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Oxidation of methoxybenzenes by manganese peroxidase and by Mn3+
ARCHIVES
Vol.
OF BlOCHEMlSTRY
288, No. 1, July,
ANU
pp. 1455148,
BIOPHYSICS
1991
Oxidation of Methoxybenzenes Peroxidase and by Mn3+ ’ Janet
L. Popp’
and T. Kent
by Manganese
Kirk3
Institute for Microbial and Biochemical Technology, USDA Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, Wisconsin 53705-2398
Received
December
28, 1990, and in revised
form
February
25, 1991
Manganese peroxidase, produced by some white-rot fungi during lignin degradation, catalyzes the oxidation of Mn2+ to Mn3+. Whereas Mn3+ is known to oxidize phenolic compounds, its role in lignin degradation is not clear. We have used a series of methoxybenzenes with Eli2 values of 1.76-0.81 V (vs saturated calomel electrode) to investigate the oxidizing ability of Mn3+ chelates generated chemically and enzymatically. Although lignin peroxidase has been shown to oxidize high potential congeners, our results show that manganese peroxidase, or physiological concentrations of Mn3’, oxidize only the lower potential congeners. In addition, Mn3+ increased the rate of decay of the cation radical of 1,2,4,5-tetramethoxybenzene. The kinetics of decay continued to be first order, so Mn3+ does not oxidize the cation radical itself, but probably oxidizes a neutral dienyl radical derived from the cation radical. This indicates a possible role for Mn3+ in lignin degradation, as neutral dienyl radicals are proposed to be products of lignin peroxidase (0 1991 Academic Press, Inc. action.
The extracellular lignin-degrading enzyme system of the white-rot fungus Phanerochaete chrysosporium has been studied extensively in recent years (l-3). The system described to date consists of lignin peroxidase (LiP),4 manganese peroxidase (MnP), the HzOz-generating enzyme glyoxal oxidase (and possibly other peroxide-producing enzymes), and the metabolite veratryl alcohol. LIP, in the presence of HzOz, is thought to catalyze the major i This work was supported by USDA competitive Grant 86.FSTY-90167 awarded to T. K. Kirk. ’ Current address: Natural Products Biology, Sterling Research Group, 81 Columbia Turnpike, Rensselaer, NY 12144. ’ To whom correspondence should be addressed. FAX (608) Z-9262. 4 Abbreviations used: Lip, lignin peroxidase; MnP, manganese peroxidase; HRP, horseradish peroxidase.
onn3-9861/91
copyright All
rights
$43.00 @I 1991 by Academic Press, of reproduction in any form
degradative reaction, the generation of aryl cation radicals by the one electron oxidation of aromatic nuclei in lignin. Subsequent nonenzymatic reactions lead to fragmentation of the polymer (1,2). Several important questions remain, however, including the role(s), if any, of MnP. This enzyme is produced, usually as a major protein, during lignin degradation by P. chrysosporium. In the lignin-degrading fungus Lentinula edodes, MnP is apparently the only peroxidase detected during lignin degradation (4). A recent report dealing with the MnP from this organism (5) indicated that Mn”+ (chemically or enzymatically produced) is capable of significant oxidation of nonphenolic ligninrelated compounds, including veratryl alcohol. This is in contrast to reports with the MnP of P. chrysosporium (6, 7). For this reason we have taken a closer look at the oxidizing ability of MnP from P. chrysosporium and of Mn”+. Kersten and colleagues (8) used a series of methoxybenzenes with E1iz values of 1.76-0.81 V (vs saturated calomel electrode) to compare the oxidizing ability of LIP, horseradish peroxidase (HRP), and fungal lactase. Here we report the results of studies of Mn3’ and MnP oxidations of these methoxybenzenes. MATERIALS
AND
METHODS
Enzyme preparation.
MnP was purified from the filtrate of nutrient nitrogen-limited P. chrysosporium cultures (9) by Mono-Q FPLC and preparative isoelectric focusing (10, 11); the isozyme used has a p1 of 4.7. MnP activity was assayed by the oxidation of vanillyl acetone (6). No LIP (assayed by veratryl alcohol oxidation as described in Ref. (9)) was detected in our MnP preparation.
