Catalytic and structural properties of surfactant-horseradish peroxidase complex in organic media

A surfactant-horseradish peroxidase (HRP) complex that is catalytically active in organic media has been successfully prepared by a method utilizing water-in-oil (W/O) emulsions. To optimize conditions for preparation of the HRP complex, the effects of some key parameters in the aqueous phase of W/O emulsions were investigated. The surfactant-HRP complex prepared with a nonionic surfactant exhibited a high catalytic activity compared to those with a cationic or anionic surfactant in anhydrous benzene. At the preparation step, the pH of the aqueous solution had a prominent effect on the enzymatic activity of the HRP complex in organic media. Several kinds of salts present in the HRP complex could be employed to enhance the catalytic performance in organic media. However, anionic ions present in the preparation process appeared to lower the catalytic activity owing to the complexation with heme iron. UV-visible absorption spectra of the HRP complex in benzene, which were prepared from a KCN solution (pH 7.0) or an alkaline solution (pH 12), were comparable with those of native HRP in aqueous solution under the same conditions. Resonance Raman spectroscopic studies also revealed that no significant change in the coordination state of the heme iron occurred even after coating the enzyme with surfactant molecules, lyophilization, and solubilization in nonaqueous media.     

Characterization and catalytic property of surfactant-laccase complex in organic media

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated. The addition of HBT at the preparation stage of the enzyme complex did not accelerate the catalytic reaction because HBT was converted to an inactive benzotriazole (BT) by laccase. However, the addition of HBT at the reaction stage enhanced the catalytic performance by a factor of five compared to that without HBT.     

Surfactant-lactoperoxidase complex catalytically active in organic media

A surfactant-lactoperoxidase (LPO) complex catalytically active in organic solvents was developed by the emulsion coating method. The oxidation of 2,6-dimethoxyphenol (2,6-DMP) was conducted by the surfactant-LPO complex in organic media. The LPO complex efficiently catalyzed the oxidation of 2,6-DMP in various organic solvents, although lyophilized LPO did not display the catalytic activity at all. To optimize the preparation and reaction conditions for the surfactant-LPO complex, we examined the effects of pH value in the water pools of W/O emulsions, kinds of oxidants, and the nature of organic solvents on the oxidation reaction. Its optimum activity was obtained when the pH value of the aqueous enzyme solution was adjusted to ca. 8 at the preparation stage. The LPO complex exhibited the highest catalytic activity in chloroform when H(2)O(2) was employed as the oxidant. Furthermore, the storage stability of the surfactant-LPO complex was far better than that of the surfactant-horseradish peroxidase complex. This high storage stability of the LPO complex will be a benefit for industrial usage of peroxidases.     

Conformational mobility in the active site of a heme peroxidase

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.     

Raman evidence that the lyoprotectant poly(ethylene glycol) does not restore nativity to the heme active site of horseradish peroxidase suspended in organic solvents

Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.     

Novel preparation method for surfactant-lipase complexes utilizing water in oil emulsions

A novel preparation method for surfactant-lipase complexes has been developed utilizing water in oil emulsions. In order to optimize the preparation conditions, we have investigated the effects of several operational parameters on the enzymatic activity of the surfactant-lipase complexes in organic media. When a nonionic surfactant was employed under optimal preparation conditions [alkaline pH 8-10, organic/aqueous = 90/10 (v/v), concentration of surfactant, 10 mM[, the surfactant-lipase complex efficiently catalyzed the esterification of benzyl alcohol with lauric acid in organic media. The esterification rate of the surfactant-lipase complex was increased over 16-fold relative to the native powder lipase. Furthermore, the lipase complex showed high storage stability. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 455-460, 1997.     

Protein control of prosthetic heme reactivity. Reaction of substrates with the heme edge of horseradish peroxidase

Incubation of horseradish peroxidase with phenylhydrazine and H2O2 markedly depresses the catalytic activity and the intensity, but not position, of the Soret band. Approximately 11-13 mol of phenylhydrazine and 25 mol of H2O2 are required per mol of enzyme to minimize the chromophore intensity. The enzyme retains some activity after such treatment, but this activity is eliminated if the enzyme is isolated and reincubated with phenylhydrazine. The prosthetic heme of the enzyme does not react with phenylhydrazine to give a sigma-bonded phenyl-iron complex, as it does in other hemoproteins, but is converted instead to the delta-mesophenyl and 8-hydroxymethyl derivatives. The loss of activity is due more to protein than heme modification, however. The inactivated enzyme reacts with H2O2 to give a spectroscopically detectable Compound I. The results imply that substrates interact with the heme edge rather than with the activated oxygen of Compounds I and II and specifically identify the region around the delta-meso-carbon and 8-methyl group as the exposed sector of the heme. Horseradish peroxidase, in contrast to cytochrome P-450, generally does not catalyze oxygen-transfer reactions. The present results indicate that oxygen-transfer reactions do not occur because the activated oxygen and the substrate are physically separated by a protein-imposed barrier in horseradish peroxidase.     

