Role of molecular oxygen in lignin peroxidase reactions

Homogeneous lignin peroxidase (diarylpropane oxygenase) oxidized veratryl alcohol to veratryl aldehyde under anaerobic conditions in the presence of either H2O2, m-chloroperoxybenzoic acid (mCPBA), or p-nitroperoxybenzoic acid (pNPBA). Lignin peroxidase also oxidized the 1-(3',4'-diethoxyphenyl)-1,2-dihydroxy-(4"-methoxyphenyl)-propane I under anaerobic conditions in the presence of mCPBA to yield 3,4-diethoxybenzaldehyde III and 1-(4'-methoxyphenyl)-1,2-dihydroxyethane IV. In contrast to what occurs under aerobic conditions, under anaerobic conditions no 2-hydroxy-1-(4'-methoxyphenyl)-1-oxoethane V was obtained. During the diarylpropane I cleavage under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of the phenylglycol IV. Lignin peroxidase also hydroxylated 1-(4'-ethoxy-3'-methoxyphenyl)propane II at the alpha-position to yield 1-(4'-ethoxy-3'-methoxyphenyl)-1-hydroxypropane VI under anaerobic conditions in the presence of mCPBA. During the phenylpropane II hydroxylation under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of VI. These results are rationalized according to a mechanism involving an initial one-electron oxidation of the diarylpropane I by the lignin peroxidase compound I to form a benzene pi cation radical which undergoes alpha, beta cleavage to produce a benzaldehyde and a C6C2 benzylic radical. The latter is then attacked by O2 to form a hydroperoxy radical which may decompose through a tetroxide to form the phenylglycol IV and phenylketol V. Under anaerobic conditions the C6C2 benzylic radical is probably oxidized to a carbonium ion which would be subsequently attacked by H2O to yield the phenylglycol V.     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium

An H2O2-requiring oxygenase found in the extracellular medium of ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium was purified by DEAE-Sepharose ion-exchange chromatography and gel filtration on Sephadex G-100. Sodium dodecyl sulfate (SDS)-disc gel electrophoresis indicated that the purified protein was homogeneous. The Mr of the enzyme as determined by gel filtration and SDS-polyacrylamide gel electrophoresis was 41,000. The absorption spectrum of the enzyme indicated the presence of a heme prosthetic group. The absorption maximum of the native enzyme (407 nm) shifted to 435 nm in the reduced enzyme and to 420 nm in the reduced-CO complex. The pyridine hemochrome absorption spectrum indicated that the enzyme contained one molecule of heme as iron protoporphyrin IX. Both CN- and N-3 bound readily to the native enzyme, indicating an available coordination site and that the heme iron was high spin. The purified enzyme generated ethylene from 2-keto-4-thiomethyl butyric acid, and oxidized a variety of lignin model compounds, including the diarylpropane, 1-(3'4'-diethoxyphenyl)1,3-dihydroxy-2-(4"-methoxyphenyl)propane (I); a beta-ether dimer, 1-(4'-ethoxy-3'-methoxyphenyl)glycerol-beta-guaiacyl ether (V); an olefin, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2 propene (III); and a diol, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2-propane diol (IV). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures.     

Inhibition of lignin peroxidase H2 by sodium azide.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was strongly inhibited by sodium azide. Inhibition was competitive with respect to veratryl alcohol (Ki = 1-2 microM) and uncompetitive with respect to H2O2. In contrast, sodium azide bound to the native enzyme at pH 6.0 with an apparent dissociation constant (KD) of 126 mM. Formation of azidyl radicals was detected by ESR spin trapping techniques. The enzymes is nearly completely inactivated in four turnovers. The H2O2-activated enzyme intermediate (compound I) reacted with sodium azide to form a new species rather than be reduced to the enzyme intermediate compound II. The new species has absorption maxima at 418, 540, and 570 nm, suggesting the formation of a ferrous-lignin peroxidase-NO complex. Confirmation of this assignment was obtained by low-temperature ESR spectroscopy. An identical complex could be simulated by the addition of nitrite to the reduced enzyme. The enzyme intermediate compound II is readily reduced by sodium azide to native enzyme with essentially no loss of activity.     