Methoxybenzenes oxidation reactions. Reactions were carried out with 2.5 mM Mn”+ (chemically generated and chelated by pyrophosphate) and with the MnP system: MnP/H20,/Mn2’. Since pyrophosphate inhibits MnP activity (Ref. 12, our unpublished results), the enzyme reactions were carried out in malonate buffer, also a good Mn3+ chelator (13). For all, stock solutions of methoxybenzenes (50 mM, synthesized or purchased as previously described; (Ref (14)) were dissolved in dimethylformamide. Mn3+/pyrophosphate was prepared fresh for each experiment by dissolving Mn3’ acetate in sodium pyrophosphate, pH 4 (1:l molar ratio, stock solution was 25 mM each). Reaction mixtures
145 Inc. reserved.
146
POPP TABLE Oxidation
I
of Methoxybenzenes Mn3+, MnP, and HRP
by Lip,
Oxidation
Compound
El/Z0
Methoxybenzene 1.3.Dimethoxybenzene 1,3,5-Trimethoxybenzene 1,2-Dimethoxybenzene 1,2,3-Trimethoxybenzene 1,4-Dimethoxybenzene 1,2,3,4-Tetramethoxybenzene
1.76 ND 1.49 1.45 1.42 1.34 1.25
Hexamethoxybenzene 1,2,4-Trimethoxybenzene 1,2,3,5-Tetramethoxybenzene Pentamethoxybenzene 1,2,4,5-Tetramethoxybenzene
1.24 1.12 1.09 1.07 0.81
AND
LIP +HzOz* -d + + + + + + t + + +
MnP system
by 2.5 mM Mr?+/PP’
-
+ + t t
HRP +H202
-
-
-
-
t + + t
+ + + +
a vs saturated calomel electrode, from Ref. (20). * From Kersten et al. (8). ’ PP, pyrophosphate. d -, no oxidation detected; +, oxidation product observed.
contained 2.5 mM M@/pyrophosphate and 2 mM of the methoxybenzene in a volume of 1 ml. In controls, Mn3+ was omitted. They were incubated 48 h and the products detected by reverse phase HPLC (Cl8 column, isocratic solvent of 30% acetonitrile in aqueous 0.1% trifluoroacetic acid, flow rate of 1 ml/min). For the enzymatic reaction, mixtures contained 0.4 U MnP, 20 mM malonate, pH 4, 10 mM MnSO,, and 2 mM methoxybenzene in a volume of 1 ml. H202 (1 mM) was added to initiate the reaction and again at 24 b. The maximum Mn3+ concentration in these reaction mixtures (determined by the absorbance of the Mn3+/malonate complex at 266 nm, t = 11,500) was 1-2 mM. Total incubation time was 48 h and products were detected by HPLC as above. Control reactions lacked MnP. Kinetics of the decay of the cation radical of 1,2,4,5-tetramethoxybenzene. Reaction mixtures to generate the cation radical intermediate contained 25 mM tartrate, pH 3.0,O.l mM 1,2,4,5-tetramethoxybenzene, and 0.01 mg/ml HRP. The reaction was initiated by the addition of 0.05 mM H202 and the cation radical monitored by its absorbance at 450 nm (8) on a Phillips PUS800 spectrophotometer. Under these conditions, the rate of formation of the cation radical is much greater than the rate of its decay. At the peak of absorbance, Mn3+ chelated by pyrophosphate (1:lO molar ratio) was added to a final concentration of 0.0-0.10 mM and the decay of the radical monitored.
