ARCHIVES
OF BIOCHEMISTRY
Vol. 298, No. 2, November
AND
BIOPHYSICS
1, pp. 480-485,
1992
Production of Hydroxyl Radical by Lignin Peroxidase from Phanerochaete chrysosporium David Utah
P. Barr, State
Received
April
Manish
University,
M. Shah,
Biotechnology
7, 1992, and in revised
form
Thomas
A. Grover,
Center,
Logan,
June
29,1992
Utah
The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme HZ (LiPHS), HzOz, veratryl alcohol, oxalate, ferric chloride, and l,lOphenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H,O,, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPHa-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, HaOz, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants. o 1992 Academic press, IN.
The white rot fungus Phanerochuete chrysosporium is one of a few microorganisms capable of degrading lignin, the structural polymer found in wood (1). Lignin degradation is catalyzed by a group of enzymes including extracellular peroxidases secreted by P. chrysosporium under nutrient nitrogen-limiting conditions (2, 3). Both lignin 1 To whom
correspondence
should
be addressed.
Fax:
(801)
and Steven
D. Au&
84322-4705
750-2755.
peroxidases (Lip)’ and manganese-dependent peroxidases are produced by the white rot fungus (4). The fungus also produces enzymes that generate hydrogen peroxide (5,6). Veratryl alcohol (3,4-dimethoxybenzyl alcohol) is a secondary metabolite of P. chrysosporium also thought to be involved in lignin degradation (7). The degradation of several environmental pollutants to carbon dioxide by the white rot fungus has also been reported (8-11). These studies have concluded that the lignin degrading system of P. chrysosporium is also involved in xenobiotic degradation. The involvement of activated oxygen species (i.e., O;, H202, and ‘OH) in lignin degradation by white rot fungi was extensively studied in the years prior to the discovery of lignin peroxidase (12-15). Studies by Faison et al. (15) demonstrated the indirect involvement of 0, and ‘OH in lignin degradation by P. chrysosporium cultured under low nutrient nitrogen conditions. These investigators showed lignin degradation was inhibited (as assayed by [14C]C0, evolution from radiolabeled lignin) upon the addition of 0; scavengers such as cytochrome c or superoxide dismutase. Addition of ‘OH scavengers such as benzoate, salicylate, and DMNA were also shown to inhibit lignin degradation. Forney et al. demonstrated ‘OH production in cultures of P. chrysosporium using ethylene gas evolution from KTBA as a method of detection (14). More recently, it has been reported that LiP catalyzes the reduction of molecular oxygen to superoxide in reaction mixtures containing H202 and the secondary metabolites of P. chrysosporium veratryl alcohol and oxalate (16, 17). These studies concluded that the CO; was gen-
2 Abbreviations used: Lip, lignin peroxidase; LiPH2, lignin peroxidase isozyme H2; KTBA, a-keto-y-thiobutyric acid; EDTA, ethylenediamine tetraacetate; DMAB, 3-dimethylamino benzoic acid; MBTH, 3-methyl2-benzothiazolinone hydrazone; SOD, superoxide dismutase; DMNA, N,N’-dimethyl-4-nitrosoaniline; DMPO, 5,5-dimethyl-1-pyrroline-iVoxide.
480 All
Copyright 0 1992 rights of reproduction
0003.9861/92 $5.00 by Academic Press, Inc. in any form reserved.
LIGNIN
PEROXIDASE
FROM
erated via a one-electron oxidation of oxalate by the cation radical of veratryl alcohol. The CO5 then reacted with molecular oxygen to produce 0; and carbon dioxide. Although evidence exists for the production of the hydroxyl radical in cultures of P. chrysosporium (12-14), a mechanism for the production of ‘OH by LiP has not been demonstrated. A study by Kirk et al. concluded that LiP does not catalyze the production of ‘OH (18). However these investigators did not include veratryl alcohol, oxalate, or iron in reaction mixtures in which Lip-catalyzed ‘OH production was being studied. In the present study the reduction of ferric iron in a reaction mixture containing LiPH2, Hz02, veratryl alcohol, and oxalate was investigated since this reaction mixture has been shown to produce 0; (16,17). The ferrous iron generated in such a reaction system can then react with H202 to produce the ‘OH. The production and mineralization of oxalate in low nitrogen cultures of P. chrysosporium was also investigated. The results of this investigation suggest that ‘OH production results from an iron-dependent Haber-Weiss reaction which is catalyzed by Lip. Such a reaction may have physiological relevance with regard to the degradation of recalcitrant environmental pollutants. MATERIALS
AND
METHODS
Chemicals. Hydrogen peroxide, oxalate, SOD, and l,lO-phenanthroline were purchased from Sigma (St. Louis, MO). Veratryl alcohol, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and oxalate were obtained from Aldrich (Milwaukee, WI). EDTA was purchased from Mallinckrodt (Paris, KY). Sodium formate was purchased from MCB (division of EM Science Gibbstown, NJ). Sodium acetate buffers were prepared using purified water (Barnstead NANOpure II system; specific resistance 18.0 Mohm cm-i). Enzyme production and purification. The culture conditions used to produce lignin peroxidases and their purification and activity assay were as described previously (19). Extracellular fluid was dialyzed overnight against 10 mM sodium acetate buffer, pH 6.0, and the proteins purified on a Mono Q HR 5/5 column (Pharmacia, Uppsula, Sweden) (19). Veratryl alcohol oxidase activity in the extracellular fluid was determined by measuring an increase in absorbance at 310 nm (veratraldehyde formation) as described previously (19). The major lignin peroxidase (LiPH2) was used in experiments throughout the study. Oxalute production. Cultures of P. chrysosporium were grown under low nitrogen conditions (1.2 mM ammonium tartrate) using the media described previously (19). The low molecular weight fraction was obtained from low nitrogen shake cultures using Centriprep filters (Amicon division W. R. Grace & Co., Beverly, MA) with a molecular weight cut off of 10,000 Da. Centrifugation was at 2000g for 20 min. The oxalate concentration in each sample was quantitated enzymatically. The assay involved the production of an indamine dye (A,, = 590 nm) from the reaction of DMAB and MBTH with horseradish peroxidase and HzOz. The HzOz in the reaction was derived from the reaction of oxalate with oxalate oxidase and molecular oxygen. The reagents were purchased from Sigma Chemical Co. Mineralization of [%]oxalate. Low nutrient nitrogen stationary cultures of P. chrysosporium were prepared using the conditions and media described by Shah et al. (20). Radioactive oxalate (0.12 pCi/culture) was added on Day 0; cultures were then flushed with oxygen (99%) every 24 h through Day 7. The liberated COz was trapped in lo-ml solutions containing ethanolamine-methanopsafety solve scintillation cocktail (1:4:5) (Research Products International Corp., Mt. Prospect,