Lignin peroxidase compound III. Mechanism of formation and decomposition

Lignin peroxidase compound III (LiPIII) was prepared via three procedures: (a) ferrous LiP + O2 (LiPIIIa), (b) ferric LiP + O2-. (LiPIIIb), and (c) LiP compound II + excess H2O2 followed by treatment with catalase (LiPIIIc). LiPIIIa, b, and c each have a Soret maximum at approximately 414 nm and visible bands at 543 and 578 nm. LiPIIIa, b, and c each slowly reverted to native ferric LiP, releasing stoichiometric amounts of O2-. in the process. Electronic absorption spectra of LiPIII reversion to the native enzyme displayed isosbestic points in the visible region at 470, 525, and 597 nm, suggesting a single-step reversion with no intermediates. The LiPIII reversion reactions obeyed first-order kinetics with rate constants of approximately 1.0 X 10(-3) s-1. In the presence of excess peroxide, at pH 3.0, native LiP, LiPII, and LiPIIIa, b, and c are all converted to a unique oxidized species (LiPIII*) with a spectrum displaying visible bands at 543 and 578 nm, but with a Soret maximum at 419 nm, red-shifted 5 nm from that of LiPIII. LiPIII* is bleached and inactivated in the presence of excess H2O2 via a biphasic process. The fast first phase of this bleaching reaction obeys second-order kinetics, with a rate constant of 1.7 X 10(1) M-1 s-1. Addition of veratryl alcohol to LiPIII* results in its rapid reversion to the native enzyme, via an apparent one-step reaction that obeys second-order kinetics with a rate constant of 3.5 X 10(1) M-1 s-1. Stoichiometric amounts of O2-. are released during this reaction. When this reaction was run under conditions that prevented further reactions, HPLC analysis of the products demonstrated that veratryl alcohol was not oxidized. These results suggest that the binding of veratryl alcohol to LiPIII* displaces O2-., thus returning the enzyme to its native state. In contrast, the addition of veratryl alcohol to LiPIII did not affect the rate of spontaneous reversion of LiPIII to the native enzyme.     

Catalytic mechanisms and regulation of lignin peroxidase.

Lignin peroxidase (LiP) is a fungal haemoprotein similar to the lignin-synthesizing plant peroxidases, but it has a higher oxidation potential and oxidizes dimethoxylated aromatic compounds to radical cations. It catalyses the degradation of lignin models but in vitro the outcome is net lignin polymerization. LiP oxidizes veratryl alcohol to radical cations which are proposed to act by charge transfer to mediate in the oxidation of lignin. Phenolic compounds are, however, preferentially oxidized, but transiently inactivate the enzyme. Analysis of the catalytic cycle of LiP shows that in the presence of veratryl alcohol the steady-state turnover intermediate is Compound II. We propose that veratryl alcohol is oxidized by the enzyme intermediate Compound I to a radical cation which now participates in charge-transfer reactions with either veratryl alcohol or another reductant, when present. Reduction of Compound II to native state may involve a radical product of veratryl alcohol or radical product of charge transfer. Phenoxy radicals, by contrast, cannot engage in charge-transfer reactions and reaction of Compound II with H2O2 ensues to form the peroxidatically inactive intermediate, Compound III. Regulation of LiP activity by phenolic compounds suggests feedback control, since many of the products of lignin degradation are phenolic. Such control would lower the concentration of phenolics relative to oxygen and favour degradative ring-opening reactions.     

Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium

The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

Biomimetic oxidation of nonphenolic lignin models by Mn(III): new observations on the oxidizability of guaiacyl and syringyl substructures

Mn(III) is a one-electron oxidant, produced in vivo by the Mn peroxidases of white-rot fungi, and thought to be involved in lignin degradation by these organisms. However, Mn(III) has not been shown to oxidize the major nonphenolic substructures of lignin under mild conditions. We have used Mn(III) acetate as a biomimetic model for enzymatically generated Mn(III), and report that low concentrations of this oxidant suffice to oxidize nonphenolic lignin models at physiological temperatures and pH values. Under these conditions, the monomeric lignin model veratryl alcohol was oxidized to veratraldehyde, and the diarylpropane model 1-(3,4-dimethoxyphenyl)-2-phenylpropanol was oxidatively cleaved to veratraldehyde, 1-phenylethanol, and acetophenone. In an attempt to identify other lignin models that might be oxidized by Mn(III) more rapidly, we compared the rates at which Mn(III) was reduced by two guaiacyl models, veratryl alcohol and 1-(3-methoxy-4-isopropoxyphenyl)ethanol, vs two syringyl models, 3,4,5-trimethoxybenzyl alcohol and 1-(3,5-dimethoxy-4-isopropoxyphenyl)ethanol. The results were the opposite of those predicted: the syringyl models were oxidized slower than the guaiacyl models by Mn(III). To investigate the basis for this unexpected result, we recorded the visible absorption spectra of charge-transfer complexes prepared between each of the lignin models and an electron acceptor, tetracyanoethylene or p-chloranil. The results, in general agreement with the kinetic findings, showed that the nonphenolic syringyl lignin models had higher ionization potentials than the guaiacyl models.     