RESULTS
AND
KIRK
Mn3+ has a high standard redox potential (1.5 V). However, standard conditions (pH 0,O.l M ionic strength) are not likely to be present in physiological situations. In addition, complexation by dicarboxylic or ol-hydroxy acids affects the potential of Mn3+. Therefore examination of the oxidizing capabilities of Mn3+ in physiologically relevant conditions is appropriate. Our results indicate that Mn3+, generated by MnP and stabilized by a small organic acid, will not efficiently oxidize high potential lignin substructures. Therefore Mn3+ does not function as a primary oxidant of nonphenolic units in lignin (i.e., it does not have the same role as LIP in lignin degradation). This is in contrast to the report of Forrester et al. (5) which states that Mn3’ is capable of oxidizing veratryl alcohol and is stimulated by reducing agents such as glutathione. Rather, our results support the contention of Wariishi et al. (15), that the oxidation of veratryl alcohol is due to thiyl radicals derived from Mn3+ oxidation of glutathione, and not to Mn3+ directly. We observed no oxidation of veratryl alcohol by 2.5 mM Mn3+ or the MnP system. Some of the methoxybenzenes give fairly stable cation radicals on oxidation (14). In particular, 1,2,4,5-tetramethoxybenzene gives a long-lived radical with significant visible absorbance with a maximum at 450 nm; its ESR spectrum has been characterized (E&14). We observed the formation of this cation radical in oxidation reactions with Mn3+ and noted that its decay was more rapid when it was generated by excess Mn3+ than when generated by HRP. Therefore, we examined the effect of the concentration of Mn3+ on the decay of this radical by using HRP and H,O, (at one half the concentration of the 1,2,4,5tetramethoxybenzene) to generate the radical. The results of this experiment are shown in Fig. 1. As the concentration of Mn3+ increased, the rate of decay of the cation radical also increased. The plot of In A450 vs time shows a linear relationship, indicating that decay of
DISCUSSION
The results of oxidation of the methoxybenzene series by Mn3+/pyrophosphate, as well as by the MnP system, are shown in Table I, together with the results for LiP and HRP from Kersten et al. (8). Whereas LiP oxidized all but the two highest potential congeners, Mn3’/pyrophosphate and the MnP system were able to oxidize only the four lowest potential compounds, which is the same result obtained with HRP. In our experiments, the use of two different Mn3+ chelators (pyrophosphate and malonate) gave indistinguishable results.
100
200 time (s)
300
FIG. 1. Decay of the cation radical of 1,2,4,5-tetramethoxybenzene in various concentrations of Mn3’. 0, no added Mn3+; 0, 10 /.LM Mn3+; 0, 30 PM Mn3’; n , 50 PM Mn3+.
MANGANESE
PEROXIDASE/Mr?+
OXIDATION
the cation radical is an apparent first-order process both in the absence and presence of Mn3+. Kersten and co-workers (8) proposed a mechanism for the decay of the cation radical of 1,2,4,5-tetramethoxybenzene that involves addition of water to form a neutral dienyl radical. This is followed by a second oxidation and loss of two equivalents of methanol. 2,5-Dimethoxy-pquinone and 4,5-dimethoxy-o-quinone were the products formed and 2 mol of methanol per mole of quinone were detected (8). They further showed (by experiments with H2180) that the quinone oxygens are derived from water. The decay of the cation radical is pseudo-first order because water is present in vast excess relative to the cation radical. Further support of this assertion is the observation that the relationship between the concentration of the enzyme and the steady-state concentration of the cat ion radical was linear (14). A square root relationship would be expected for a second-order dismutation of the cation radical. We propose that Mn3+ oxidizes the neutral dienyl radical as illustrated in Fig. 2. Though only the p-quinone product is shown, both 2,5-dimethoxy-p-quinone and 4,5dimethoxy-o-quinone were formed by Mn3+ oxidation (detected by reverse phase HPLC as in (Ref. (8)) and the ratio of the two products (p-:o-quinone = 1:4) was the same in the presence and absence of Mn3+. In the presence of Mn3+, the decay of the cation radical