Phunerochaete
481
chrysosporium
IL). The radioactivity in the COs was quantitated using scintillation spectroscopy (Beckman LS-5801). The average amount of radioactive CO2 evolved each day was obtained from four replicate culture bottles. Radioactive CO2 evolution from a control bottle containing formaldehyde was subtracted from each bottle before calculating the average. Oxygen consumption experiments. Oxygen consumption was measured using a Gilson oxygraph monitor equipped with a 1.8-ml reaction chamber and a Clark type oxygen-sensitive electrode. Reaction mixtures contained 100 mM acetate buffer (pH 5.0), 0.63 PM LiPH2,500 pM HzOz, 2 mM veratryl alcohol, and 5 mM oxalate. Additions of ferric chloride are described in figure legends. All reactions were performed at room temperature. Ferric iron reduction assay. The reduction of ferric iron was measured as an increase in absorbance at 510 nm using the ferrous-specific chelate l,lO-phenanthroline (E, = 12.11 mM-’ cm-‘). Reaction mixtures contained 100 mM acetate buffer (pH 5.0), 0.63 PM LiPHP, 100 aM HzOz, 2 mM veratryl alcohol, 100 pM FeCls, 1.5 mM l,lO-phenanthroline, and EDTA or oxalate at concentrations as described in the figure legends. In separate experiments 30 units/ml SOD were added to reaction mixtures. SOD activity was calculated using the xanthine-xanthine oxidasedependent reduction of cytochrome c (21). Spin trapping of the hydroxyl radical. Formation of the DMPO-OH spin adduct was used to verify the production of ‘OH by LiPH2. Reaction mixtures contained 100 mM acetate buffer (pH 5.0), 0.63 pM LiPH2, 500 pM HzOz, 500 pM veratryl alcohol, 500 pM EDTA, 100 fiM FeCls, and 40 mM DMPO. Controls involved reactions in which one of the above-mentioned reactants was removed from the complete reaction mixture. All spectral recordings were made 1 min following the initiation of the reaction with Hz02. A Varian E-109 spectrometer operating at 9.5 GHz with a lOO-kHz modulation frequency was used for all recordings. Other spectrometer settings are shown in the figure legends. Oxidation and reduction of 1,2,4,5-tetramethoxybenzene. The synthesis of 1,2,4,5-tetramethoxybenzene was performed as described by Kersten et al. (22). The formation and reduction of the cation radical of 1,2,4,5-tetramethoxybenzene was monitored at 450 nm (23). Experimental conditions are described in the figure legend.
RESULTS Figure 1 shows the relationship between oxalate concentration and veratryl alcohol oxidase activity in nutrient
-3
-2
-1
-- 0 012345678
Days FIG. 1. Relationship between oxalate concentration and lignin peroxidase activity in nutrient nitrogen-limited shake cultures of P. chrysosporium. Incubations were carried out in quadruplicate at 37°C. LiP activity (Cl) and oxalate concentration (m). Values are expressed as the mean of quadruplicate samples with standard deviation error bars.
482
BARR
ET
AL. TABLE
I
Effect of Ferric Iron on Oxygen Consumption Catalyzed by LiPH2 Oxalate
FeCls
bM)
(PM)
(W/mid
0 125 250 0 250 500
3.9 2.5 1.6 5.4 4.3 2.3
2.5
5.0
z
0
2
4
6
8
Day FIG. 2. Mineralization of sosporium. Incubations were values are expressed as the COP each day. The error bars symbols in some cases.
oxalate by stationary cultures of P. chrycarried out in quadruplicate at 37°C. The mean percentage of oxalate converted to (standard deviations) are within the figure
nitrogen-limited cultures of P. chrysosporium. The onset of oxalate production was observed 2 days after inoculation. A maximum oxalate concentration of about 2.5 mM was observed on Day 3. The decrease in oxalate concentration between Days 4 and 7 corresponded with the period of maximum veratryl alcohol oxidase activity. The mineralization of ‘*C-labeled oxalate in nutrient nitrogen-limited stationary cultures of P. chrysosporium is presented in Fig. 2. Evolution of [‘*C]C02 began on Day 3. By Day 6 over 80% of the initial [‘*C]oxalate was converted to COa. The addition of ferric iron to a reaction mixture containing LiPH2, H202, veratryl alcohol, and oxalate inhibited oxygen consumption (Table 1). Inhibition was presumably due to the reaction of 0~ with ferric iron to yield oxygen and ferrous iron. The overall extent of oxygen consumption was also decreased by the addition of ferric chloride (data not shown). In control experiments in which no ferric chloride was added, total oxygen consumption was 75 PM. However, in experiments in which 500 PM ferric chloride was added to the reaction mixture, total oxygen consumption was only 29 PM. Figure 3 shows the effect of oxalate concentration on the rate of ferric iron reduction in a reaction mixture containing LiPH2, H202, veratryl alcohol, ferric chloride, and l,lO-phenanthroline. The addition of SOD (30 units/ml) to the reaction mixture resulted in a 60% decrease in the rate of ferric reduction (data not shown). Difficulties were encountered in experiments designed to spin trap ‘OH produced in reaction mixtures containing LiPH2, H202, veratryl alcohol, oxalate, and ferric chloride. The oxalate concentrations used in this study were relatively high and could have effectively scavenged ‘OH generated by the reaction system. To overcome this prob-
O2 consumption
(20.1) (kO.0) (kO.1) (kO.1) (+o.o) (fO.l)
Note. The reaction mixtures contained 0.63 pM LiPH2,500 pM HzOz, 2 mM veratryl alcohol, and the indicated concentrations of oxalate and FeC&. The total reaction volumes were 1.8 ml. All reactions were initiated by the addition of HzOx and were performed at room temperature. Values represent the mean k SD of triplicate measurements.
lem, spin trapping experiments were designed using a more efficient electron donor, EDTA, in place of oxalate. Ferric reduction in a reaction mixture containing LiPH2, H202, veratryl alcohol, ferric chloride, and various concentrations of EDTA is shown in Fig. 4. LiPHB-catalyzed ferric reduction rates were approximately twofold higher at an EDTA concentration (500 PM) that was 20 times lower than that used for oxalate (10 mM) (Figs. 3 and 4). The higher iron reduction rate obtained using the EDTA reaction system may have increased the production of ‘OH. The ESR spectrum of the DMPO-OH (AN = 14.9 G, A& = 14.9 G) adduct was observed in a reaction mixture containing LiPH2, HzOz, veratryl alcohol, EDTA, and ferric chloride (Fig. 5a). The addition of SOD to the re-
6~
0
2
4
6 Oxalate
8
10
12
14
(mM)
FIG. 3. Effect of oxalate on the rate of LiPHB-catalyzed ferric iron reduction. Reaction mixtures contained 100 mM sodium acetate (pH 5.0), 0.63 j&M LiPH2,500 pM HxOx, 2 mM veratryl alcohol, 106 PM FeCl,, 1.5 mM l,lO-phenanthroline, and various concentrations of oxalate. All reactions were performed at 25°C and were initiated by the addition of H202. Values represent the mean of triplicate measurements with standard deviation error bars.