Vibrational structure of the formyl group on heme a. Implications on the properties of cytochrome c oxidase.

Resonance Raman spectra have been recorded for heme a derivatives in which the oxygen atom of the formyl group has been isotopically labeled and for Schiff base derivatives of heme a in which the Schiff base nitrogen has been isotopically labeled. The 14N-15N isotope shift in the C = N stretching mode of the Schiff base is close to the theoretically predicted shift for an isolated C = N group for both the ferric and ferrous oxidation states and in both aqueous and nonaqueous solutions. In contrast, the 16O-18O isotope shift of the C = O stretching mode of the formyl group is significantly smaller than that predicted for an isolated C = O group and is also dependent on whether the environment is aqueous or nonaqueous. This differences between the theoretically predicted shifts and the observed shifts are attributed to coupling of the C = O stretching mode to as yet unidentified modes of the heme. The complex behavior of the C = O stretching vibration precludes the possibility of making simple interpretations of frequency shifts of this mode in cytochrome c oxidase.     

Key roles of Arg,Tyr and His residues in Aβ–heme peroxidase: Relevance to Alzheimer’s disease

Recent reports show that heme binds to amyloid β-peptide (Aβ) in the brain of Alzheimer’s disease (AD) patients and forms Aβ–heme complexes, thus leading a pathological feature of AD. However, the important biological relevance to AD etiology, resulting from human Aβ–heme peroxidase formation, was not well characterized. In this study, we used wild-type and mutated human Aβ_1–16 peptides and investigated their Aβ–heme peroxidase activities. Our results indicated that both histidine residues (His¹³, His¹⁴) in Aβ_1–16 and free histidine enhanced the peroxidase activity of heme, hence His residues were essential in peroxidase activity of Aβ–heme complexes. Moreover, Arg⁵ was found to be the key residue in making the Aβ_1–16–heme complex as a peroxidase. Under oxidative and nitrative stress conditions, the Aβ_1–16–heme complexes caused oxidation and nitration of the Aβ Tyr¹⁰ residue through promoting peroxidase-like reactions. Therefore, these residues (Arg⁵, Tyr¹⁰ and His) were pivotal in human Aβ–heme peroxidase activity. However, three of these residues (Arg⁵, Tyr¹⁰ and His¹³) identified in this study are all absent in rodents, where rodent Aβ–heme complex lacks peroxidase activity and it does not show AD, implicating the novel significance of these residues as well as human Aβ–heme peroxidase in the pathology of AD.     

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor <Emphasis Type="Italic">Af</Emphasis>GcHK

AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O₂ or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H₂S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O₂, CO, and NO. To address this knowledge gap, we investigated the effects of H₂S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O₂-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na₂S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na₂S, the initially formed Fe(III)–SH heme complex was quickly converted into Fe(II) and Fe(II)–O₂ complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)–SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)–OH complex), whereas in the case of EcDOS the changes in coordination caused by Na₂S treatment led to remarkable increases in catalytic activity.     

Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase

The human heme oxygenase-1 crystal structure suggests that Gly-139 and Gly-143 interact directly with iron-bound ligands. We have mutated Gly-139 to an alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate. All of these mutants bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mutants, giving rise to mixtures of hexacoordinate and pentacoordinate ligation states. The active site perturbation is greatest when large amino acid side chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, but most of them exhibit a new H(2)O(2)-dependent guaiacol peroxidation activity. The Gly-143 mutants, all of which have lost the water ligand, have no heme oxygenase or peroxidase activity. The results establish the importance of Gly-139 and Gly-143 in maintaining the appropriate environment for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase active site may be to suppress the ferryl species formation responsible for peroxidase activity.     

Preparation and Characterization of Water-in-Oil-in-Water Emulsions Containing a High Concentration of L-Ascorbic Acid

This study sought to encapsulate a high concentration of L-ascorbic acid, up to 30% (w/v), in the inner aqueous phase of water-in-oil-water (W/O/W) emulsions with soybean oil as the oil phase. Two-step homogenization was conducted to prepare W/O/W emulsions stabilized by a hydrophobic emulsifier and 30% (v/v) of W/O droplets stabilized by a hydrophilic emulsifier. First-step homogenization prepared W/O emulsions with an average aqueous droplet diameter of 2.0 to 3.0 μm. Second-step homogenization prepared W/O/W emulsions with an average W/O droplet diameter of 14 to 18 μm and coefficients of variation (CVs) of 18% to 25%. The results indicated that stable W/O/W emulsions containing a high concentration of L-ascorbic acid were obtained by adding gelatin and magnesium sulfate in the inner aqueous phase and glucose in both aqueous phases. L-Ascorbic acid retention in the W/O/W emulsions was 40% on day 30 and followed first-order kinetics.     