is still pseudofirst order. Therefore, Mn3+ cannot be reacting directly with the cation radical because that would be a secondorder reaction. The concentration of the cation radical in our experiments was 0.1 mM based on the complete oxidation of the tetramethoxybenzene under our reaction conditions (excess HRP) and on the cd50of 9800 M-l cm-l (8). The concentration of Mn3+ added was 0.01-0.05 mM. While with the lower concentrations of Mn”+, a secondorder reaction of MnR+ with the cation radical might be masked by the pseudo-first order decay, at 0.05 mM Mn3+ (one half the concentration of the cation radical) we would expect to observe deviation from first-order kinetics if, in fact, Mn3+ reacted with the cation radical. Thus, Mnsi probably reacts with the neutral dienyl radical. In order for this reaction to affect the decay of the cation radical, the addition of water to the cation radical must be a reversible reaction as shown in Fig. 2. The effect of Mn3+ is then to disturb the equilibrium of the cation radical/ neutral radical couple by oxidizing the neutral radical which leads to an increased decay of the cation radical. In other words, accelerating the decay of the neutral radical leads to acceleration of the decay of the cation radical. The oxidation of neutral dienyl radicals by Mn3+ may have implications for the biodegradation of lignin. Such radicals are proposed to be intermediates in the decay of compounds oxidized by LIP. This includes veratryl alcohol (16) as well as more complex lignin model compounds (17). LIP and MnP are produced simultaneously in lignin-
147
OF METHOXYBENZENES OCH,
HO
OCH,
OCH,
OCH,
CH,OH m2’
B
CH,OH
&?H,
FIG. 2. Proposed mechanism for the Mn3’ oxidation of the neutral dienyl radical derived from the cation radical of 1,2,4,5tetramethoxybenzene.
degrading cultures of P. chrysosporium (lo), and manganese is prevalent in wood. Thus it is conceivable that the two enzymes act in concert in the degradation of lignin. The Mn”+ (lo), and manganese is prevalent in wood. Thus it is conceivable that the two enzymes act in concert in the degradation of lignin. The Mn3+ oxidation of LiPderived neutral radicals is one possible point of interaction of these two enzymes. Some studies of LIP reactions have included the investigation of the effect of Mn’+. Schmidt et al. (16) reported that in the presence of MnS04 the yields of some of the products of LIP oxidation of veratryl alcohol methyl ether are altered. They did not speculate on the mechanism of the Mn2+ effect. Another report (18) suggests that Mn*+ scavenges superoxide formed during LIP oxidation of veratryl alcohol. This would generate Mn3+. In recent work, we showed that LIP, in the presence of malonate or oxalate, can oxidize Mn” to Mn3+ (19). An activated oxygen species is likely to be involved in this reaction also. Thus, it seems probable that in uivo both Mn*+ and Mn3+ (produced enzymatically or chemically) are present. Further work is needed to determine whether Mn3+ influences LIP oxidation reactions and to clarify the role of manganese and MnP in lignin degradation. ACKNOWLEDGMENT The authors thank Philip d. Kersten for helpful discussions.
REFERENCES I. Kirk, T. K., and Farrell, R. L. (1987) Annu. Rev. Microbid. 41, 465-505. 2. Tien, M. (1987) CRC Crit. Rev. J. Microbid. 15, 141-168. 3. Schoemaker, H. E., and Leisola, M. S. A. (1990) J. BiotechnoL 13, 101-109.
148 4. Leatham, G. F. (1986) Appl. Microbial.
POPP Biotechnol.
AND
24, 51-58.
5. Forrester, I. T., Grabski, A. C., Burgess, R. R., and Leatham, G. F. (1988) Biochem. Biophys. Res. Common. 157, 992-999. 6. Paszczynski, A., Huynh, V.-B., and Crawford, Biochem. Biophys. 244, 750-765. 7. Paszczynski, A., Huynh, V.-B., and Crawford, crobial. Lett. 29, 37-41.
R. (1986) Arch.
R. (1985) FEMS Mi-
8. Kersten, P. J., Kalyanaraman, B., Hammel, K. E., Reinhammar, B., and Kirk, T. K. (1990) Biochem. J. 268, 475-480. 9. Kirk, T. K., Croan, S., Tien, M., Murtagh, K. E., and Farrell, R. L. (1986) Enzyme Microb. Technol. 8, 27-32. 10. Farrell, R. L., Murtagh, K. E., Tien, M., Mozuch, M. D., and Kirk, T. K. (1989) Enzyme Microb. Technol. 11, 322-328. 11. Leisola, M. S. A., Kozulic, B., Meussdoerffer, (1987) J. Biol. Chem. 262,419-424.