LIGNIN
0
100
200
300 EDTA
PEROXIDASE
400
500
FROM
600
(uh4)
FIG. 4. Effect of EDTA on the rate of LiPHa-catalyzed ferric iron reduction. Reaction mixtures contained 100 mM sodium acetate (pH 5.0), 0.63 pM LiPH2,500 pM H,Oz, 2 mM veratryl alcohol, 100 pM FeCl,, and various concentrations of EDTA. Reactions were performed at 25°C. The assays were performed as described in the legend to Fig. 3. Values are expressed as the mean of triplicate measurements with standard deviation error bars. Some of the error bars are within the data points.
action mixture inhibited the formation of DMPO-OH (Fig. 5b). When ferric chloride was left out of the reaction mixture, the DMPO-OOH (AN = 14.3 G, A& = 11.7 G, A& = 1.3 G) ESR spectrum was observed (Fig. 5~). Control experiments revealed that all reactants (i.e., LiPH2, H202, veratryl alcohol, EDTA, and ferric chloride) were required to obtain the DMPO-OH ESR spectrum. The addition of formate to the above reaction mixture resulted in the formation of a DMPO-CO2 adduct (AN = 15.7, A& = 18.7) (Fig. 5D). This adduct was not observed when EDTA was removed from the reaction mixture. Presumably the oxidation of oxalate (or EDTA) resulted from reduction of the veratryl alcohol cation radical. Unfortunately, it has been impossible to observe this radical. However, the cation radical of 1,2,4,5-tetramethoxybenzene can be detected since it has a distinct absorption at 450 nm (23). This radical, generated with LiPH2 and HzOz, was reduced upon the addition of EDTA (Fig. 6).
Phanerochete
chrysosporium
483
has been no direct evidence to confirm the formation of veratryl alcohol cation radical in Lip-catalyzed reactions. However, the reduction of the tetramethoxybenzene radical by EDTA, observed in the present study, supports such a mechanism. In the study by Popp et al. (16), the CO; formed was proposed to react with molecular oxygen to produce 0; and carbon dioxide. This mechanism was further supported by Akamatsu et al. who demonstrated the evolution of [14C]C02 from radiolabeled oxalate in a reaction mixture containing Lip, HzOz, veratryl alcohol, and oxalate. These investigators also demonstrated that oxalate is a noncompetitive inhibitor of Lip-catalyzed veratryl alcohol oxidation (17). In the present study, oxygen consumption was inhibited by the addition of ferric iron to a reaction mixture containing LiPH2, HzOz, veratryl alcohol, and oxalate. Inhibition was likely caused by the reaction of 0; with ferric iron to produce oxygen and ferrous iron. The fact that LiPH2-catalyzed ferric reduction was dependent, at least in part, on 05 supports this theory. Thus, the role of LiP generated 0; in the degradation of chemicals may be, in
B
DISCUSSION
Previous studies have established a Lip-catalyzed mechanism for the production of 0;. Popp et al. (16) showed oxygen consumption in reaction mixtures containing Lip, HzOz, veratryl alcohol, and oxalate. These investigators also observed the ESR spectrum of the DMPO-CO2 and DMPO-OOH spin adducts when DMPO was added to the same reaction mixture. All of the reactants (i.e., Lip, HzOz, veratryl alcohol, and oxalate) were required to obtain the ESR spectra of the DMPO-COP and DMPO-OOH spin adducts. It was concluded that the CO; was produced via a one-electron oxidation of oxalate by the cation radical of veratryl alcohol. To date, there
FIG. 5.
ESR spectrum of the DMPO-OH radical adduct formed by LiPH2. The reaction mixture contained 100 mM sodium acetate (pH 5.0), 0.63 pM LiPH2, 500 pM HxOr, 500 fiM veratryl alcohol, 500 fiM EDTA, and 100 pM FeC& . All reactions were initiated by the addition of HzO,. Recordings were taken 1 min after initiation. Spectrometer settings were: modulation amplitude, 1.0 G; time constant, 0.128 s; scan time, 2 min; receiver gain, 1.0 X ld; and microwave power, 50 mW. Spectrum A was obtained using the complete reaction mixture as described above. Spectrum B was obtained when 60 units/ml of SOD was added. Spectrum C was obtained when FeC& was left out of the reaction mixture. Spectrum D was obtained when 200 mM formate was added to the complete reaction mixture.
484
BARR
0.0
2.0
1.0
Time(min) FIG. 6. Reduction of the cation radical of l&4,5-tetramethoxybenzene by EDTA. The reaction mixture contained 0.1 M sodium acetate buffer, pH 5.5, 40 /IM 1,2,4,5-tetramethoxybenzene, and 0.1 M LiPH2. The reactions were initiated by the addition of 100 gM H202. Cation radical formation was monitored by measuring the increase in absorbance at 450 nm. At t = 1.2 min, EDTA was added at different concentrations, 0 mM (D), 0.1 mM (m), 0.3 mM (+), 0.6 mM (O), 1.2 mM (0).
part, to reduce ferric iron and promote subsequent ‘OH production. This could also explain the inhibition of lignin degradation observed by Faison et al. when SOD was added to low nitrogen cultures of P. chrysosporium (15). Alternatively, ferric iron could be reduced by the CO;. However, this was not observed, at least under atmospheric oxygen conditions. The proposed mechanism for ‘OH production by LIP is presented in Fig. 7. The results of this investigation demonstrate that LiP can catalyze ‘OH production using veratryl alcohol and an electron donor such as EDTA. Although ‘OH was not detected when oxalate was used as the electron donor, iron reduction was observed. Therefore, oxalate may be the physiological electron donor involved in Lip-catalyzed ‘OH production. The fact that veratryl alcohol and oxalate are metabolites of P. chrysosporium suggests an in vivo significance to the reactions observed in this study. The concentration of oxalate in low nitrogen cultures reached a maximum on the day prior to detection of LiP activity. A significant decrease in oxalate concentration was observed between Days 4 and 7, which corresponds to the period of maximum LIP activity. In addition, the highest rates of oxalate mineralization occurred during this period
FIG. 7. Proposed can replace oxalate
mechanism for the production in the proposed scheme.
