Thiol-mediated Oxidation of Nonphenolic Lignin Model Compounds by Manganese Peroxidase of Phanerochaete chrysosporium*

In the presence of Mn", H202, and glutathione (GSH), manganese peroxidase oxidized veratryl alcohol (I) to veratraldehyde (IV). Anisyl alcohol (11) and benzyl al- cohol (111) were also oxidized by this system to their corresponding aldehydes (V and VI). In the presence of GSH, chemically prepared Mn"' or y-irradiation also catalyzed the oxidation of I, 11, and I11 to IV, V, and VI, respectively. GSH and dithiothreitol rapidly reduced Mn"' to Mn" in the absence of aromatic substrates and the dithiothreitol was oxidized to its disul- fide (4,5-d.ihydroxyl-l,Z-dithiane). These results indicate that the thiol is oxidized by enzyme-generated Mn"' to a thiyl radical. The latter abstracts a hydrogen from the substrate, forming a benzylic radical which reacts with another thiyl radical to yield an interme- diate which decomposes to the benzaldehyde product. In the presence of Mn", GSH, and HzOz, manganese peroxidase also oxidized 1-(4-ethoxy-3-methoxy-phenyl)-2-(4'-hydroxymethyl-2'-methoxyphenoxy)- 1,3-dihydroxypropane (XII) to yield

white rot basidiomycete Phanerochuete chrysosporium produces two heme peroxidases (3)(4)(5)(6)(7)(8)(9)(10) which along with an Hz02 generating system (3) appear to be the major components of its lignin degradative system. The structure and mechanism of lignin peroxidase have been studied extensively (3-5, 7, 8, 11-13). Manganese peroxidase (MnP)' has also been purified and characterized (5,6,9,(13)(14)(15). The enzyme exists as a series of isozymes (13), contains one iron protoporphyrin IX prosthetic group (9), and is a glycoprotein of M, -46,000 (6,9,15). MnP catalyzes the H20zand Mn"-dependent oxidation of a variety of phenols, amines, and organic dyes ( 5 , 9, Electronic absorption (5,9,16), EPR, and resonance Raman spectral evidence (17) indicates that the heme environment of native MnP has features which are similar to those of other plant peroxidases (18). The nucleotide sequence of a cDNA encoding an MnP isozyme has been determined and confirms the presence of a proximal and distal histidine at the active center of the enzyme (19). In addition, spectral and kinetic evidence (16,20) indicates that the HzOz-oxidized states (compounds I and 11) and the catalytic cycle of MnP are similar to those of lignin and horseradish peroxidases (11,18). Most importantly, it has been demonstrated that MnP oxidizes Mn" to Mn"' and that the Mn"' in turn oxidizes monomeric phenols (9,(14)(15)(16) and phenolic lignin dimers (21) via the formation of a phenoxy radical. Transient state kinetic analysis (20) has confirmed that Mn"/Mn"' acts as a redox couple rather than as an enzyme binding activator. Chelation by certain organic acids such as lactate and malonate stabilizes the Mn"' at a high redox potential (0.9-1.2 V) facilitating the oxidation of organic substrates (22,23).
The one-electron oxidation of phenols (14, 16,24) and thiols (24)(25)(26) to phenoxy or thiyl radicals by Mn" ' and other transition metals has been well studied. In contrast, Mn"' complexes with organic acids such as malonate apparently are not capable of easily oxidizing most nonphenolic lignin model compounds such as veratryl alcohol under normal physiological conditions (5,6,9,21). In contrast, a recent report by Forrester et al. (27) claims that nonphenolic lignin model compounds are oxidized directly by Mn"'-pyrophosphate in the presence of glutathione via the initial formation of aryl cation radicals. However, since the mechanism of Forrester et al. (27) appeared unlikely, we have re-examined the oxidation of nonphenolic lignin models by MnP-generated and chemically prepared Mn"' in the presence of thiols. Herein, we demonstrate that in this system, Mn"' oxidizes thiols to thiyl radicals which in turn react with the lignin models to form carbon-centered radicals. The latter undergo a variety of reactions to yield the final products. 14-16).
The abbreviations used are: MnP, manganese peroxidase; DTT, dithiothreitol; DTE, dithioerythritol; GCMS, gas chromatography mass spectrometry. Product Analyses-Upon completion of the reactions, mixtures were acidified to pH 3.0 with HC1, extracted with chloroform (2 X 2 ml), dried over Na2SO4, evaporated with Nz, and analyzed either directly or following derivatization (N,O-bis(trimethylsi1yl)trifluoroacetamide/pyridine 2:l v/v). Products were identified by comparison of their retention times on GC and by comparison of their MS spectra with those of chemically synthesized standards. GCMS was performed at 70 eV on a VG Analytical 7070E mass spectrometer fitted with an HP5790A GC and a 25-m fused silica column (DB-5, J&W Scientific).