F., and Fiechter,
A.
KIRK 12. Glenn, J. K., and Gold, M. H. (1985) Archiu. Biochem. Biophys. 242,329-341. 13. Bullock, J. I., Patel, M. M., and Salmon, J. E. (1969) J. Inorg. Nucl.
Chem.31,415-423. 14. Kersten, P. J., Tien, M., Kalyanaraman, B., and Kirk, T. K. (1985) J. Biol. Chem. 260, 2609-2612. 15. Wariishi, H., Valli, K., Renganathan, V., and Gold, M. H. (1989) J. Biol. Chem. 264, 14,185-14,191. 16. Schmidt, H. W. H., Haemmerli, S. D., Schoemaker, H. E., and Leisola, M. S. A. (1989) Biochemistry 28, 1776-1783. 17. Umezawa, T., and Higuchi, T. (1987) FEBS Lett. 218, 255-260. 18. Bono, J.-J., Goulas, P., Boe, J.-F., Portet, N., and Seris, J.-L. (1990) Eur. J. Biochem. 192, 189-193. 19. Popp, J. L., Kalyanaraman, B., and Kirk, T. K. (1990) Biochemistry 29, 10,475-10,480. 20. Zweig, A., Hodgson, W. G., and Jura, W. H. (1964) J. Am. Chem.
Sac. 86,4124-4129.
Vol.
OF BlOCHEMlSTRY
288, No. 1, July,
ANU
pp. 1455148,
BIOPHYSICS
1991
Oxidation of Methoxybenzenes Peroxidase and by Mn3+ ’ Janet
L. Popp’
and T. Kent
by Manganese
Kirk3
Institute for Microbial and Biochemical Technology, USDA Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, Wisconsin 53705-2398
Received
December
28, 1990, and in revised
form
February
25, 1991
Manganese peroxidase, produced by some white-rot fungi during lignin degradation, catalyzes the oxidation of Mn2+ to Mn3+. Whereas Mn3+ is known to oxidize phenolic compounds, its role in lignin degradation is not clear. We have used a series of methoxybenzenes with Eli2 values of 1.76-0.81 V (vs saturated calomel electrode) to investigate the oxidizing ability of Mn3+ chelates generated chemically and enzymatically. Although lignin peroxidase has been shown to oxidize high potential congeners, our results show that manganese peroxidase, or physiological concentrations of Mn3’, oxidize only the lower potential congeners. In addition, Mn3+ increased the rate of decay of the cation radical of 1,2,4,5-tetramethoxybenzene. The kinetics of decay continued to be first order, so Mn3+ does not oxidize the cation radical itself, but probably oxidizes a neutral dienyl radical derived from the cation radical. This indicates a possible role for Mn3+ in lignin degradation, as neutral dienyl radicals are proposed to be products of lignin peroxidase (0 1991 Academic Press, Inc. action.
The extracellular lignin-degrading enzyme system of the white-rot fungus Phanerochaete chrysosporium has been studied extensively in recent years (l-3). The system described to date consists of lignin peroxidase (LiP),4 manganese peroxidase (MnP), the HzOz-generating enzyme glyoxal oxidase (and possibly other peroxide-producing enzymes), and the metabolite veratryl alcohol. LIP, in the presence of HzOz, is thought to catalyze the major i This work was supported by USDA competitive Grant 86.FSTY-90167 awarded to T. K. Kirk. ’ Current address: Natural Products Biology, Sterling Research Group, 81 Columbia Turnpike, Rensselaer, NY 12144. ’ To whom correspondence should be addressed. FAX (608) Z-9262. 4 Abbreviations used: Lip, lignin peroxidase; MnP, manganese peroxidase; HRP, horseradish peroxidase.