of ‘OH by lignin
ET
AL.
of maximum LIP activity. Together, these results suggest an in vivo mechanism by which LIP catalyzes the degradation of oxalate. The metabolism of oxalate via this mechanism provides reducing equivalents (i.e., CO;) for the production of 0; and the subsequent reduction of ferric iron. Forney et al. obtained evidence for the production of ‘OH in nutrient nitrogen-limited cultures of P. chrysosporium (14). These investigators also observed a lOOfold lower ‘OH concentration (as detected by ethylene gas formation from KTBA) in high nutrient nitrogen cultures. Several reports have shown that LiP activity is absent in high nitrogen cultures of P. chrysosporium (24-26). The loo-fold decrease in the ‘OH concentration and the absence of LiP activity in high nutrient nitrogen cultures support the hypothesis that ‘OH production by P. chrysosporium involves lignin peroxidases. An earlier study by Kirk et al. (18) reported that the ‘OH is not an integral component of the lignin degrading system. These investigators did not detect ‘OH in LiP reaction mixtures. However, their LiP reaction mixtures did not include veratryl alcohol, oxalate, or ferric iron. The results of the present investigation demonstrate that LiP can catalyze the production of-OH. This is supported by the spin trapping of ‘OH by DMPO in reaction mixtures containing LiPH2, HzOz, veratryl alcohol, ferric chloride, and EDTA. The addition of the ‘OH scavenger formate to this reaction mixture resulted in the formation of a DMPO-CO2 adduct. Thus, it was concluded that ‘OH was produced by the iron-dependent Haber-Weiss reaction and not the homolytic cleavage of a DMPO-OOH adduct. The hyperfine coupling constants obtained for the DMPO-OH, DMPO-OOH, and DMPO-CO2 adducts were in agreement with those from previous literature (27). The absence of a DMPO-OH ESR spectrum in LiPH2 reaction mixtures containing oxalate is likely due to a limitation in detection and not the actual absence of ‘OH. In conclusion, the results of this study demonstrate that LiP can catalyze iron reduction via a mechanism that involves secondary metabolites of P. chrysosporium (i.e., veratryl alcohol and oxalate). The reduction of iron through the proposed mechanism could promote the production of ‘OH in vivo. A closer evaluation of these reactions may provide insight into some of the unexplained mechanisms involved in xenobiotic degradation by P.
peroxidase.
(VA)
veratryl
alcohol,
(VA+‘)
veratryl
alcohol
cation
radical.
EDTA
LIGNIN
chrysosporium. The involvement the metabolism of environmental under investigation.
PEROXIDASE
FROM
of Lip-generated-OH in pollutants is currently
12. Kelley,
13.
485
chrysosporium R. L., and Reddy,
Kutsuki, H., and Gold, mun. 109,320-328.
C. A. (1982)
Maughan for by NIH Grant
secretarial ES04922.
assistance.
This
REFERENCES 1. Kirk, T. K., Schultz, E., Connors, W. J., Lorenz, C. F., and Ziekus, J. G. (1978) Arch. Microbial. 117, 277-285. 2. Gold, M. H., Kuwahara, M., Chin, A. A., and Glenn, J. K. (1984) Arch. Biochem. Biophys. 234,353-362. 3. Tien, M., and Kirk, T. K. (1984) Proc. Natl. Acad. Sci. USA 81, 2280-2284. 4. Glenn, J. K., and Gold, M. H. (1985) Arch. Biochem. Biophys. 242, 329-341. 144, 2485. Kelley, R. L., and Reddy, C. A. (1986) Arch. Microbial. 253. P. J. (1990) Biochemistry 87, 2936-2940. 6. Kersten, P. J., Shoemaker, H. E., and Palmer, J. M. (1985) FEBS 7. Harvey, Lett. 196.242-246. J. A., Tien, M., Wright, D., and Aust, S. D. (1985) Science 8. Bumpus, 228,1434-1436. 9. Ryan, T. P., and Bumpus, J. A. (1989) Appl. Microbial. Biotechnol. 3 1,302-307. 10. Fernando, T., Bumpus, J. A., and Aust, S. D. (1990) Appl. Enuiron. Microbial. 66, 1666-1671. 11. Kennedy, D. W., Aust, S. D., and Bumpus, J. A. (1990) Appl. Enuiron. Microbial. 56, 2346-2353.
15. Faison, B. D., and Kirk, 1140-1145.
T. K. (1983)
Akamatsu, Y., Ma, D. B., Higuchi, Lett. 269,261-263.
18. Kirk, T. K., Mozuch, 455-460.
B&hem.
Appl.
J. 206,423-425. Biophys.
M., and Aust,
16. Popp, J. L., and Kalyanaraman, chemistry 29,10475-10480.
-Ii.
Biochem.
M. H. (1982)
L. J., Reddy, C. A., Tien, 14. Forney, Chem. 257,11455-11462.
ACKNOWLEDGMENTS The authors thank Terri work was supported in part
Phawrochete
Res. Com-
S. D. (1982)
Enuiron.
J. Biol.
Microbial.
B., and Kirk,
T. K. (1990)
T., and Shimada,
M. (1990)
M. D., and Tien,
M. (1985)
Biochem.
46, BioFEBS
J. 226,
19. Tuisel, H., Sinclair, R., Bumpus, J. A., Ashbaugh, W., Brock, B. J., and Aust, S. D. (1990) Arch. Biochem. Biophys. 279, 158-166. 20. Shah, M. M., Grover, Biophys. 290,173-178. 21. McCord, 6055.
T. A., and Aust,
J. M., and Fridovich,
22. Kersten, P. J., Tien, J. Biol. Chem. 260,
24. Faison, B. D., and Kirk, 299-304.
26. Keyser,
T. W., Choi, 42, 290-296. P., Kirk,
I. (1969)
J. Biol.
M., Kalyanaraman, 2609-2612.
23. Popp, J. L., and Kirk, 145-148.
25. Jefferies, Microbial.
S. D. (1991)
T. K. (1991) T. K. (1985) S., and Kirk,
T. K., and Zeikus,
Arch.
Chem.
B., and Kirk, Arch. Appl.
Biochem. Environ.
244,6049T. K. (1985)
Biophys. Microbial.
T. K. (1981) J. G. (1978)
Appl.
G. R. (1987)
Free Radicals
Biol.
Med.
3, 259-303.
288, 49,
Enuiron.
J. Bacterial.
790-797. 27. Buettner,
Biochem.