Veratryl alcohol oxidases from the lignin-degrading basidiomycete Pleurotus sajor-caju.

The basidiomycete Pleurotus sajor-caju mineralizes ring-14C-labelled lignin (dehydrogenative polymer) when grown in mycological broth. Under these conditions, two veratryl alcohol oxidase (VAO) enzymes were found in the culture medium. They oxidized a number of aromatic alcohols to aldehydes and reduced O2 to H2O2. The enzymes were purified by ion-exchange and gel-permeation chromatography. The final step of purification on Mono Q resolved the activity into two peaks (VAO I and VAO II). Both enzymes had the same Mr, approx. 71,000, but their isoelectric points differed slightly, 3.8 for VAO I and 4.0 for VAO II. Their amino acid compositions were similar except for aspartic acid/asparagine and glycine. Both enzymes are glycoproteins and contain flavin prosthetic groups. Their pH optima were around 5, and kinetic constants and specificities were similar. 4-Methoxybenzyl alcohol was oxidized the most rapidly, followed by veratryl alcohol. Not all aromatic alcohols were oxidized, neither were non-aromatic alcohols. Cinnamyl alcohol was oxidized at the gamma position. The VAO enzymes thus represent a significantly different route for veratryl alcohol oxidation from that catalysed by the previously found lignin peroxidases from Phanerochaete chrysosporium. The role of the oxidases in biodegradation might be to produce H2O2 during oxidation of lignin fragments.     

Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N-3 readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 microM. The enzyme is also stimulated to varying degrees by alpha-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl-6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism.     

Separation and identification of the regioisomers of verdoheme by reversed-phase ion-pair high-performance liquid chromatography, and characterization of their complexes with heme oxygenase

We report an HPLC method for separating the four regioisomers of verdoheme formed in the coupled oxidation of hemin with oxygen and ascorbate in aqueous pyridine. The reversed-phase ion-pair system uses hexafluoroacetone and pyridine as ion-pair agents. The regiochemistry of the separated isomers was established both by HPLC of the corresponding biliverdin IX derivatives and by 1H NMR of each isomer. Optical spectra of the pyridine verdohemochrome isomers were similar to each other, but showed differences in the absorption maxima in the red region, which appear at 680, 663, 648 and 660 nm for the alpha, beta, gamma, and delta-isomers, respectively. Each of the four isomers was incorporated anaerobically into heme oxygenase-1, yielding the corresponding verdoheme-enzyme complex. The ferrous forms had absorption maxima at 690, 667, 655, and 663 nm, and their CO-bound forms had maxima at 638, 624, 616, and 626 nm for alpha, beta, gamma, and delta-isomer, respectively. Addition of ferricyanide to the alpha-verdoheme-heme oxygenase complex brought about a ferric low-spin heme-like signal, which is identical with the ferric alpha-verdoheme complexed with the heme oxygenase that was observed in the heme oxygenase reaction.     

Oxidation of monomethoxylated aromatic compounds by lignin peroxidase: role of veratryl alcohol in lignin biodegradation

Lignin peroxidase (LiP), an extracellular heme enzyme from the lignin-degrading fungus Phanerochaete chrysosporium, catalyzes the H2O2-dependent oxidation of a variety of nonphenolic lignin model compounds. The oxidation of monomethoxylated lignin model compounds, such as anisyl alcohol (AA), and the role of veratryl alcohol (VA) in LiP reactions were studied. AA oxidation reached a maximum at relatively low H2O2 concentrations, beyond which the extent of the reactions decreased. The presence of VA did not affect AA oxidation at low molar ratios of H2O2 to enzyme; however, at ratios above 100, the presence of VA abolished the decrease in AA oxidation. Addition of stoichiometric amounts of AA to LiP compound II (LiPII) resulted in its reduction to the native enzyme at rates that were significantly faster than the spontaneous rate of reduction, indicating that AA and other monomethoxylated aromatics are directly oxidized by LiP, albeit slowly. Under steady-state conditions in the presence of excess H2O2 and VA, a visible spectrum for LiPII was obtained. In contrast, under steady-state conditions in the presence of AA a visible spectrum was obtained for LiPIII*, a noncovalent complex of LiPIII and H2O2. AA competitively inhibited the oxidation of VA by LiP; the Ki for AA inhibition was 32 microM. Addition of VA to LiPIII* resulted in its conversion to the native enzyme. In contrast, AA did not convert LiPIII* to the native enzyme; instead, LiPIII* was bleached in the presence of AA. Thus, AA does not protect LiP from inactivation by H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.     

