Proc. Natl. Sci. Counc. ROC(B) H.C. Chang and J.A. Bumpus Vol. 25, No. 1, 2001. pp. 26-33

Inhibition of -Mediated Oxidation Activity by Ethylenediamine Tetraacetic Acid and N-N-N’-N’-Tetramethylenediamine

HEBRON C. CHANG* AND JOHN A. BUMPUS**

*Division of Biochemical Toxicology National Center for Toxicology Research Food and Drug Administration Jefferson, AR, U.S.A. **Department of Chemistry University of Northern Iowa Cedar Fall, IA, U.S.A

(Received April 11, 2000; Accepted June 15, 2000)

ABSTRACT

The mineralization rate of 14C-[1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] (DDT) was reduced by 90% on the 18th day in fungal cultures of Phanerochaete chrysosporium in the presence of 8 mM ethylenediamine tetraacetic acid (EDTA). In the presence of 8 mM N-N-N’-N’-tetramethylenediamine (TEMED), the mineralization rate of 14C-DDT was reduced by 80%. In the presence of 2 mM or 10 mM EDTA, 95% inhibition of lignin peroxidase (LiP) mediated veratryl alcohol oxidase activity and 97% inhibition of LiP mediated iodide oxidase activity occurred. TEMED caused 79% inhibition of veratryl alcohol oxidase activity and 92% inhibition of iodide oxidase activity when the amount used was 2 mM and 10 mM, respectively. In the presence of Zn(II) with slight molar excess of the EDTA concentration, reversed the EDTA mediated non-competitive inhibition of LiP catalyzed veratryl alcohol or iodide oxidation. Zn(II) also reversed the inhibition of LiP catalyzed veratryl alcohol oxidase activity caused by chelators other than EDTA and TEMED. In addition to Zn(II), several other metal ions also relieved EDTA mediated inhibition of veratryl alcohol and iodide oxidase activity catalyzed by LiP. The ability of veratryl alcohol to inhibit iodide oxidation catalyzed by LiP showed that veratryl alcohol could inhibit LiP mediated iodide oxidase activity. Increasing the concentration of iodide was also shown to inhibit veratryl alcohol oxidation. Kinetic analysis showed that the reaction was competitive inhibition.

Key Words: lignin peroxidase (LiP), EDTA, TEMED, chelator, kinetic study, competitive inhibition, non-competitive inhibition

I. Introduction oxide requiring oxygenases (Tien and Kirk, 1984), subsequent studies confirmed that these are similar to other White rot fungi and a few species of bacteria are the , such as (HRP) and lac- only micro-organisms that are able to cause extensive biodeg- toperoxidase (LPO) (Andrawis et al., 1988; Kuila et al., 1985; radation of lignin (Crawford, 1981). Most of the available Renganathan and Gold, 1986). LiP has been shown to medi- information concerning fungal biodegradation of lignin has ate the biodegradation of many environmental pollutants, come from studies on the white rot fungus Phanerochaete including [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] chrysosporium. This fungus is able to degrade lignin by se- (DDT) in fungal cultures (Bumpus and Aust, 1987; Fernando creting peroxidases that are able to catalyze the initial oxida- et al., 1989; Kersten et al., 1985; Haemmerli et al., 1986; tion involved in lignin degradation (Glenn et al., 1993; Harvey Hammel et al., 1986; Bumpus and Brock, 1988; Hammel and et al., 1986; Kuwahara et al., 1984; Tien and Kirk, 1983; Tien, Tardone, 1988; Mileski et al., 1988; Schreiner et al., 1988). 1987). Lignin peroxidases (LiP) are secreted by P. chry- LiP and other peroxidases were also found to mediate oxida- sosporium during idiophase (Tien and Kirk, 1983). Although tion of a number of nitrogen-containing compounds (Chang, lignin peroxidases were originally described as hydrogen per- 1994). Ethylenediamine tetraacetic acid (EDTA) was shown

Abbreviations used: LiP, Lignin peroxidase; DDT, [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane]; EDTA, ethylenediamine tetraacetic acid; TEMED, N- N-N’-N’-tetramethylenediamine; detapac, diethylenetriamine pentaacetic acid; EGTA, ethylenenglycol-bis-(β-aminoethylether) N,N,N’,N’-tetraacetic acid; PDTA, 1,2-diaminopropane-N,N,N’,N’-tetraacetic acid; Desferal, 1-amino-6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22- tetraazaheptaeicosane; Dexrazoxane, 4,4-(1-methyl-1,2-ethanediyl)bis-2,6-piperazinedione .