135,
OF BIOCHEMISTRY
Vol. 298, No. 2, November
AND
BIOPHYSICS
1, pp. 480-485,
1992
Production of Hydroxyl Radical by Lignin Peroxidase from Phanerochaete chrysosporium David Utah
P. Barr, State
Received
April
Manish
University,
M. Shah,
Biotechnology
7, 1992, and in revised
form
Thomas
A. Grover,
Center,
Logan,
June
29,1992
Utah
The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme HZ (LiPHS), HzOz, veratryl alcohol, oxalate, ferric chloride, and l,lOphenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H,O,, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPHa-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, HaOz, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants. o 1992 Academic press, IN.
The white rot fungus Phanerochuete chrysosporium is one of a few microorganisms capable of degrading lignin, the structural polymer found in wood (1). Lignin degradation is catalyzed by a group of enzymes including extracellular peroxidases secreted by P. chrysosporium under nutrient nitrogen-limiting conditions (2, 3). Both lignin 1 To whom
correspondence
should
be addressed.
Fax:
(801)
and Steven
D. Au&
84322-4705
750-2755.
peroxidases (Lip)’ and manganese-dependent peroxidases are produced by the white rot fungus (4). The fungus also produces enzymes that generate hydrogen peroxide (5,6). Veratryl alcohol (3,4-dimethoxybenzyl alcohol) is a secondary metabolite of P. chrysosporium also thought to be involved in lignin degradation (7). The degradation of several environmental pollutants to carbon dioxide by the white rot fungus has also been reported (8-11). These studies have concluded that the lignin degrading system of P. chrysosporium is also involved in xenobiotic degradation. The involvement of activated oxygen species (i.e., O;, H202, and ‘OH) in lignin degradation by white rot fungi was extensively studied in the years prior to the discovery of lignin peroxidase (12-15). Studies by Faison et al. (15) demonstrated the indirect involvement of 0, and ‘OH in lignin degradation by P. chrysosporium cultured under low nutrient nitrogen conditions. These investigators showed lignin degradation was inhibited (as assayed by [14C]C0, evolution from radiolabeled lignin) upon the addition of 0; scavengers such as cytochrome c or superoxide dismutase. Addition of ‘OH scavengers such as benzoate, salicylate, and DMNA were also shown to inhibit lignin degradation. Forney et al. demonstrated ‘OH production in cultures of P. chrysosporium using ethylene gas evolution from KTBA as a method of detection (14). More recently, it has been reported that LiP catalyzes the reduction of molecular oxygen to superoxide in reaction mixtures containing H202 and the secondary metabolites of P. chrysosporium veratryl alcohol and oxalate (16, 17). These studies concluded that the CO; was gen-
2 Abbreviations used: Lip, lignin peroxidase; LiPH2, lignin peroxidase isozyme H2; KTBA, a-keto-y-thiobutyric acid; EDTA, ethylenediamine tetraacetate; DMAB, 3-dimethylamino benzoic acid; MBTH, 3-methyl2-benzothiazolinone hydrazone; SOD, superoxide dismutase; DMNA, N,N’-dimethyl-4-nitrosoaniline; DMPO, 5,5-dimethyl-1-pyrroline-iVoxide.
480 All
Copyright 0 1992 rights of reproduction
0003.9861/92 $5.00 by Academic Press, Inc. in any form reserved.
LIGNIN
PEROXIDASE
FROM
erated via a one-electron oxidation of oxalate by the cation radical of veratryl alcohol. The CO5 then reacted with molecular oxygen to produce 0; and carbon dioxide. Although evidence exists for the production of the hydroxyl radical in cultures of P. chrysosporium (12-14), a mechanism for the production of ‘OH by LiP has not been demonstrated. A study by Kirk et al. concluded that LiP does not catalyze the production of ‘OH (18). However these investigators did not include veratryl alcohol, oxalate, or iron in reaction mixtures in which Lip-catalyzed ‘OH production was being studied. In the present study the reduction of ferric iron in a reaction mixture containing LiPH2, Hz02, veratryl alcohol, and oxalate was investigated since this reaction mixture has been shown to produce 0; (16,17). The ferrous iron generated in such a reaction system can then react with H202 to produce the ‘OH. The production and mineralization of oxalate in low nitrogen cultures of P. chrysosporium was also investigated. The results of this investigation suggest that ‘OH production results from an iron-dependent Haber-Weiss reaction which is catalyzed by Lip. Such a reaction may have physiological relevance with regard to the degradation of recalcitrant environmental pollutants. MATERIALS
AND
METHODS
Chemicals. Hydrogen peroxide, oxalate, SOD, and l,lO-phenanthroline were purchased from Sigma (St. Louis, MO). Veratryl alcohol, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and oxalate were obtained from Aldrich (Milwaukee, WI). EDTA was purchased from Mallinckrodt (Paris, KY). Sodium formate was purchased from MCB (division of EM Science Gibbstown, NJ). Sodium acetate buffers were prepared using purified water (Barnstead NANOpure II system; specific resistance 18.0 Mohm cm-i). Enzyme production and purification. The culture conditions used to produce lignin peroxidases and their purification and activity assay were as described previously (19). Extracellular fluid was dialyzed overnight against 10 mM sodium acetate buffer, pH 6.0, and the proteins purified on a Mono Q HR 5/5 column (Pharmacia, Uppsula, Sweden) (19). Veratryl alcohol oxidase activity in the extracellular fluid was determined by measuring an increase in absorbance at 310 nm (veratraldehyde formation) as described previously (19). The major lignin peroxidase (LiPH2) was used in experiments throughout the study. Oxalute production. Cultures of P. chrysosporium were grown under low nitrogen conditions (1.2 mM ammonium tartrate) using the media described previously (19). The low molecular weight fraction was obtained from low nitrogen shake cultures using Centriprep filters (Amicon division W. R. Grace & Co., Beverly, MA) with a molecular weight cut off of 10,000 Da. Centrifugation was at 2000g for 20 min. The oxalate concentration in each sample was quantitated enzymatically. The assay involved the production of an indamine dye (A,, = 590 nm) from the reaction of DMAB and MBTH with horseradish peroxidase and HzOz. The HzOz in the reaction was derived from the reaction of oxalate with oxalate oxidase and molecular oxygen. The reagents were purchased from Sigma Chemical Co. Mineralization of [%]oxalate. Low nutrient nitrogen stationary cultures of P. chrysosporium were prepared using the conditions and media described by Shah et al. (20). Radioactive oxalate (0.12 pCi/culture) was added on Day 0; cultures were then flushed with oxygen (99%) every 24 h through Day 7. The liberated COz was trapped in lo-ml solutions containing ethanolamine-methanopsafety solve scintillation cocktail (1:4:5) (Research Products International Corp., Mt. Prospect,
