Chloroperoxidase Research Articles

Selectivity in the peroxidase catalyzed oxidation of phenolic sulfides

The catalytic oxidations of ortho - and para -alkylthiophenols by peroxidases exhibit various types of selectivities. Horseradish peroxidase and chloroperoxidase are active toward these substrates, although the latter enzyme addresses the oxidation preferentially on the sulfide function. Lactoperoxidase is only active with the ortho -substituted compounds, and the lack of reactivity toward the para -isomers depends on improductive binding to the protein. The catalytic oxidation of ortho - and para -alkylthiophenols, carrying methyl or ethyl substituents at sulfur, by lactoperoxidase (LPO), horseradish peroxidase (HRP) or chloroperoxidase (CPO), in the presence of hydrogen peroxide, has been investigated. HRP and CPO are active toward these substrates, whereas LPO is only active with the ortho -substituted compounds. The enzymatic solutions containing ortho -alkylthiophenols develop an intense blue color (with optical absorptions near 400 and 600 nm) that is attributed to the formation of relatively stable dimeric three-electron bonded complexes resulting from the association of enzyme-derived radical cations with the phenolic sulfide. The products of the enzymatic reactions by HRP and LPO are oligomers resulting from phenol oxidative coupling reactions and the sulfoxide, with minor amounts of oligomers containing mono or disulfoxide functionalities. With CPO the major product is always the sulfoxide, while phenol coupling products are formed in minor amounts. The selectivity exhibited by LPO toward 2- and 4-methylthiophenol has been investigated through binding experiments, NMR relaxation measurements of LPO-substrate complexes and docking calculations. The para -isomer binds much more strongly than the ortho -isomer to the enzymes. The stronger binding depends on the establishment of hydrogen bonding interactions with protein residues in the active site.

Two-dimensional NMR Study of the Heme Active Site Structure of Chloroperoxidase

The heme active site structure of chloroperoxidase (CPO), a glycoprotein that displays versatile catalytic activities isolated from the marine mold Caldariomyces fumago, has been characterized by two-dimensional NMR spectroscopic studies. All hyperfine shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues including the heme iron ligand (Cys(29)) attributable to the unique catalytic properties of CPO have been firmly assigned through (a) measurement of nuclear Overhauser effect connectivities, (b) prediction of the Curie intercepts from both one- and two-dimensional variable temperature studies, (c) comparison with assignments made for cyanide derivatives of several well characterized heme proteins such as cytochrome c peroxidase, horseradish peroxidase, and manganese peroxidase, and (d) examination of the crystal structural parameters of CPO. The location of protein modification that differentiates the signatures of the two isozymes of CPO has been postulated. The function of the distal histidine (His(105)) in modulating the catalytic activities of CPO is proposed based on the unique arrangement of this residue within the heme cavity. Contrary to the crystal state, the high affinity Mn(II) binding site in CPO (in solution) is not accessible to externally added Mn(II). The results presented here provide a reasonable explanation for the discrepancies in the literature between spectroscopists and crystallographers concerning the manganese binding site in this unique protein. Our study indicates that results from NMR investigations of the protein in solution can complement the results revealed by x-ray diffraction studies of the crystal form and thus provide a complete and better understanding of the actual structure of the protein.

Toward the development of a biocatalytic system for oxidation of p-xylene to terephthalic acid: oxidation of 1,4-benzenedimethanol

The oxidation of hydrocarbons is an important value-enhancing chemical transformation. Current inorganic catalysts often operate under harsh conditions and produce large amounts of heavy-metal waste. Enzymatic oxidations, on the other hand, operate under relatively mild conditions and produce little if any waste. In the present work, we describe several steps in the oxidative pathway leading from p-xylene to terephthalic acid that are catalyzed by enzymes. Chloroperoxidase (CPO) from Caldariomyces fumago was used to oxidize p-xylene. However, only one of the two aromatic methyl groups was oxidized. To examine the route from 1,4-benzenedimethanol (1,4-BDM) to terephthalic acid, we investigated numerous peroxidase and oxidase enzyme systems. A combination of two enzymes, CPO and xanthine oxidase (XO), was found to produce the highest yield of terephthalic acid from 1,4-BDM. Oxidation of 1,4-BDM to a mixture of predominantly terepthaldicarboxaldehyde, 4-carboxybenzaldehyde, and 4-hydroxymethylbenzaldehyde was carried out by CPO with the continuous addition of hydrogen peroxide as an oxidant. Subsequent addition of XO resulted in a 65% yield of terephthalic acid. A tandem system in which both CPO and XO were present enabled the initial H 2O 2 to be enzymatically regenerated an average of 1.6 times. However, much lower final yields (ca. 2%) of terephthalic acid were obtained.

