poly-extremophiles, i.e., taxa growing optimally at a Na⁺ concentration at or above 1.7 M (approximately 10% w/v NaCl); alkaline pH, at or above 8.5; and elevated temperature optima, at or above 50°C. So far, only a very few extreme halophiles that are able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated. The distribution of extremely halophilic archaea growing optimally at 3.4 M Na⁺ (approximately 20% w/v NaCl) is bifurcated with respect to pH optima, either they are neutrophilic, with a pH_opt of approximately 7, or strongly alkaliphilic, with pH_opt at or above 8.5. Amongst these extreme halophiles which have elevated pH optima, only four taxa have an optimum temperature above 50°C: Haloarcula quadrata (52°C), Haloferax elongans (53°C), Haloferax mediterranei (51°C) and Natronolimnobius ‘aegyptiacus’ (55°C).     

Protective effect of L-carnitine on hyperammonemia

The diheme cytochrome c-554 which participates in ammonia oxidation in the chemoautotroph , Nitrosomonas europaea has been studied by Soret excitation resonance Raman spectroscopy. The Raman spectrum of reduced cytochrome c-554 at neutral pH is similar classical 6-coordinate low-spin ferrous mammalian cytochrome c. In contrast, the spectrum of ferric cytochrome c-554 suggests a 5-coordinate state which is unusual for c hemes. The oxidized spectrum closely resemble that of horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP) at pH 6.4. The narrow linewidth of the heme core-size vibrations indicates that both heme irons of c-554 have similar geometries.     

The Development of a Phytoremediation Technique for the Detoxification of Soils Contaminated with Phenolic Compounds Using Horseradish Peroxidase (Armoracia rusticana): Preliminary Results

The proposed phytoremediation technique is based on the successful exploitation and optimization of oxidative coupling, mediated by horseradish peroxidase. Susceptibility to degradation of a selection of phenolic compounds, in solution, by horseradish peroxidase appears to be structurally related and was found to be of the order 2,4-dichlorophenol (2,4-DCP) > 4-chlorophenol (4-CP) > 2-chlorophenol (2-CP). Only 1.89% of 2,4-DCP, at an initial concentration of 5 mM, remained unchanged at the end of the experiment. Reaction rates between purified horseradish peroxidase and 2,4-DCP were found to be extremely rapid with 74% of the substrate removed from solution during the first 30 s. Inhibition of the reaction by the heavy metals Cd, Zn, Ni, and Pb at concentrations of 100 mg/l is of concern because these metals are often present in contaminated soils. H₂O₂ has a dominant role in optimizing peroxidase activity in crude horseradish extracts. Fluctuations in temperature and pH, normally experienced in soils, did not appear to have a detrimental impact on peroxidase activity. However, the functioning of the enzyme is seriously affected at a pH ≤ 3. All reactions in this study were carried out in solution.     

Resonance Raman evidence for Fe(IV) in compound II of horseradish peroxidase

Resonance Raman spectra have been obtained for Compound II of horseradish peroxidase. Its prophyrin vibrational frequencies are consistent with a planar low-spin heme containing Fe(IV). The oxidation-state marker band is found at the unprecedentedly high value of 1382 cm^?1. This band was also observed in solutions of myoglobin and cytochrome c peroxidase to which H₂O₂ had been added. No evidence was found for an actual FeO double bond in Compound II.     

Purification and characterisation of lignin peroxidase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K _m, k _cat and k _cat/K _m values of the enzyme using veratryl alcohol and H₂O₂ as the substrate were 61 M, 2.13 s⁻¹, 3.5 × 10⁴ M⁻¹s⁻¹ and 71 M, 2.13 s⁻¹, 3.0 × 10⁴ M⁻¹ s⁻¹ respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C.     

Inhibition of horseradish peroxidase byN-ethylamide ofo-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence ofN-ethylamide ofo-sulfobenzoylacetic acid (EASBA) at pH 5.0–7.5. In the oxidation ofo-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases bothK _m andV _m in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.