The temperature was programmed from 80-320 "C a t 10 "C/min. Quantitative analysis of substituted benzaldehyde products was carried out by high pressure liquid chromatography (HP LiChosphere 100 RP-18 column) using a solvent gradient system from 5% MeOH in H20 to 100% MeOH.

Oxidation of Aryl Alcohols by the MnP/Mn"/Thiol System-As shown in
In the presence of thiol, under anaerobic conditions, chemically prepared Mn"'-malonate was also capable of oxidizing these substituted benzyl alcohols to the corresponding aldehyde and coupled dimer products (Table I).
When the Mn"'-malonate or Mn"'-pyrophosphate reaction was carried out in the absence of thiol at either pH 4.5 or 3.0, no products were observed.
In order to test the possibility that Mn"'-generated thiyl radicals (24)(25)(26) were involved in the oxidation of the benzyl alcohols I, 11, and 111, we examined the oxidation of I, 11, and I11 in a y-irradiation system consisting of substrates, GSH, and buffer. The generation of thiyl radicals from thiols by yirradiation has previously been well studied (37,38). As shown in Table I, when the alcohols were irradiated in the presence of GSH under anaerobic conditions, the same aldehyde and coupled dimer products were obtained. Furthermore, in the absence of GSH, y-irradiation did not lead to oxidation of the substrates.
Since the formation of the coupled dimer (IX) was negligible, the initial rate of veratryl alcohol oxidation could be followed spectrophotometrically by measuring the rate of formation of veratraldehyde at 310 nm (7,8,10) (Table 11). Under anaerobic conditions GSH, DTE, and DTT were all effective thiols. However, activity was considerably reduced when cysteine was used as the thiol. In the presence of GSH, both malonate and lactate were effective as Mn"' chelators. Activity was considerably reduced when oxalate or pyrophosphate were substituted for malonate. Very low activity was detected in succinate buffer, confirming that succinate is unable to form an Mn"' complex (23). With all thiols and organic acids used, the initial rate of veratraldehyde formation was -2-fold greater under anaerobic conditions than under aerobic conditions. Oxidation of Thiol by Mn' "-As shown in Fig. lA, Mn"'malonate was effectively reduced by GSH in the absence of substituted benzyl alcohols. Addition of 10 equivalents of GSH to Mn"'-malonate resulted in the rapid formation of a featureless spectrum between 250-500 nm, characteristic of Mn" organic acid complexes (Fig. lA ) (9, 14). In contrast, in the absence of thiol, less that 10% of the Mn"'-malonate was reduced upon addition of 10 equivalents of veratryl alcohol (data not shown). Furthermore, Fig. 1B shows that in the absence of veratryl alcohol, Mn"'-malonate accumulation in the enzyme system is suppressed by the addition of thiol. The kinetic curve obtained in the presence of 1.0 mM GSH (Fig.  1B) suggests that there is an initial burst of Mn"'-malonate followed by a plateau when the rate of Mn"' formation approximately equals the rate of Mn"' reduction. After Mn"'malonate was reduced by DTT (1,4-dimercapto-2,3-dihydroxybutane), the mixtures were extracted with chloroform, derivatized, and analyzed by GCMS. The MS spectrum of the  101 (241, 73 (92). These results indicate that the Mn"'-malonate is capable of effectively oxidizing the thiol to a thiyl radical which subsequently undergoes radical coupling to form an intramolecular disulfide bond.
The pH dependence from pH 3.1-6.1 for the oxidation of veratryl alcohol by MnP/Mn"/GSH is shown in Fig. 2. Activity increased with increasing pH. Fig. 2 also shows the pH dependence of the lignin peroxidase-catalyzed oxidation of veratryl alcohol (39). Here, activity increased with decreasing pH.
When the substrates XII, XIV, and XVI were irradiated under anaerobic conditions in the presence of GSH, the same products were obtained as with the enzyme system (Fig. 3). In the absence of GSH, no products were obtained and substrate was recovered nearly quantitatively after irradiation for 6 h.
Oxidation of Substrates under Aerobic Conditions-Product analysis revealed that with all of the substrates examined using each of the three systems ((i) MnP/Mn"/thiol; (ii) Mn"'/thiol; (iii) y-irradiation/thiol), identical products were obtained under aerobic conditions but in reduced yield (-50% of anaerobic samples).