Mixed peroxides from the chloroperoxidase-catalyzed oxidation of conjugated dienoic esters with a trisubstituted terminal double bond

The chloroperoxidase (CPO)-catalyzed oxidations of conjugated dienoic esters with a trisubstituted terminal double bond were studied by using tert-butyl hydroperoxide as the terminal oxidant. Most of the substrates gave disubstituted mixed peroxides as the major products.

Chloroperoxidase-catalyzed cyclodimerization of methyl (2 E)-2,4-pentadienoate: a [4+2] cycloaddition product

The chloroperoxidase (CPO)-catalyzed oxidation of the methyl (2 E)-2,4-pentadienoate gives the terminal double bond epoxide (25%) and a cyclodimerization compound (63%) as the major products.

Resonance Raman detection of the Fe-S bond in endothelial nitric oxide synthase.

We report the first low-frequency resonance Raman spectra of ferric endothelial nitric oxide synthase (eNOS) holoenzyme, including the frequency of the Fe-S vibration in the presence of the substrate L-arginine. This is the first direct measurement of the strength of the Fe-S bond in NOS. The Fe-S vibration is observed at 338 cm(-1) with excitation at 363.8 nm. The assignment of this band to the Fe-S stretching vibration was confirmed by the observation of isotopic shifts in eNOS reconstituted with 54Fe- and 57Fe-labeled hemin. Furthermore, the frequency of this vibration is close to those observed in cytochrome P450(cam) and chloroperoxidase (CPO). The frequency of this vibration is lower in eNOS than in P450(cam) and CPO, which can be explained by differences in hydrogen bonding to the proximal cysteine heme ligand. On addition of substrate to eNOS, we also observe several low-frequency vibrations, which are associated with the heme pyrrole groups. The enhancement of these vibrations suggests that substrate binding results in protein-mediated changes of the heme geometry, which may provide the protein with an additional tuning element for the redox potential of the heme iron. The implications of our findings for the function of eNOS will be discussed by comparison with P450(cam) and model compounds.

Biocatalytic oxidation of S-alkylcysteine derivatives by chloroperoxidase and Beauveria species

Treatment of N-methoxycarbonyl C-carboxylate ester derivatives of S-methyl- l-cysteine by chloroperoxidase (CPO)/hydrogen peroxide resulted in oxidation at sulfur to produce the ( R S) sulfoxide in moderate to high diastereomeric excess (DE). The ( S S) natural product sulfoxide chondrine was obtained via biotransformation of the N-t.boc derivative of l-4-S-morpholine-2-carboxylic acid using Beauveria bassiana or Beauveria caledonica.

4-chlorophenol degradation by chloroperoxidase from Caldariomyces fumago: formation of insoluble products.

This study investigated the degradation of 4-chlorophenol (4-CP) by Caldariomyces fumago chloroperoxidase (CPO). Enzymatic oxidations were studied in reaction mixtures at pH 3.0, 4.0, and 6.0 in the presence and absence of Cl- containing 3.5 IU of CPO and 4-CP and hydrogen peroxide concentrations within the range of 0.5-50 and 0.005-50 mM, respectively. Distinct patterns of products regarding color, concentration, and solubility were observed. Reaction mixtures at pH 6.0 containing 3.5 IU of CPO and 5.0 mM 4-CP and H2O2 (1:1 stoichiometry) showed the highest 4-CP removal of 95% and the highest formation of a dark precipitate.

Highly enantioselective oxidation of cis-cyclopropylmethanols to corresponding aldehydes catalyzed by chloroperoxidase.