Phunerochaete
481
chrysosporium
IL). The radioactivity in the COs was quantitated using scintillation spectroscopy (Beckman LS-5801). The average amount of radioactive CO2 evolved each day was obtained from four replicate culture bottles. Radioactive CO2 evolution from a control bottle containing formaldehyde was subtracted from each bottle before calculating the average. Oxygen consumption experiments. Oxygen consumption was measured using a Gilson oxygraph monitor equipped with a 1.8-ml reaction chamber and a Clark type oxygen-sensitive electrode. Reaction mixtures contained 100 mM acetate buffer (pH 5.0), 0.63 PM LiPH2,500 pM HzOz, 2 mM veratryl alcohol, and 5 mM oxalate. Additions of ferric chloride are described in figure legends. All reactions were performed at room temperature. Ferric iron reduction assay. The reduction of ferric iron was measured as an increase in absorbance at 510 nm using the ferrous-specific chelate l,lO-phenanthroline (E, = 12.11 mM-’ cm-‘). Reaction mixtures contained 100 mM acetate buffer (pH 5.0), 0.63 PM LiPHP, 100 aM HzOz, 2 mM veratryl alcohol, 100 pM FeCls, 1.5 mM l,lO-phenanthroline, and EDTA or oxalate at concentrations as described in the figure legends. In separate experiments 30 units/ml SOD were added to reaction mixtures. SOD activity was calculated using the xanthine-xanthine oxidasedependent reduction of cytochrome c (21). Spin trapping of the hydroxyl radical. Formation of the DMPO-OH spin adduct was used to verify the production of ‘OH by LiPH2. Reaction mixtures contained 100 mM acetate buffer (pH 5.0), 0.63 pM LiPH2, 500 pM HzOz, 500 pM veratryl alcohol, 500 pM EDTA, 100 fiM FeCls, and 40 mM DMPO. Controls involved reactions in which one of the above-mentioned reactants was removed from the complete reaction mixture. All spectral recordings were made 1 min following the initiation of the reaction with Hz02. A Varian E-109 spectrometer operating at 9.5 GHz with a lOO-kHz modulation frequency was used for all recordings. Other spectrometer settings are shown in the figure legends. Oxidation and reduction of 1,2,4,5-tetramethoxybenzene. The synthesis of 1,2,4,5-tetramethoxybenzene was performed as described by Kersten et al. (22). The formation and reduction of the cation radical of 1,2,4,5-tetramethoxybenzene was monitored at 450 nm (23). Experimental conditions are described in the figure legend.
RESULTS Figure 1 shows the relationship between oxalate concentration and veratryl alcohol oxidase activity in nutrient
-3
-2
-1
-- 0 012345678
Days FIG. 1. Relationship between oxalate concentration and lignin peroxidase activity in nutrient nitrogen-limited shake cultures of P. chrysosporium. Incubations were carried out in quadruplicate at 37°C. LiP activity (Cl) and oxalate concentration (m). Values are expressed as the mean of quadruplicate samples with standard deviation error bars.
482
BARR
ET
AL. TABLE
I
Effect of Ferric Iron on Oxygen Consumption Catalyzed by LiPH2 Oxalate
FeCls
bM)
(PM)
(W/mid
0 125 250 0 250 500
3.9 2.5 1.6 5.4 4.3 2.3
2.5
5.0
z
0
2
4
6
8
Day FIG. 2. Mineralization of sosporium. Incubations were values are expressed as the COP each day. The error bars symbols in some cases.
oxalate by stationary cultures of P. chrycarried out in quadruplicate at 37°C. The mean percentage of oxalate converted to (standard deviations) are within the figure
nitrogen-limited cultures of P. chrysosporium. The onset of oxalate production was observed 2 days after inoculation. A maximum oxalate concentration of about 2.5 mM was observed on Day 3. The decrease in oxalate concentration between Days 4 and 7 corresponded with the period of maximum veratryl alcohol oxidase activity. The mineralization of ‘*C-labeled oxalate in nutrient nitrogen-limited stationary cultures of P. chrysosporium is presented in Fig. 2. Evolution of [‘*C]C02 began on Day 3. By Day 6 over 80% of the initial [‘*C]oxalate was converted to COa. The addition of ferric iron to a reaction mixture containing LiPH2, H202, veratryl alcohol, and oxalate inhibited oxygen consumption (Table 1). Inhibition was presumably due to the reaction of 0~ with ferric iron to yield oxygen and ferrous iron. The overall extent of oxygen consumption was also decreased by the addition of ferric chloride (data not shown). In control experiments in which no ferric chloride was added, total oxygen consumption was 75 PM. However, in experiments in which 500 PM ferric chloride was added to the reaction mixture, total oxygen consumption was only 29 PM. Figure 3 shows the effect of oxalate concentration on the rate of ferric iron reduction in a reaction mixture containing LiPH2, H202, veratryl alcohol, ferric chloride, and l,lO-phenanthroline. The addition of SOD (30 units/ml) to the reaction mixture resulted in a 60% decrease in the rate of ferric reduction (data not shown). Difficulties were encountered in experiments designed to spin trap ‘OH produced in reaction mixtures containing LiPH2, H202, veratryl alcohol, oxalate, and ferric chloride. The oxalate concentrations used in this study were relatively high and could have effectively scavenged ‘OH generated by the reaction system. To overcome this prob-
O2 consumption
(20.1) (kO.0) (kO.1) (kO.1) (+o.o) (fO.l)
Note. The reaction mixtures contained 0.63 pM LiPH2,500 pM HzOz, 2 mM veratryl alcohol, and the indicated concentrations of oxalate and FeC&. The total reaction volumes were 1.8 ml. All reactions were initiated by the addition of HzOx and were performed at room temperature. Values represent the mean k SD of triplicate measurements.
lem, spin trapping experiments were designed using a more efficient electron donor, EDTA, in place of oxalate. Ferric reduction in a reaction mixture containing LiPH2, H202, veratryl alcohol, ferric chloride, and various concentrations of EDTA is shown in Fig. 4. LiPHB-catalyzed ferric reduction rates were approximately twofold higher at an EDTA concentration (500 PM) that was 20 times lower than that used for oxalate (10 mM) (Figs. 3 and 4). The higher iron reduction rate obtained using the EDTA reaction system may have increased the production of ‘OH. The ESR spectrum of the DMPO-OH (AN = 14.9 G, A& = 14.9 G) adduct was observed in a reaction mixture containing LiPH2, HzOz, veratryl alcohol, EDTA, and ferric chloride (Fig. 5a). The addition of SOD to the re-
6~
0
2
4
6 Oxalate
8
10
12
14
(mM)
FIG. 3. Effect of oxalate on the rate of LiPHB-catalyzed ferric iron reduction. Reaction mixtures contained 100 mM sodium acetate (pH 5.0), 0.63 j&M LiPH2,500 pM HxOx, 2 mM veratryl alcohol, 106 PM FeCl,, 1.5 mM l,lO-phenanthroline, and various concentrations of oxalate. All reactions were performed at 25°C and were initiated by the addition of H202. Values represent the mean of triplicate measurements with standard deviation error bars.