onn3-9861/91
copyright All
rights
$43.00 @I 1991 by Academic Press, of reproduction in any form
degradative reaction, the generation of aryl cation radicals by the one electron oxidation of aromatic nuclei in lignin. Subsequent nonenzymatic reactions lead to fragmentation of the polymer (1,2). Several important questions remain, however, including the role(s), if any, of MnP. This enzyme is produced, usually as a major protein, during lignin degradation by P. chrysosporium. In the lignin-degrading fungus Lentinula edodes, MnP is apparently the only peroxidase detected during lignin degradation (4). A recent report dealing with the MnP from this organism (5) indicated that Mn”+ (chemically or enzymatically produced) is capable of significant oxidation of nonphenolic ligninrelated compounds, including veratryl alcohol. This is in contrast to reports with the MnP of P. chrysosporium (6, 7). For this reason we have taken a closer look at the oxidizing ability of MnP from P. chrysosporium and of Mn”+. Kersten and colleagues (8) used a series of methoxybenzenes with E1iz values of 1.76-0.81 V (vs saturated calomel electrode) to compare the oxidizing ability of LIP, horseradish peroxidase (HRP), and fungal lactase. Here we report the results of studies of Mn3’ and MnP oxidations of these methoxybenzenes. MATERIALS
AND
METHODS
Enzyme preparation.
MnP was purified from the filtrate of nutrient nitrogen-limited P. chrysosporium cultures (9) by Mono-Q FPLC and preparative isoelectric focusing (10, 11); the isozyme used has a p1 of 4.7. MnP activity was assayed by the oxidation of vanillyl acetone (6). No LIP (assayed by veratryl alcohol oxidation as described in Ref. (9)) was detected in our MnP preparation.
Methoxybenzenes oxidation reactions. Reactions were carried out with 2.5 mM Mn”+ (chemically generated and chelated by pyrophosphate) and with the MnP system: MnP/H20,/Mn2’. Since pyrophosphate inhibits MnP activity (Ref. 12, our unpublished results), the enzyme reactions were carried out in malonate buffer, also a good Mn3+ chelator (13). For all, stock solutions of methoxybenzenes (50 mM, synthesized or purchased as previously described; (Ref (14)) were dissolved in dimethylformamide. Mn3+/pyrophosphate was prepared fresh for each experiment by dissolving Mn3’ acetate in sodium pyrophosphate, pH 4 (1:l molar ratio, stock solution was 25 mM each). Reaction mixtures
145 Inc. reserved.
146
POPP TABLE Oxidation
I
of Methoxybenzenes Mn3+, MnP, and HRP
by Lip,
Oxidation
Compound
El/Z0
Methoxybenzene 1.3.Dimethoxybenzene 1,3,5-Trimethoxybenzene 1,2-Dimethoxybenzene 1,2,3-Trimethoxybenzene 1,4-Dimethoxybenzene 1,2,3,4-Tetramethoxybenzene
1.76 ND 1.49 1.45 1.42 1.34 1.25
Hexamethoxybenzene 1,2,4-Trimethoxybenzene 1,2,3,5-Tetramethoxybenzene Pentamethoxybenzene 1,2,4,5-Tetramethoxybenzene
1.24 1.12 1.09 1.07 0.81
AND
LIP +HzOz* -d + + + + + + t + + +
MnP system
by 2.5 mM Mr?+/PP’
-
+ + t t
HRP +H202
-
-
-
-
t + + t
+ + + +
a vs saturated calomel electrode, from Ref. (20). * From Kersten et al. (8). ’ PP, pyrophosphate. d -, no oxidation detected; +, oxidation product observed.