Chloroperoxidase (CPO) catalyzes the enantioselective oxidation of cyclopropylmethanols, such as 2-methylcyclopropylmethanol, to cyclopropyl aldehydes using tert-butyl hydroperoxide as the terminal oxidant. In all cases, CPO oxidation of cis-cyclopropanes shows much higher enantioselectivity than with the trans isomers, although CPO gives similar catalytic activity on both isomers. This presents the first example for a heme enzyme that catalyzes the enantioselective oxidation of cyclopropylmethanols. This finding enables a novel route to the synthesis of optically active cyclopropane derivatives, which occur widely in natural products and compounds of pharmaceutical interest. In addition, chiral cyclopropane molecules may be useful model substrates to investigate reaction mechanisms of CPO and the related cytochromes P450.

Catalytic activity of mesoporous silicate-immobilized chloroperoxidase

A versatile enzyme, FeHeme chloroperoxidase (CPO) from Caldariomyces fumago, is immobilized in the mesoporous silicate material, mesocellular foam (MCF). MCF is a promising material for immobilizing enzymes, due to its large pore structure and high loading capacity compared to other mesoporous materials, such as MCM-48, SBA-16 and SBA-15. The immobilized CPO in MCF retains its activity. The optimal pH at which the maximum amount of enzyme is immobilized was determined to be pH 3.4, slightly below the isoelectric point of the enzyme. A weak ionic interaction between the enzyme and the surface of the inorganic substrate is thought to be critical in maintaining the activity of the immobilized enzyme. The loading capacity of MCF is 122 mg protein per 1 g of MCF. We demonstrate the advantage of MCF as an inorganic substrate for immobilization of enzymes.

Chloroperoxidase-catalyzed oxidation of conjugated dienoic esters

The chloroperoxidase (CPO)-catalyzed oxidations of dienes conjugated to an ester group were studied using tert-butyl hydroperoxide as the terminal oxidant.

C-terminal propeptide of the Caldariomyces fumago chloroperoxidase: an intramolecular chaperone?

The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a 372-aa precursor which undergoes two proteolytic processing events: removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal propeptide. The Aspergillus niger expression system developed for CPO was used to get insight into the function of this C-terminal propeptide. A. niger transformants expressing a CPO protein from which the C-terminal propeptide was deleted failed in producing any extracellular CPO activity, although the CPO polypeptide was synthesised. Expression of the full-length gene in an A. niger strain lacking the KEX2-like protease PclA also resulted in the production of CPO cross-reactive material into the culture medium, but no CPO activity. Based on these results, a function of the C-terminal propeptide in CPO maturation is indicated.

Directed Evolution of Chloroperoxidase for Improved Epoxidation and Chlorination Catalysis

Chloroperoxidase (CPO) from the fungus Caldariomyces fumago is undoubtedly the most versatile member of the heme protein family. In addition to functioning as a halogenating enzyme and a classical peroxidase, CPO catalyzes the dismutation of peroxides in a catalase-type reaction and carries out cytochrome P450 oxygen insertion reactions. From the viewpoint of biocatalysis the most important CPO reactions are chiral epoxidations, hydroxylations, and sulfoxidations. CPO catalyzes a variety of chiral epoxidation reactions with high yields and high enantioselectivities. However, the industrial use of native CPO for the synthesis of chiral epoxides is limited because of its relatively low epoxidation rates in comparison to its high catalase activity, which robs the epoxidation reaction of oxidant. The use of CPO is also restricted by its poor reactivities in organic solvents. Directed evolution technology has been used to address these problems. After three rounds of PCR-based random mutagenesis, we have isolated mutants of chloroperoxidase having greatly enhanced epoxidation activity compared to the wild-type enzyme. In addition, in the screening of a first generation library of random mutation transformants, we have isolated three CPO mutant clones having improved chlorination activity and enhanced stability in a ternary solvent microemulsion comprised of toluene, isopropanol and water. Surprisingly, all three recombinant variants carry a single mutation in the cysteine residue that functions as the proximal heme ligand in the native enzyme. Two of these mutant clones are identical, having the proximal cysteine heme-ligand replaced with a tyrosine residue. The third mutant has the cysteine-29 replaced with a histidine residue. The cysteine mutation in the three mutants is the only amino acid replacement. All other mutations in the three clones were silent mutations. These data suggest that ‘‘directed evolution’’ can be successfully applied to the engineering of chloroperoxidase in the quest for a better industrial biocatalyst.