The cytochrome c peroxidase-cytochrome c electron transfer complex. The role of histidine residues

The histidine-selective reagent diethyl pyrocarbonate and dye-sensitized photooxidation have been used to study the functional role of histidines in cytochrome c peroxidase. Of the 6 histidines in cytochrome c peroxidase, 5 are modified by diethyl pyrocarbonate at alkaline pH and 4 by photooxidation. The sixth histidine serves as the proximal heme ligand and is unavailable for reaction. Both modification reactions result in the loss of enzymic activity. However, photooxidized peroxidase retains its ability to react with H2O2 and to form a 1:1 cytochrome c peroxidase-cytochrome c complex. It is, therefore, concluded that the extra histidine modified by diethyl pyrocarbonate is the catalytic site distal histidine, His 52. In the presence of cytochrome c, no enzymic activity is lost by photooxidation and a single histidine, His 181, is protected from oxidative destruction. This finding provides strong support for the hypothetical model of the cytochrome c peroxidase-cytochrome c complex in which His 181 lies near the center of the intermolecular interface where it seems to provide an important link in the electron transfer process.     

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.     

Engineering the proximal heme cavity of catalase-peroxidase

Catalase-peroxidases (KatGs) are prokaryotic heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs, CCP and APXs contain identical amino acid triads in the heme pocket (distal Arg/Trp/His and proximal His/Trp/Asp), but differ dramatically in their reactivities towards hydrogen peroxide and various one-electron donors. Only KatGs have high catalase activity in addition to a peroxidase activity of broad specificity. Here, we investigated the effect of mutating the conserved proximal triad on KatG catalysis. With the exception of W341F, all variants (H290Q, W341A, D402N, D402E) exhibited a catalase activity <1% of wild-type KatG and spectral properties indicating alterations in heme coordination and spin states. Generally, the peroxidase activity was much less effected by these mutations. Compared with wild-type KatG the W341F variant had a catalase and halogenation activity of about 40% and an even increased overall peroxidase activity. This variant, for the first time, allowed to monitor the hydrogen peroxide mediated transitions of ferric KatG to compound I and back to the resting enzyme. Compound I reduction by aromatic one-electron donors (o-dianisidine, pyrogallol, aniline) was not influenced by exchanging Trp by Phe. The findings are discussed in comparison with the data known from CCP and APX and a reaction mechanism for the multifunctional activity of the W341F variant is suggested.     

Purification of glycoproteins by selective transport using concanavalin-mediated reverse micellar extraction.

A novel methodology for coupling liquid-liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar-binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT-isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin-glycoprotein complex (96,000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water-pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.     

Effect of water activity control on the catalytic performance of surfactant–Arthromyces ramosus peroxidase complex in toluene

Arthromyces ramosus peroxidase (ARP) was successfully modified with a synthetic surfactant for one-electron oxidation reaction of a hydrophobic substrate in toluene. Although UV–visible absorption spectrum of surfactant–ARP complex in toluene showed slight red shift of Soret band compared to that in water, the complex can catalyze oxidation reaction of o-phenylenediamine (o-PDA) with hydrogen peroxide. It appeared that thermodynamic water activity in the reaction system has dominant effect on either the catalytic activity or the stability in the catalytic cycle. Steady-state kinetics under the optimal condition revealed that the specific constant (k_cat/K_m) of ARP complex for o-PDA was 2 orders of magnitude lower than that in aqueous media, while only 13-fold lower for hydrogen peroxide. The reduction of catalytic activity caused by altering the reaction media from water to toluene was found to be mainly due to the low specific constant of ARP complex for o-PDA rather than hydrogen peroxide.     

Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.     

Modulation of the NO<Emphasis Type="Italic"> trans</Emphasis> effect in heme proteins: implications for the activation of soluble guanylate cyclase

The binding of NO to the iron heme in guanylate cyclase and other heme proteins induces the cleavage of the proximal histidine bonded to the metal. In this study we assess by means of density functional theory (DFT) electronic structure calculations the role of H-bonding to histidine in the modulation of this effect. We have considered in the first place a model of the isolated active site coordinated with imidazole and imidazolate to mimic the effects of a very strong H-bond. We have also investigated four selected ferrous heme proteins with different proximal histidine environments: the O(2) sensing FixL, horseradish peroxidase C, and the alpha and beta subunits of human hemoglobin. Our results indicate that polarization and charge transfer effects associated with H-bonding to the proximal histidine play a fundamental role in the modulation of the NO trans effect in heme proteins. We also find computational evidence suggesting that protein structural constraints may affect significantly the cleavage of the Fe-His bond.     

Isolation of a heme crevice peptide from affinity-labeled horseradish peroxidase

Apo-horseradish peroxidase was affinity-labeled with the monosulfuric anhydride derivative of mesoheme. The stoichiometry of heme anhydride binding was 1.1 moles of the anhydride per mole of apo-peroxidase.Tryptic digestion of the affinity-labeled peroxidase yielded a major lysine peptide which corresponded in composition to peptides T8 and T9a in the sequence of horseradish peroxidase (Welinder, K. G., Eur. J. Biochem. 96: 483–502, 1979) which contained one mole of histidine (histidine 170) per mole of peptide.