Thiol and Mn(2+)-mediated oxidation of veratryl alcohol by horseradish peroxidase.

Horseradish peroxidase has been shown to catalyze the oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) and benzyl alcohol to the respective aldehydes in the presence of reduced glutathione, MnCl2, and an organic acid metal chelator such as lactate. The oxidation is most likely the result of hydrogen abstraction from the benzylic carbon of the substrate alcohol leading to eventual disproportionation to the aldehyde product. An aromatic cation radical intermediate, as would be formed during the oxidation of veratryl alcohol in the lignin peroxidase-H2O2 system, is not formed during the horseradish peroxidase-catalyzed reaction. In addition to glutathione, dithiothreitol, L-cysteine, and beta-mercaptoethanol are capable of promoting veratryl alcohol oxidation. Non-thiol reductants, such as ascorbate or dihydroxyfumarate (known substrates of horseradish peroxidase), do not support oxidation of veratryl alcohol. Spectral evidence indicates that horseradish peroxidase compound II is formed during the oxidation reaction. Furthermore, electron spin resonance studies indicate that glutathione is oxidized to the thiyl radical. However, in the absence of Mn2+, the thiyl radical is unable to promote the oxidation of veratryl alcohol. In addition, Mn3+ is unable to promote the oxidation of veratryl alcohol in the absence of glutathione. These results suggest that the ultimate oxidant of veratryl alcohol is a Mn(3+)-GSH or Mn(2+)-GS. complex (where GS. is the glutathiyl radical).     

Modification of the heme·heme oxygenase system with iron and cobalt tetrasulfonated phthalocyanines

Modification of heme·heme oxygenase by iron(III) and cobalt(II) tetrasulfonated phthalocyanines has been performed. New compounds have been isolated and their properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, electron paramagnetic resonance (EPR) and carboxymethylation at histidyl groups. Spectrophotometric titration data indicate the ratio of the reagents in this process to be 1:1. The visible absorption spectra show the main peak at 650 nm for the iron compound and 682 nm for the cobalt one. Electrophoresis and molecular weight estimation show both complexes to be monomers. Cobalt(II) tetrasulfonated phthalocyanine, under aerobic conditions with heme oxygenase protein, undergoes autooxidation to the cobalt(III) complex, as has been proved by EPR and spectroscopic data. Iron and cobalt phthalocyanine modified heme·heme oxygenase with excess dithionite is reduced at the phthalocyanine ligand. In the presence of oxygen, the reduction product transforms into oxygenated Fe(III)Lheme oxygenase or Co(III)heme oxygenase, respectively. Reduction of the iron(III) model complex with ascorbic acid under anaerobic conditions leads to degradation of the phthalocyanine moiety, while Co(III)heme oxygenase with ascorbic acid is reduced to Co(II)Lheme oxygenase. As has been shown by carboxymethylation of the heme oxygenase protein at the histidine residues, the predominant binding site of both phthalocyanine complexes is the heme-binding histidyl residue. There is evidence that there is a second binding site with lower affinity towards Co(II)L on the heme oxygenase protein. Iron and cobalt tetrasulfonated phthalocyanines are not able to displace heme from the heme·heme oxygenase complex. In this reaction the iron complex undergoes degradation and the cobalt one gives a hybrid compound with heme·heme oxygenaseHeme oxygenase protein complexes with iron and cobalt tetrasulfonated phthalocyanines do not exhibit activity in their oxidative degradation.     

Spectrophotometric investigations with hexa-coordinate ferric lignin peroxidase: does water retention at the active site influence catalysis?