Utilization of peroxide and its relevance in oxygen insertion reactions catalyzed by chloroperoxidase

Chloroperoxidase (CPO) catalyzed oxygen insertions are highly enantioselective and hence of immense biotechnological potential. A peroxide activation step is required to give rise to the compound I species that catalyzes this chiral reaction. A side reaction, a catalase type peroxide dismutation, is another feature of CPO’s versatility. This work systematically investigates the utilization of different peroxides for the two reactions, i.e. the catalase type reaction and the oxygen insertion reaction. For the oxygen insertion reaction, indene and phenylethyl sulfide were chosen as substrate models for epoxidation and sulfoxidation respectively. The results clearly show that CPO is stable towards hydrogen peroxide and has a total number of turnovers near one million prior to deactivation. The epoxidation reactions terminate before completion because the enzyme functioning in its catalatic mode quickly removes all of the hydrogen peroxide from the reaction mixture. Sulfoxidation reactions are much faster than epoxidation reactions and thus are better able to compete with the catalase reaction for hydrogen peroxide utilization. A preliminary study towards optimizing the reaction system components for a laboratory scale synthetic epoxidation is reported.

Proximal ligand effects on electronic structure and spectra of compound I of peroxidases

Computational studies exploring the extent to which differences in proximal axial ligands modulate structure, spectra, and function of peroxidases have been performed. To this end, three heme models of compound I were characterized differing only in the axial ligand. The axial ligands considered were L = ImH , Im-, that are alternative protonation models for a typical peroxidase with an imidazole ligand such as horseradish peroxidase (HRP-I), and L = SCH - that is a model for an unsual peroxidase, chloroperoxidase (CPO-I). Density functional calculations (DFTs) were performed to determine the optimized geometries and electronic structure of each of these three species. Their electronic spectra were also calculated at the DFT optimized geometries, using the INDO/S/CI method. The results of these studies led to the following conclusions: (1) the presence of the nearby Asp in a typical peroxidase does indeed decrease the energy required to deprotonate the imidazole making the two forms essentially degenerate, (2) neither the state of protonation of the imidazole ligand nor the change in axial ligand from an imidazole in typical peroxidases such as HRP to a mercaptide in CPO significantly alters the characteristics of the lowest energy spin state or the electronic structure of compound I in a way that can obviously affect function, (3) both the Im-and ImH forms of the peroxidase compound I (HRP-I) lead to the same dramatic reduction in intensity relative to the ferric resting form observed experimentally. However, only in the ImH form of HRP-I does the calculated relative shift of one component of the Soret bands relative to CPO-I agree with that observed in the transient spectra of HRP-I compared to CPO-I. These results taken together strongly indicate that factors other than the nature of the proximal axial ligand are the main determinants of function.

Synthetic active site analogues of heme–thiolate proteins: Characterization and identification of intermediates of the catalytic cycles of cytochrome P450 cam and chloroperoxidase

In view of recent results from different sources, the reaction mechanisms of two heme–thiolate proteins, cytochrome P450 cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H 2O 2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl + donor chlorinating activated C–H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450 cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.

Prochiral selectivity in H(2)O(2)-promoted oxidation of arylalkanols catalysed by chloroperoxidase. The role of the interactions between the OH group and the amino-acid residues in the enzyme active site.

The H(2)O(2)-promoted oxidations of (R)-[alpha-(2)H(1)]-and (S)-[alpha-(2)H(1)]-arylalkanols catalysed by chloroperoxidase (CPO) from Caldariomyces fumago have been investigated. It has been found that with (R)-[alpha-(2)H(1)]-alcohols, the oxidation involves almost exclusively the cleavage of the C-H bond, whereas in the case of the oxidation of (S)-[alpha-(2)H(1)]-alcohols, the C-D bond is preferentially broken. These results clearly indicate that the reactions of corresponding undeuterated arylalkanols are characterized by a high prochiral selectivity, involving the cleavage of the pro-S C-H bond. This prochiral selectivity is poorly influenced by the electronic effect of ring substituents, whereas it decreases with the length of the carbon lateral chain, in the order: benzyl alcohol > 2-phenylethanol > 3-phenylpropanol. Molecular binding studies showed that the main factor directing the docking of the substrate in such a specific orientation in the enzyme active site is the interaction between the alcoholic OH group and the residue Glu183. This interaction is likely to drive both the stereochemistry and the regiochemistry of these reactions. A bifurcated hydrogen bond involving the OH group, the carboxylate oxygen of Glu183 and the oxoferryl oxygen might also be operating.