DISCUSSION
Spectral and kinetic studies have indicated that the principal function of MnP is the oxidation of Mn" to Mn"' (9, 14) via a typical peroxidase catalytic cycle (9,14,16,18,20). The enzymatically generated Mn"' in turn oxidizes a variety of organic substrates (9, 14-16, 20, 21). A number of organic acids are capable of forming complexes with Mn"' (9,16,20,(22)(23)(24)(25). For example, malonate and lactate chelate Mn"' to form distorted octahedral complexes, containing two water molecules (23). These complexes are relatively stable in aqueous solution but still possess a high redox potential (0.9-1.2 V) (22,23). We have recently demonstrated that chelation of Mn"' by organic acids also facilitates its release from the enzyme-Mn complex (20). Mn"'-organic acid complexes are well-studied one-electron oxidants which are capable of oxidizing numerous substrates including phenols and thiols (9,14,22,24,25). We recently reported that MnP catalyzes C,-Cp and alkylphenyl bond cleavage of a phenolic diarylpropane dimer and that these cleavage reactions are initiated by the oxidation of the dimer to a phenoxy radical by enzymatically generated Mn"' (21). Nonphenolic lignin models are not oxidized by either the MnP/Mn"-malonate system or by Mn"'malonate complexes (21) under physiological conditions. In contrast, lignin peroxidase catalyzes the one-electron oxidation of non-phenolic lignin model compounds to form aryl cation radicals which subsequently undergo a variety of nonenzymatic reactions including C,-Co cleavage and ring cleavage (3-5, 11,12,29,33).
Recently, the oxidation of veratryl alcohol and nonphenolic P-aryl-ether-type lignin dimers by MnP in the presence of thiols has been reported (27). In that report (27) the authors claim that in the presence of thiols the Mn"'-pyrophosphate complex is capable of oxidizing aromatic substrates to their corresponding aryl cation radicals and that GSH stimulates the reaction by reducing oxygen to superoxide. We considered this to be an unlikely mechanism (27) for several reasons.   (22,23). Therefore, the oxidation to aryl cation radicals of veratryl alcohol and other dimethoxy benzenes with higher redox potentials (41) by Mn"'-pyrophosphate is not energetically favorable.
To clarify the mechanism of the MnP-catalyzed thiolmediated oxidation of nonphenolic lignin models, we have reexamined the reaction using spectroscopic, kinetic, and product analyses.
As shown in Tables I and 11, veratryl alcohol, anisyl alcohol, and benzyl alcohol were oxidized by (i) MnP/Mn"/thiol, (ii) Mn"'-malonate/thiol, and (iii) yirradiation in the presence of thiol to yield identical products. In each system the benzyl alcohols were oxidized to yield the corresponding aldehyde as the major product and a coupled dimer as a minor product. These reactions were absolutely dependent on the presence of thiol. Initial rates of reactions conducted under anaerobic conditions were -2 X greater than the rates of reactions conducted under aerobic conditions. Table I shows that veratryl alcohol, anisyl alcohol, and benzyl alcohol were oxidized with approximately equal efficiency by all three systems, indicating that aromatic methoxy groups do not influence these reactions. The addition of aromatic methoxy groups lowers the redox potential of methoxy benzenes (41); for example, the redox potentials of monomethoxy and dimethoxy benzenes have been determined to be -1.76 and 1.34-1. 45 V, respectively (41). Thus, the oxidation of methoxy benzenes to aryl cation radicals by peroxidases is facilitated by the addition of methoxy groups (33, 42). For example, lignin peroxidase easily oxidizes veratryl alcohol, but oxidizes anisyl alcohol slowly, and is not capable of oxidizing benzyl alcohol.' It is therefore unlikely that the reactions reported herein proceed through a cation radical intermediate.
The results in Fig. 1A indicate that Mn"' is rapidly reduced to Mn" in the presence of GSH. Furthermore, accumulation of Mn"' in the enzyme system is suppressed in the presence of GSH (Fig. 1B). XPI an intramolecular disulfide product. All of these results indicate that the Mn"' oxidizes the thiol to a thiyl radical which undergoes radical coupling to form the disulfide. The oxidation of thiols to thiyl radicals by Mn"' and other transition metals has been reported previously (23-26,43). The existence of free thiyl radicals has been confirmed by electron spin resonance spectroscopy (44, 45).
As further proof of the involvement of thiyl radicals, we utilized y-irradiation in the presence of thiols as a source of these radicals. Generation of thiyl radicals from thiols by yirradiation via .OH mediation has been well established (37,38,46). Table I shows that the thiolly-irradiation system oxidizes all of the benzyl alcohols to yield the same products as the MnP/Mn"/thiol system. None of the reactions occurred in the absence of thiol. These results also suggest that the enzyme-generated Mn"' oxidizes thiols to thiyl radicals which, in turn, mediate the dehydrogenation of the substituted benzyl alcohols. Hydrogen abstraction from active hydrogen donors by thiyl radicals to yield carbon-centered radicals has been previously reported (47, 48).