– 26 – Inhibition of Oxidation Activity by Chelators to affect LiP oxidase activities (Aust et al., 1989; Shah et al., (β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), o- 1992; Barr and Aust, 1993; Shah and Aust, 1993; Chang, phenanthroline, 1,2-diaminopropane-N,N,N’,N’-tetraacetic 1994). In this study, we examined the effects of EDTA and acid (PDTA), MgCl2, CaCl2, NaCl, FeSO4, CuSO4, CdSO4, N-N-N’-N’-tetramethylenediamine (TEMED) in fungal cul- and KI were purchased from Mallinckrodt (St. Louis, MO, tures of P. chrysosporium to determine the biodegradation U.S.A.). 4,4-(1-methyl-1,2-ethanediyl)bis-2,6-piperazine- 14 ability of C-DDT, and studied the effects of EDTA and dione (Dexrazoxane), NiSO4, V2(SO4)3, Al2(SO4)3, GaSO4, TEMED to understand their inhibition activities towards LiP Li2SO4, InSO4, and AgSO4, CoSO4 were purchased from catalyzed veratryl alcohol oxidase activity and iodide oxidase Aldrich (Milwaukee, WI, U.S.A.). HgCl2 was purchased from activity. The reversal activities of Zn(II) with respect to the EM Science (Darmstadt, Germany). ZnSO4 and MnSO4 were inhibition of LiP veratryl alcohol oxidase activity mediated purchased from Baker (Phillipsburg, NJ, U.S.A.). by EDTA and other metal chelators (Fig. 1) was also moni- tored. We report here the results of kinetic studies on the 2. Incubation of 14C-DDT in the Fungal Cultures of inhibition of LiP veratryl alcohol and iodide oxidase activity Phanerochaete chrysosporium mediated by EDTA. We also report on the competitive mecha- nism of veratryl alcohol and iodide with regard to LiP-medi- P. chrysosporium cultures were incubated with 14C- ated oxidation activity. DDT as described by Bumpus and Aust (1987), and with vari- ous concentrations (1 – 8 mM) of EDTA or TEMED at 39°C 14 14 II. Materials and Methods for 20 days. Biodegradation of C-DDT to CO2 was mea- sured as described else where (Bumpus and Aust, 1987; Fer- 1. Chemicals nando et al., 1989).

Hydrogen peroxide solutions were prepared by dilu- 3. Assay and Kinetic Studies tion of a 3% stock solution purchased from Sigma (St. Louis, MO, U.S.A.). 14C-DDT (ring labeled, 10–30 mCi/mmol), and LiP was purified from nutrient nitrogen-limited agitated 1-amino-6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetyl- culture of P. chrysosporium as described by Tuisel et al. (1990). hydroxylamino)-6,11,17,22-tetraazaheptaeicosane (desferal) Veratryl alcohol oxidase activity of LiP was monitored at 310 –1 –1 were also purchased from Sigma. EDTA, TEMED, diethyl- nm (ε310nm = 9.3 µM cm ) (Tien and Kirk, 1984). Reaction enetriamine pentaacetic acid (detapac), ethylenenglycol-bis- mixtures for veratryl alcohol oxidase activity contained 0.05 µM LiP, 1.5 mM veratryl alcohol and 250 µM H2O2 in 0.1 M – HOOCH2C CH2COOH sodium tartrate buffer, pH 3.5. Oxidation of iodide (I ) by NNCH2 CH2 LiP was monitored to determine the absorbance change of HOOCH C CH2COOH 2 EDTA – triiodide (I3 ) at 353 nm (Huwiler and Kohler, 1985). Reac- H3C CH3 NNCH2 CH2 tion mixtures for iodide oxidation contained 1.5 mM potas- CH H3C 3 µ µ TEMED sium iodide, 0.05 M LiP and 250 M H2O2 in 0.1M sodium tartrate buffer, pH 3.5. In several experiments, relatively high HOOCH2C CH2COOH NNCH2 CH2 OCH2 CH2 OCH2 CH2 (2 – 10 mM) concentrations of EDTA (or TEMED) were used CH COOH HOOCH2C EGTA 2 to inhibit veratryl alcohol oxidase and/or iodide oxidase ac- tivities mediated by LiP. Also, a variety of metal ions were CH3 HOOCH2C CH2COOH added to determine their relative ability to relieve enzyme in- NNCH CH2 CH COOH hibition caused by EDTA. In general, sulfate salts are essen- HOOCH2C 2 PDTA tially insoluble in water, so HgCl2 and CaCl2 were used instead, HOOCH C 2 CH2COOH and NaCl was used as a control to study the possible effect of NCH2 CH2 NNCH2 CH2 CH2COOH chloride ion. Kinetic parameters were measured following HOOCH2C CH2COOH Detapac the methods described by Chang (1994).