Engineering chloroperoxidase for activity and stability

Random mutagenesis of Caldariomyces fumago was carried out to produce a chloroperoxidase (CPO) with enhanced activity in mixtures of aqueous buffers and organic cosolvents. A mutant CPO with a 3.4-fold increased activity in 40% aqueous tert-butyl alcohol was obtained.A CPO-surfactant conjugate, prepared by colyophilisation, mediated the oxidation of thioanisole to its (R)-sulfoxide by TBHP in water-saturated isooctane. Copolymerisation of CPO and a toluenediisocyanate prepolymer resulted in a stable preparation that mediated the oxidation of indole to 2-oxindole in a range of organic solvents. The highest yield was obtained in 1-octanol.

Effects of a thiolate axial ligand on the pi-->pi* electronic states of oxoferryl porphyrins: a study of the optical and resonance Raman spectra of compounds I and II of chloroperoxidase.

Optical absorption and resonance Raman spectra have been investigated for enzymatic intermediates, compounds I and II, of chloroperoxidase (CPO) which contains a thiolate-ligated iron porphyrin. Compound I of CPO (CPO-I), an oxoferryl porphyrin pi cation radical, gave an apparently asymmetric single-peaked Soret band at 367 nm, for which band fitting analyses revealed the presence of two transition bands around 365 and 415 nm. Compound II of CPO (CPO-II), an oxoferryl neutral porphyrin, gave a split Soret spectrum with two bands (blue and red Soret bands) at 373 and 436 nm. Thus both CPO-I and CPO-II can be categorized as hyperporphyrins. The maximum extinction coefficients (epsilon(b) and epsilon(r)) and energies (Eb and Er) of the blue and red Soret bands of CPO-II were found to fall on an epsilon(b)/epsilon(r) versus Eb-Er correlation line derived from data reported for six-coordinate ferrous derivatives of cytochrome P450 and CPO. Corresponding data for CPO-I did not fall on the correlation line. Resonance enhancement of the FeIV=O stretching (vFeO) Raman band was found for CPO-I when Raman scattering was excited at wavelengths within both transition bands around 365 and 415 nm, while the vFeO Raman band was not identified for CPO-II at any of the excitation wavelengths examined here. These findings suggest that the thiolate axial ligand causes Soret band splitting of CPO-II through configuration interaction between the sulfur-->porphyrin e(g)* charge transfer and porphyrin a1u,a2u-->e(g)* transitions, while the FeO portion is important in determining the shape of the Soret band of CPO-I.

The electronic and vibrational structures of iron–oxo porphyrin with a methoxide or cysteinate axial ligand

Hybrid density functional theory (DFT) calculations for the electronic and vibrational structures of compound I species with a methoxide (MeO −) ( 1) or cysteinate (CysS −) ( 2) axial ligand are carried out in order to elucidate the natures of a methoxide-coordinating new type of compound I species (Bull. Chem. Soc. Jpn. 71 (1998) 1343) and cysteinate-coordinating compound I species of chloroperoxidase (CPO-I) and cytochrome P450s (P450-I). DFT computations of 1 and 2 demonstrate that these “anionic” ligands are a spin carrier; 70% (80%) of a spin density resides on the O (S) atom of the axial ligand and 30% (20%) is distributed on the porphyrin ring. These results suggest that for the generation of the compound I species, one electron is removed from the iron centers and the rest of the one electron is supplied from the oxidizable axial ligands instead of the iron centers or the porphyrin ring. Vibrational analyses demonstrate that the FeO bond is more strongly activated in 1 compared with 2 with the stretching mode at 849 cm −1 (878 cm −1) for the doublet state 1a ( 2a) and at 814 cm −1 (875 cm −1) in the quartet state 1b ( 2b). This reverse order of the FeO bond strength with respect to the axial donor strength should have relevance to the significantly oxidized character of the CysS − axial ligand. In conjunction with the recent results of the extensive resonance Raman (RR) studies, some interpretations of unsettled RR results for compound I of chloroperoxidase (CPO-I) and a synthetic compound I species [OFe IV(TMP ⋅+)(alcohol)] (J. Am. Chem. Soc. 113 (1991) 6542) concerning the OFe stretching frequencies are discussed.