The initial rate of the reactions conducted under anaerobic conditions is twice the reaction rate under aerobic conditions (Table 11). As described previously (26), molecular oxygen reacts with thiyl radicals to form superoxide anion, thereby lowering the effective concentration of the reactive thiyl radicals. These results contradict the proposal by Forrester et al.  Table I1 shows that malonate and lactate stimulate the oxidation of veratryl alcohol in these systems more effectively than either oxalate or pyrophosphate. The results in Fig. 2, demonstrating that activity increases with increasing pH, suggest that protonation of the thiol inhibits its oxidation (26).
Mechanism of Benzyl Alcohol Oxidation-Oxidation of thiol by Mn"' generates a thiyl radical, which in turn can abstract
OH I rn bEt X E a benzylic hydrogen from benzyl alcohol (47, 48) to form a benzylic radical. Under anaerobic conditions, the benzylic radical probably couples with another thiyl radical to produce an unstable thiohemiacetal (Fig. 4). The latter would decompose to form a free thiol and a benzaldehyde. The formation of stable GSH conjugates from thiyl radicals has been reported previously (45, 49). Radical coupling of two benzylic radicals would yield the coupled dimers IX, X, and XI, which were observed in this study. Although these coupled dimers are obtained in only trace amounts, their formation strongly supports this mechanism. Under aerobic conditions the benzylic radical could be scavenged by molecular oxygen or a hydroperoxy radical to yield a peroxy intermediate which would decompose to yield the benzaldehyde product (50). Formation of the dimeric a-carbonyl (XIII) and the @-vanillin ether (XIV) from the P-vanillyl alcohol ether (XII) (Fig. 3) and the benzylic oxidation of XIV and XVI to yield XV and XIX, respectively (Fig. 3) can be explained in a similar manner.
Mechanism of P-Ether Cleauage-The P-vanillyl alcohol ether dimer (XII) has two benzylic hydrogens available for abstraction by thiyl radicals. Abstraction of the C, (A ring) hydrogen yields a benzylic radical, leading to Cp oxygen ether bond cleavage. This results in the formation of the unstable phenylpropene and phenoxy radical intermediates (Fig. 5,  left). The phenylpropene would be converted to the phenylpropane-1-oxo-3-01 (XIX). The phenoxy radical could abstract a hydrogen from GSH as previously proposed (43) to yield vanillyl alcohol (VII) and another thiyl radical (Fig. 5).
When the reaction was conducted in DzO, no deuterium was incorporated into the vanillyl alcohol (data not shown), suggesting that the phenolic hydrogen derived from GSH. The phenoxy radical of vanillyl alcohol may also be oxidized to vanillin via the transient formation of a quinone methide intermediate (21,51).
Alternatively, when the benzylic radical is formed at the CL (ring B) (Fig. 5, right), the ensuing radical cleavage yields a quinone methide and a Cp radical intermediate. The quinone methide spontaneously rearranges to vanillin. The Cp radical may abstract a proton from GSH to generate the phenylpropane-1-3-diol (XVI). The fact that when the reaction was conducted in DzO, no deuterium was incorporated into the diol (XVI) supports this mechanism. Exogenous phenylpropane-1,3-diol was oxidized to the corresponding ketone (XIX) by the enzyme system (Figs. 3 and 5).

2RS. + RSSR
where RSH and AH represent thiol and nonphenolic aromatic substrates, respectively. The key steps in the system are the oxidation of thiols to thiyl radicals by enzymically generated Mn"' (reaction 1) and hydrogen abstraction at the a-carbon by thiyl radicals to form a benzylic radical (reaction 2). These  (27). Product and kinetic analyses (Tables I and 11) demonstrate that molecular oxygen is not required for these reactions. Indeed, oxygen probably inhibits the reaction by competing for the thiyl radical (26) (reactions 5 and 6): RS. + 0 2 + RSO; RSO; + RSH + RSSR + H+ + 0;. (6) Although nonphenolic B-aryl ether lignin dimers were effectively cleaved at the CrO bond subsequent to the thiolmediated formation of benzylic radicals, it seems unlikely that extracellular thiols could play a significant role in the process of lignin degradation by white rot fungi since (a) lignin degradation is greatly stimulated under aerobic conditions, and ( b ) there is no evidence for free thiols in the extracellular culture media of several white rot fungi.3 Nevertheless, the effective thiol-mediated degradation of dimeric model compounds and of polymeric lignin (27) by MnP suggests that this system may have potential applications in the degradation of industrial lignins. Further studies on the degradation of polymeric lignin are in progress.