N o-phenanthroline N III. Results O O CH 3 N 14 Dexrazoxane In vivo study showed that C-DDT was mineralized NNCH CH2 N O by P. chrysosporium as described by Bumpus and Aust (1987). O 14 H H In the presence of 8 mM EDTA, mineralization of C-DDT

H2NN(CH2)5 CC(CH2)2 N(CH2)5 NNN CC(CH2)2 (CH2)5 CCH3 was reduced by ~90% on 18th day (Fig. 2). In the presence of OHOC OH OO OH O 8 mM TEMED, the mineralization rate of 14C-DDT was re- Desferal duced by ~80% (Fig. 3). In vitro experiments also showed Fig. 1. Structures of chelators. that EDTA caused 95% inhibition of veratryl alcohol oxidase

– 27 – H.C. Chang and J.A. Bumpus

1200 1.0 0 mM TEMED 1000 1 mM TEMED 2 mM TEMED 0.8 8 mM TEMED 800 0.6 +2 mM EDTA 600 0.4 400

200 0.2 +2.5 mM Zn(II) Absorbance at 310 nm C-DDT mineralized (dpm) 14 0 0 3 6 9 12 15 18 0.0 incubation days 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time (min) Fig. 2. 14C-DDT mineralization by Phanerochaete chrysosporium in the 14 3.0 presence of EDTA (1 – 8 mM). CO2 evolution was monitored every three days. 2.5

1200 2.0 0 mM TEMED +15 mM EDTA 1 mM TEMED 1000 2 mM TEMED 1.5 8 mM TEMED 800 1.0

600 +18 mM Zn(II)

Absorbance at 353 nm 0.5

400 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 200

C-DDT mineralized (dpm) Time (min) 14 0 Fig. 4. Ability of Zn(II) to relieve inhibition of LiP catalyzed veratryl al- 0 3 6 9 12 15 18 incubation days cohol oxidase activity (top) and iodide oxidase activity (bottom) mediated by EDTA. The reaction mixtures contained 0.05 µM LiP Fig. 3. 14C-DDT mineralization by Phanerochaete chrysosporium in the and 1.5 mM veratryl alcohol (or iodide) in sodium tartrate buffer. 14 Veratryl alcohol and iodide oxidase activity was initiated by adding presence of TEMED (1 – 8 mM). CO2 evolution was monitored µ every three days. 250 M H2O2 and were monitored for 4 minutes. Activity was inhibited by the addition of 2 mM (or 15 mM) EDTA. Recovery of activities was achieved by adding of 2.5 mM (or 18 mM) Zn(II). activity and 97% inhibition of iodide oxidase activity when the EDTA concentrations were 2 mM and 10 mM, respectively. Other chelators besides EDTA and TEMED were also TEMED caused 79% inhibition of veratryl alcohol oxidase tested to determine their inhibition activities for LiP catalyzed activity and 92% inhibition of iodide oxidase activity when veratryl alcohol oxidation. Results showed that dexrazoxane TEMED was at 2 mM and 10 mM (data not shown). had a highest inhibition rate (99.1%), and that o-phenanthroline Results showed that in the presence of Zn(II) in slight had the lowest inhibition rate (27.8%) at 0.5 mM. Excess molar excess of the EDTA concentration, reversed EDTA molar of Zn(II) (1 mM) could not recover the inhibition of mediated inhibition of LiP catalyzed veratryl alcohol and io- LiP catalyzed veratryl alcohol oxidation by dexrazoxane and dide oxidation (Fig. 4). In addition of Zn(II), several other desferal. The results also showed that 1 mM of Zn(II) recov- metal ions were also analyzed to determine their ability to ered 66%, 56%, 27%, 26%, 11% and 11% of the inhibition relieve EDTA mediated inhibition of veratryl alcohol oxidase rates mediated by 0.5 mM of EDTA , PDTA, detapac, TEMED, activity catalyzed by LiP (Table 1). A similar experiment was EGTA and o-phenanthroline, respectively (Table 2). performed to assess the ability of metal ions to relieve EDTA Our kinetic study results suggest that EDTA is a non- inhibition of iodide oxidase activity catalyzed by LiP. competitive inhibitor for veratryl alcohol oxidase activity cata- However, 10 mM EDTA was required to achieved ~95% lyzed by LiP (Fig. 5). EDTA was able to inhibit LiP mediated inhibition, and a 20 mM concentration of metal ion was used iodide oxidase activity, but the inhibition pattern remains un- to assess the ability of each metal to relieve EDTA inhibition clear (Fig. 6). The ability of veratryl alcohol to inhibit iodide of iodide oxidase activity (Table 1). oxidation catalyzed by LiP showed that veratryl alcohol in-