LIGNIN
0
100
200
300 EDTA
PEROXIDASE
400
500
FROM
600
(uh4)
FIG. 4. Effect of EDTA on the rate of LiPHa-catalyzed ferric iron reduction. Reaction mixtures contained 100 mM sodium acetate (pH 5.0), 0.63 pM LiPH2,500 pM H,Oz, 2 mM veratryl alcohol, 100 pM FeCl,, and various concentrations of EDTA. Reactions were performed at 25°C. The assays were performed as described in the legend to Fig. 3. Values are expressed as the mean of triplicate measurements with standard deviation error bars. Some of the error bars are within the data points.
action mixture inhibited the formation of DMPO-OH (Fig. 5b). When ferric chloride was left out of the reaction mixture, the DMPO-OOH (AN = 14.3 G, A& = 11.7 G, A& = 1.3 G) ESR spectrum was observed (Fig. 5~). Control experiments revealed that all reactants (i.e., LiPH2, H202, veratryl alcohol, EDTA, and ferric chloride) were required to obtain the DMPO-OH ESR spectrum. The addition of formate to the above reaction mixture resulted in the formation of a DMPO-CO2 adduct (AN = 15.7, A& = 18.7) (Fig. 5D). This adduct was not observed when EDTA was removed from the reaction mixture. Presumably the oxidation of oxalate (or EDTA) resulted from reduction of the veratryl alcohol cation radical. Unfortunately, it has been impossible to observe this radical. However, the cation radical of 1,2,4,5-tetramethoxybenzene can be detected since it has a distinct absorption at 450 nm (23). This radical, generated with LiPH2 and HzOz, was reduced upon the addition of EDTA (Fig. 6).
Phanerochete
chrysosporium
483
has been no direct evidence to confirm the formation of veratryl alcohol cation radical in Lip-catalyzed reactions. However, the reduction of the tetramethoxybenzene radical by EDTA, observed in the present study, supports such a mechanism. In the study by Popp et al. (16), the CO; formed was proposed to react with molecular oxygen to produce 0; and carbon dioxide. This mechanism was further supported by Akamatsu et al. who demonstrated the evolution of [14C]C02 from radiolabeled oxalate in a reaction mixture containing Lip, HzOz, veratryl alcohol, and oxalate. These investigators also demonstrated that oxalate is a noncompetitive inhibitor of Lip-catalyzed veratryl alcohol oxidation (17). In the present study, oxygen consumption was inhibited by the addition of ferric iron to a reaction mixture containing LiPH2, HzOz, veratryl alcohol, and oxalate. Inhibition was likely caused by the reaction of 0; with ferric iron to produce oxygen and ferrous iron. The fact that LiPH2-catalyzed ferric reduction was dependent, at least in part, on 05 supports this theory. Thus, the role of LiP generated 0; in the degradation of chemicals may be, in
B
DISCUSSION
Previous studies have established a Lip-catalyzed mechanism for the production of 0;. Popp et al. (16) showed oxygen consumption in reaction mixtures containing Lip, HzOz, veratryl alcohol, and oxalate. These investigators also observed the ESR spectrum of the DMPO-CO2 and DMPO-OOH spin adducts when DMPO was added to the same reaction mixture. All of the reactants (i.e., Lip, HzOz, veratryl alcohol, and oxalate) were required to obtain the ESR spectra of the DMPO-COP and DMPO-OOH spin adducts. It was concluded that the CO; was produced via a one-electron oxidation of oxalate by the cation radical of veratryl alcohol. To date, there
FIG. 5.
ESR spectrum of the DMPO-OH radical adduct formed by LiPH2. The reaction mixture contained 100 mM sodium acetate (pH 5.0), 0.63 pM LiPH2, 500 pM HxOr, 500 fiM veratryl alcohol, 500 fiM EDTA, and 100 pM FeC& . All reactions were initiated by the addition of HzO,. Recordings were taken 1 min after initiation. Spectrometer settings were: modulation amplitude, 1.0 G; time constant, 0.128 s; scan time, 2 min; receiver gain, 1.0 X ld; and microwave power, 50 mW. Spectrum A was obtained using the complete reaction mixture as described above. Spectrum B was obtained when 60 units/ml of SOD was added. Spectrum C was obtained when FeC& was left out of the reaction mixture. Spectrum D was obtained when 200 mM formate was added to the complete reaction mixture.
484
BARR
0.0
2.0
1.0
Time(min) FIG. 6. Reduction of the cation radical of l&4,5-tetramethoxybenzene by EDTA. The reaction mixture contained 0.1 M sodium acetate buffer, pH 5.5, 40 /IM 1,2,4,5-tetramethoxybenzene, and 0.1 M LiPH2. The reactions were initiated by the addition of 100 gM H202. Cation radical formation was monitored by measuring the increase in absorbance at 450 nm. At t = 1.2 min, EDTA was added at different concentrations, 0 mM (D), 0.1 mM (m), 0.3 mM (+), 0.6 mM (O), 1.2 mM (0).
part, to reduce ferric iron and promote subsequent ‘OH production. This could also explain the inhibition of lignin degradation observed by Faison et al. when SOD was added to low nitrogen cultures of P. chrysosporium (15). Alternatively, ferric iron could be reduced by the CO;. However, this was not observed, at least under atmospheric oxygen conditions. The proposed mechanism for ‘OH production by LIP is presented in Fig. 7. The results of this investigation demonstrate that LiP can catalyze ‘OH production using veratryl alcohol and an electron donor such as EDTA. Although ‘OH was not detected when oxalate was used as the electron donor, iron reduction was observed. Therefore, oxalate may be the physiological electron donor involved in Lip-catalyzed ‘OH production. The fact that veratryl alcohol and oxalate are metabolites of P. chrysosporium suggests an in vivo significance to the reactions observed in this study. The concentration of oxalate in low nitrogen cultures reached a maximum on the day prior to detection of LiP activity. A significant decrease in oxalate concentration was observed between Days 4 and 7, which corresponds to the period of maximum LIP activity. In addition, the highest rates of oxalate mineralization occurred during this period
FIG. 7. Proposed can replace oxalate
mechanism for the production in the proposed scheme.