Native lignin peroxidase (LIP) can adopt either a stable penta- or hexa-coordinate state. We have examined catalysis with hexa-coordinate ferric LIP as the starting material, using rapid scanning spectrophotometry. Initial two-electron oxidation of hexa-coordinate native LIP by H(2)O(2) (Compound I formation) was accompanied by a shifting isosbestic point (419-->416 nm), consistent with displacement of a resident water molecule, prior to the reaction of the ferric iron with H(2)O(2). The Compound I species derived from a hexa-coordinate ferric state shows an unusual peak at 520 nm, which may be due to water retention in the vicinity of the heme active site. Compound I reduction by veratryl alcohol showed saturation kinetics, which contrasts with the situation observed when Compound I is derived from a penta-coordinate ferric state. The data inferred that water can interfere with heme access by electron donors, altering the mechanism of Compound I reduction.     

Visible light decomposition of ammonia to N2 with Ru(bpy)3(2+) sensitizer.

Visible light decomposition of aqueous NH3 to N2 was investigated using a photocatalyst aqueous solution based on molecular photoelectron relay systems of either sensitizer (tris(2,2'-bipyridine)ruthenium(II), (Ru(bpy)3(2+))/potassium peroxodisulfate(K(2)S(2)O(8)) or Ru(bpy)3(2+)/methylviologen dichloride(MV2+)/O2, capable of using visible light instead of UV-driven semiconductors such as TiO2. It was confirmed by using an in situ visible absorption spectral change under irradiation that the Ru(II) complex is oxidized to the Ru(III) complex by K(2)S(2)O(8), and that the Ru(III) complex formed is stable without NH3, while the added NH3 was oxidized by the Ru(III) complex to produce the Ru(II) complex. In the presence of 1 mM NH3 aqueous solution, the Ru(III) complex was the predominant species under the photostationary state, but in the presence of 100 mM NH3, Ru(II) predominated. Gas-chromatographic analysis of the gaseous phase in the presence of 8.1 M NH3 showed that the photochemical oxidation of ammonia yielded N2. It was also demonstrated by using the in situ visible absorption spectrum under irradiation of the NH3 (1 M)/Ru(bpy)3(2+) (0.1 mM)/MV2+ (10 mM) system under Ar that MV+* is accumulated, showing that NH3 works as an electron donor for MV+* accumulation with simultaneous formation of the oxidized product of ammonia ((NH3)ox) without producing N2. It was suggested that the reduced product (MV+*) and the oxidized product ((NH3)ox) are in a kind of dynamic equilibrium prohibiting further oxidation of (NH3)ox by Ru(bpy)3(3+) to N2. In the O2 atmosphere, the oxidation of MV+* to MV2+ takes place to accumulate Ru(III) complex, so that (NH3)ox was further oxidized to N2. The high activity of IrO2 as a cocatalyst in this system was demonstrated.     

The coprophilous mushroom Coprinus radians secretes a haloperoxidase that catalyzes aromatic peroxygenation

Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 microM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.     

Spectroscopic insights into axial ligation and active-site H-bonding in substrate-bound human heme oxygenase-2

Heme oxygenases (HOs) are monooxygenases that catalyze the first step in heme degradation, converting heme to biliverdin with concomitant release of Fe(II) and CO from the porphyrin macrocycle. Two heme oxygenase isoforms, HO-1 and HO-2, exist that differ in several ways, including a complete lack of Cys residues in HO-1 and the presence of three Cys residues as part of heme-regulatory motifs (HRMs) in HO-2. HRMs in other heme proteins are thought to directly bind heme, or to otherwise regulate protein stability or activity; however, it is not currently known how the HRMs exert these effects on HO-2 function. To better understand the properties of this vital enzyme and to elucidate possible roles of its HRMs, various forms of HO-2 possessing distinct alterations to the HRMs were prepared. In this study, variants with Cys265 in a thiol form are compared with those with this residue in an oxidized (part of a disulfide bond or existing as a sulfenate moiety) form. Absorption and magnetic circular dichroism spectroscopic data of these HO-2 variants clearly demonstrate that a new low-spin Fe(III) heme species characteristic of thiolate ligation is formed when Cys265 is reduced. Additionally, absorption, magnetic circular dichroism, and resonance Raman data collected at different temperatures reveal an intriguing temperature dependence of the iron spin state in the heme–HO-2 complex. These findings are consistent with the presence of a hydrogen-bonding network at the heme’s distal side within the active site of HO-2 with potentially significant differences from that observed in HO-1.