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Inhibition of Oxidation Activity by Chelators

Table 1. Ability of Metal Ions to Reverse EDTA Mediated Inhibition of 2.0 Veratryl Alcohol Oxidase Activity and Iodide Oxidase Activity of control LiP 50 µ M EDTA 100 µ M EDTA Remaining veratryl alcohol Remaining iodide 1.5 150 µ M EDTA µ oxidation activity (%) oxidation activity (%) 250 M EDTA Control 100 ± 1 100 ± 1 EDTA 21 ± 25 ± 2 1.0

Zn 81 ± 3 84 ± 2 1/V Cd 78 ± 2 54 ± 3 Ga 75 ± 3 61 ± 3 Cu 72 ± 3 63 ± 5 0.5 In 62 ± 5 38 ± 6 Mn 42 ± 3 48 ± 3 Al 38 ± 2 27 ± 2 0.0 V 38 ± 50Ð10 Ð5 0 5 10 15 20 25 30 35 Hg 16 ± 40 1/[veratryl aicohol] (mM) Co 15 ± 3 78 ± 5 Na 13 ± 1 26 ± 3 0.3 Mg 10 ± 2 18 ± 4 Ca 8 ± 2 40 ± 4 Li 6 ± 32 ± 1 Ni 4 ± 2 70 ± 5 Fe N/A N/A 0.2 Note: Also see Aust et al. (1989).

Table 2. Effects of Various Metal Chelators on the Inhibition of LiP Medi- 0.1 ated Veratryl Alcohol (VA) Oxidase Activity and Its Recovery through the Addition of Zn(II) Intercept Vertical % of VA activity % of VA activity (+ 0.5 mM (+ 0.5 mM chelator) chelator + 1 mM Zn(II) ) 0.0 dexrazoxane 0.9 0.5 Ð50 0 50 100 150 200 250 300 desferal 1.5 1.7 [EDTA] ( µ M) EDTA 11.6 77.4 PDTA 26.5 82.7 Fig. 5. Double reciprocal plots of LiP catalyzed veratryl alcohol oxidase detapac 35.3 62.4 activity and its inhibition by EDTA. The reaction mixtures con- TEMED 42.0 68.0 tained 10 mM sodium tartrate buffer, pH 3.5, 0.07 µM LiP and EGTA 64.0 74.8 various concentrations of EDTA. The reactions were initiated by o-phenanthroline 72.8 83.4 hte addition of 250 µM H2O2. The y-axis of the top plot represents the reciprocal of the amount of veratryl aldehyde formed (Km = 150 –1 –1 µM, Vmax = 4.95 µM min , Vmax/Km = 0.033 min ). The bottom plot is a plot of the top plot based on intercept vs [EDTA] (KI = 35 hibited LiP mediated iodide oxidase activity, and kinetic analy- µM). sis showed that the inhibition was competitive (Fig. 7). An attempt was also made to determine the pattern of iodide inhi- bition of veratryl oxidation activity (Fig. 8). In the presence ties were 0.13 mM and 5.0 mM, respectively. The EC50 of of iodide, only 7 – 18% of the initial rate of absorbance change veratryl alcohol for LiP catalyzed iodide oxidation was 50 at 310 nm was observed. However, controls showed that in- mM, and the EC50 of iodide for LiP catalyzed veratryl alcohol hibition was even greater since the observed absorbance change oxidation was 0.1 mM (Table 3). due to edge absorption of iodide oxidation activity was mea- sured at 353 nm. Furthermore, after 2 min, the absorbance IV. Discussion change at 310 nm completely stopped. However, upon con- tinued incubation, between 27% and 84% of the initial veratryl Banerjee (1989) showed that addition of selected metal alcohol oxidase activity was restored. The rate of restored ions in slight molar excess of the EDTA concentrations re- veratryl oxidase activity was inversely dependent on the ini- versed EDTA mediated inhibition of peroxidase catalyzed tial concentration of iodide and appeared to be due to deple- iodide oxidase activity. Metal ion Zn(II) chelated with tion of the iodide concentration to levels so low that they no EDTA and released EDTA from active sites. This releasing longer completely inhibited veratryl alcohol oxidation. The effect reversed the inhibition of LiP catalyzed iodide oxidation. inhibitor concentrations for 50% inhibition (EC50) of EDTA Figure 4 shows that Zn(II) was able to relieve EDTA medi- for LiP catalyzed veratryl alcohol and iodide oxidase activi- ated inhibition of LiP catalyzed veratryl alcohol oxidase and