of ‘OH by lignin
ET
AL.
of maximum LIP activity. Together, these results suggest an in vivo mechanism by which LIP catalyzes the degradation of oxalate. The metabolism of oxalate via this mechanism provides reducing equivalents (i.e., CO;) for the production of 0; and the subsequent reduction of ferric iron. Forney et al. obtained evidence for the production of ‘OH in nutrient nitrogen-limited cultures of P. chrysosporium (14). These investigators also observed a lOOfold lower ‘OH concentration (as detected by ethylene gas formation from KTBA) in high nutrient nitrogen cultures. Several reports have shown that LiP activity is absent in high nitrogen cultures of P. chrysosporium (24-26). The loo-fold decrease in the ‘OH concentration and the absence of LiP activity in high nutrient nitrogen cultures support the hypothesis that ‘OH production by P. chrysosporium involves lignin peroxidases. An earlier study by Kirk et al. (18) reported that the ‘OH is not an integral component of the lignin degrading system. These investigators did not detect ‘OH in LiP reaction mixtures. However, their LiP reaction mixtures did not include veratryl alcohol, oxalate, or ferric iron. The results of the present investigation demonstrate that LiP can catalyze the production of-OH. This is supported by the spin trapping of ‘OH by DMPO in reaction mixtures containing LiPH2, HzOz, veratryl alcohol, ferric chloride, and EDTA. The addition of the ‘OH scavenger formate to this reaction mixture resulted in the formation of a DMPO-CO2 adduct. Thus, it was concluded that ‘OH was produced by the iron-dependent Haber-Weiss reaction and not the homolytic cleavage of a DMPO-OOH adduct. The hyperfine coupling constants obtained for the DMPO-OH, DMPO-OOH, and DMPO-CO2 adducts were in agreement with those from previous literature (27). The absence of a DMPO-OH ESR spectrum in LiPH2 reaction mixtures containing oxalate is likely due to a limitation in detection and not the actual absence of ‘OH. In conclusion, the results of this study demonstrate that LiP can catalyze iron reduction via a mechanism that involves secondary metabolites of P. chrysosporium (i.e., veratryl alcohol and oxalate). The reduction of iron through the proposed mechanism could promote the production of ‘OH in vivo. A closer evaluation of these reactions may provide insight into some of the unexplained mechanisms involved in xenobiotic degradation by P.
peroxidase.
(VA)
veratryl
alcohol,
(VA+‘)
veratryl
alcohol
cation
radical.
EDTA
LIGNIN
chrysosporium. The involvement the metabolism of environmental under investigation.
PEROXIDASE
FROM
of Lip-generated-OH in pollutants is currently
12. Kelley,
13.
485
chrysosporium R. L., and Reddy,
Kutsuki, H., and Gold, mun. 109,320-328.
C. A. (1982)
Maughan for by NIH Grant
secretarial ES04922.
assistance.
This
REFERENCES 1. Kirk, T. K., Schultz, E., Connors, W. J., Lorenz, C. F., and Ziekus, J. G. (1978) Arch. Microbial. 117, 277-285. 2. Gold, M. H., Kuwahara, M., Chin, A. A., and Glenn, J. K. (1984) Arch. Biochem. Biophys. 234,353-362. 3. Tien, M., and Kirk, T. K. (1984) Proc. Natl. Acad. Sci. USA 81, 2280-2284. 4. Glenn, J. K., and Gold, M. H. (1985) Arch. Biochem. Biophys. 242, 329-341. 144, 2485. Kelley, R. L., and Reddy, C. A. (1986) Arch. Microbial. 253. P. J. (1990) Biochemistry 87, 2936-2940. 6. Kersten, P. J., Shoemaker, H. E., and Palmer, J. M. (1985) FEBS 7. Harvey, Lett. 196.242-246. J. A., Tien, M., Wright, D., and Aust, S. D. (1985) Science 8. Bumpus, 228,1434-1436. 9. Ryan, T. P., and Bumpus, J. A. (1989) Appl. Microbial. Biotechnol. 3 1,302-307. 10. Fernando, T., Bumpus, J. A., and Aust, S. D. (1990) Appl. Enuiron. Microbial. 66, 1666-1671. 11. Kennedy, D. W., Aust, S. D., and Bumpus, J. A. (1990) Appl. Enuiron. Microbial. 56, 2346-2353.
15. Faison, B. D., and Kirk, 1140-1145.
T. K. (1983)
Akamatsu, Y., Ma, D. B., Higuchi, Lett. 269,261-263.
18. Kirk, T. K., Mozuch, 455-460.
B&hem.
Appl.
J. 206,423-425. Biophys.
M., and Aust,
16. Popp, J. L., and Kalyanaraman, chemistry 29,10475-10480.
-Ii.
Biochem.
M. H. (1982)
L. J., Reddy, C. A., Tien, 14. Forney, Chem. 257,11455-11462.
ACKNOWLEDGMENTS The authors thank Terri work was supported in part
Phawrochete
Res. Com-
S. D. (1982)
Enuiron.
J. Biol.
Microbial.
B., and Kirk,
T. K. (1990)
T., and Shimada,
M. (1990)
M. D., and Tien,
M. (1985)
Biochem.
46, BioFEBS
J. 226,
19. Tuisel, H., Sinclair, R., Bumpus, J. A., Ashbaugh, W., Brock, B. J., and Aust, S. D. (1990) Arch. Biochem. Biophys. 279, 158-166. 20. Shah, M. M., Grover, Biophys. 290,173-178. 21. McCord, 6055.
T. A., and Aust,
J. M., and Fridovich,
22. Kersten, P. J., Tien, J. Biol. Chem. 260,
24. Faison, B. D., and Kirk, 299-304.
26. Keyser,
T. W., Choi, 42, 290-296. P., Kirk,
I. (1969)
J. Biol.
M., Kalyanaraman, 2609-2612.
23. Popp, J. L., and Kirk, 145-148.
25. Jefferies, Microbial.
S. D. (1991)
T. K. (1991) T. K. (1985) S., and Kirk,
T. K., and Zeikus,
Arch.
Chem.
B., and Kirk, Arch. Appl.
Biochem. Environ.
244,6049T. K. (1985)
Biophys. Microbial.
T. K. (1981) J. G. (1978)
Appl.
G. R. (1987)
Free Radicals
Biol.
Med.
3, 259-303.
288, 49,
Enuiron.
J. Bacterial.
790-797. 27. Buettner,
Biochem.
135,