contained 2.5 mM M@/pyrophosphate and 2 mM of the methoxybenzene in a volume of 1 ml. In controls, Mn3+ was omitted. They were incubated 48 h and the products detected by reverse phase HPLC (Cl8 column, isocratic solvent of 30% acetonitrile in aqueous 0.1% trifluoroacetic acid, flow rate of 1 ml/min). For the enzymatic reaction, mixtures contained 0.4 U MnP, 20 mM malonate, pH 4, 10 mM MnSO,, and 2 mM methoxybenzene in a volume of 1 ml. H202 (1 mM) was added to initiate the reaction and again at 24 b. The maximum Mn3+ concentration in these reaction mixtures (determined by the absorbance of the Mn3+/malonate complex at 266 nm, t = 11,500) was 1-2 mM. Total incubation time was 48 h and products were detected by HPLC as above. Control reactions lacked MnP. Kinetics of the decay of the cation radical of 1,2,4,5-tetramethoxybenzene. Reaction mixtures to generate the cation radical intermediate contained 25 mM tartrate, pH 3.0,O.l mM 1,2,4,5-tetramethoxybenzene, and 0.01 mg/ml HRP. The reaction was initiated by the addition of 0.05 mM H202 and the cation radical monitored by its absorbance at 450 nm (8) on a Phillips PUS800 spectrophotometer. Under these conditions, the rate of formation of the cation radical is much greater than the rate of its decay. At the peak of absorbance, Mn3+ chelated by pyrophosphate (1:lO molar ratio) was added to a final concentration of 0.0-0.10 mM and the decay of the radical monitored.
RESULTS
AND
KIRK
Mn3+ has a high standard redox potential (1.5 V). However, standard conditions (pH 0,O.l M ionic strength) are not likely to be present in physiological situations. In addition, complexation by dicarboxylic or ol-hydroxy acids affects the potential of Mn3+. Therefore examination of the oxidizing capabilities of Mn3+ in physiologically relevant conditions is appropriate. Our results indicate that Mn3+, generated by MnP and stabilized by a small organic acid, will not efficiently oxidize high potential lignin substructures. Therefore Mn3+ does not function as a primary oxidant of nonphenolic units in lignin (i.e., it does not have the same role as LIP in lignin degradation). This is in contrast to the report of Forrester et al. (5) which states that Mn3’ is capable of oxidizing veratryl alcohol and is stimulated by reducing agents such as glutathione. Rather, our results support the contention of Wariishi et al. (15), that the oxidation of veratryl alcohol is due to thiyl radicals derived from Mn3+ oxidation of glutathione, and not to Mn3+ directly. We observed no oxidation of veratryl alcohol by 2.5 mM Mn3+ or the MnP system. Some of the methoxybenzenes give fairly stable cation radicals on oxidation (14). In particular, 1,2,4,5-tetramethoxybenzene gives a long-lived radical with significant visible absorbance with a maximum at 450 nm; its ESR spectrum has been characterized (E&14). We observed the formation of this cation radical in oxidation reactions with Mn3+ and noted that its decay was more rapid when it was generated by excess Mn3+ than when generated by HRP. Therefore, we examined the effect of the concentration of Mn3+ on the decay of this radical by using HRP and H,O, (at one half the concentration of the 1,2,4,5tetramethoxybenzene) to generate the radical. The results of this experiment are shown in Fig. 1. As the concentration of Mn3+ increased, the rate of decay of the cation radical also increased. The plot of In A450 vs time shows a linear relationship, indicating that decay of
DISCUSSION
The results of oxidation of the methoxybenzene series by Mn3+/pyrophosphate, as well as by the MnP system, are shown in Table I, together with the results for LiP and HRP from Kersten et al. (8). Whereas LiP oxidized all but the two highest potential congeners, Mn3’/pyrophosphate and the MnP system were able to oxidize only the four lowest potential compounds, which is the same result obtained with HRP. In our experiments, the use of two different Mn3+ chelators (pyrophosphate and malonate) gave indistinguishable results.
100
200 time (s)
300
FIG. 1. Decay of the cation radical of 1,2,4,5-tetramethoxybenzene in various concentrations of Mn3’. 0, no added Mn3+; 0, 10 /.LM Mn3+; 0, 30 PM Mn3’; n , 50 PM Mn3+.
MANGANESE
PEROXIDASE/Mr?+
OXIDATION
the cation radical is an apparent first-order process both in the absence and presence of Mn3+. Kersten and co-workers (8) proposed a mechanism for the decay of the cation radical of 1,2,4,5-tetramethoxybenzene that involves addition of water to form a neutral dienyl radical. This is followed by a second oxidation and loss of two equivalents of methanol. 2,5-Dimethoxy-pquinone and 4,5-dimethoxy-o-quinone were the products formed and 2 mol of methanol per mole of quinone were detected (8). They further showed (by experiments with H2180) that the quinone oxygens are derived from water. The decay of the cation radical is pseudo-first order because water is present in vast excess relative to the cation radical. Further support of this assertion is the observation that the relationship between the concentration of the enzyme and the steady-state concentration of the cat ion radical was linear (14). A square root relationship would be expected for a second-order dismutation of the cation radical. We propose that Mn3+ oxidizes the neutral dienyl radical as illustrated in Fig. 2. Though only the p-quinone product is shown, both 2,5-dimethoxy-p-quinone and 4,5dimethoxy-o-quinone were formed by Mn3+ oxidation (detected by reverse phase HPLC as in (Ref. (8)) and the ratio of the two products (p-:o-quinone = 1:4) was the same in the presence and absence of Mn3+. In the presence of Mn3+, the decay of the cation radical is still pseudofirst order. Therefore, Mn3+ cannot be reacting directly with the cation radical because that would be a secondorder reaction. The concentration of the cation radical in our experiments was 0.1 mM based on the complete oxidation of the tetramethoxybenzene under our reaction conditions (excess HRP) and on the cd50of 9800 M-l cm-l (8). The concentration of Mn3+ added was 0.01-0.05 mM. While with the lower concentrations of Mn”+, a secondorder reaction of MnR+ with the cation radical might be masked by the pseudo-first order decay, at 0.05 mM Mn3+ (one half the concentration of the cation radical) we would expect to observe deviation from first-order kinetics if, in fact, Mn3+ reacted with the cation radical. Thus, Mnsi probably reacts with the neutral dienyl radical. In order for this reaction to affect the decay of the cation radical, the addition of water to the cation radical must be a reversible reaction as shown in Fig. 2. The effect of Mn3+ is then to disturb the equilibrium of the cation radical/ neutral radical couple by oxidizing the neutral radical which leads to an increased decay of the cation radical. In other words, accelerating the decay of the neutral radical leads to acceleration of the decay of the cation radical. The oxidation of neutral dienyl radicals by Mn3+ may have implications for the biodegradation of lignin. Such radicals are proposed to be intermediates in the decay of compounds oxidized by LIP. This includes veratryl alcohol (16) as well as more complex lignin model compounds (17). LIP and MnP are produced simultaneously in lignin-
147
OF METHOXYBENZENES OCH,
HO
OCH,
OCH,
OCH,
CH,OH m2’
B
CH,OH
&?H,
FIG. 2. Proposed mechanism for the Mn3’ oxidation of the neutral dienyl radical derived from the cation radical of 1,2,4,5tetramethoxybenzene.
degrading cultures of P. chrysosporium (lo), and manganese is prevalent in wood. Thus it is conceivable that the two enzymes act in concert in the degradation of lignin. The Mn”+ (lo), and manganese is prevalent in wood. Thus it is conceivable that the two enzymes act in concert in the degradation of lignin. The Mn3+ oxidation of LiPderived neutral radicals is one possible point of interaction of these two enzymes. Some studies of LIP reactions have included the investigation of the effect of Mn’+. Schmidt et al. (16) reported that in the presence of MnS04 the yields of some of the products of LIP oxidation of veratryl alcohol methyl ether are altered. They did not speculate on the mechanism of the Mn2+ effect. Another report (18) suggests that Mn*+ scavenges superoxide formed during LIP oxidation of veratryl alcohol. This would generate Mn3+. In recent work, we showed that LIP, in the presence of malonate or oxalate, can oxidize Mn” to Mn3+ (19). An activated oxygen species is likely to be involved in this reaction also. Thus, it seems probable that in uivo both Mn*+ and Mn3+ (produced enzymatically or chemically) are present. Further work is needed to determine whether Mn3+ influences LIP oxidation reactions and to clarify the role of manganese and MnP in lignin degradation. ACKNOWLEDGMENT The authors thank Philip d. Kersten for helpful discussions.
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