– 29 – H.C. Chang and J.A. Bumpus

0.5 0.8 control 0 mM VA 0.1 mM EDTA 10 mM VA 0.4 0.2 mM EDTA 50 mM VA 0.3 mM EDTA 0.6 100 mM VA 0.3

1/V 0.4 0.2 1/V

0.1 0.2

0.0 Ð4 Ð3 Ð2 Ð1 0 1 2 3 4 5 6 0.0 Ð2 Ð1 0 1 2 3 4 1/[KI] (mM) 1/[KI] (mM) 0.3 Fig. 7. Double reciprocal plots of LiP catalyzed iodide oxidase activity and its inhibition by veratryl alcohol. The reaction mixture con- tained 10 mM tartrate buffer, pH 3.5, 0.07 µM LiP, 0, 10, 50 or 100 mM veratryl alcohol (VA), and 0.33, 0.5, 1, or 4 mM potassium 0.2 iodide. The reactions were initiated by the addition of 250 µM H2O2. The y-axis represents the reciprocal of the amount of triiodide formed (KI = 10 mM).

0.1 0.8 Vertical Intercept Vertical control 0.7 50 µ M KI 100 µ M KI 0.0 0.6 150 µ M KI 200 µ M KI Ð1.00 Ð0.75 Ð0.50 Ð0.25 0.0 0.25 0.50 0.5 [EDTA] (mM) 0.4 Fig. 6. Double reciprocal plots of LiP catalyzed iodide oxidase activity and its inhibition by EDTA. The reaction mixtures contained 10 0.3 mM sodium tartrate buffer, pH 3.5, 0.07 µM LiP and various con- 0.2 centrations of EDTA. The reactions were initiated by the addition

µ Absorbance at 310 nm of 250 M H2O2. The y-axis of the top plot represents the recipro- 0.1 cal of the amount of triiodide formed. (Km = 67 µM, Vmax = 162.6 µM min–1, V /K = 2.43 min–1). The bottom plot is a plot of the 0.0 max m 0 2 4 6 8 10 12 top plot based on intercept vs [EDTA] (KI = 0.95 mM). Time (min) iodide oxidase activities. Fig. 8. Inhibition of LiP catalyzed veratryl alcohol oxidase activity by iodide. The reaction mixtures contained 0.05 µM LiP, 1.5 mM In addition to Zn(II), several other metal ions were as- veratryl alcohol in 0.1 M sodium tartrate buffer, pH 3.5, and vari- sayed to determine their ability to relieve EDTA mediated in- ous concentrations of iodide (0 – 200 µM). Veratryl alcohol oxi- hibition of veratryl alcohol oxidase activity catalyzed by LiP. dase activity was initiated by adding 250 µM H2O2 The results showed that in the presence of 2 mM EDTA, and 4 mM Cd(III), Ga(II), Cu(II), In(II), Mn(II), Al(III), V(III), Hg(III) and Co(II) were all able to at least partially relieve to relieve EDTA mediated iodide oxidase activity. Table 1 inhibition of veratryl alcohol oxidase activity mediated by 2 also shows that Zn(II), Cd(II), Ga(II), Cu(II), In(II), Mn(II), mM EDTA. The rest of the metal ions possessed little or no Al(III), Co(II), Na+, Mg(II), Ca(II), and Ni+ were all able to at ability to reverse EDTA mediated inhibition of veratryl alco- least partially relieve inhibition of iodide oxidase activity hol oxidase activity catalyzed by LiP (Table 1) (Aust et al., mediated by EDTA. The other metals were not able to relieve 1989). inhibition of activity. V(III) and Hg(II) were inhibitors at a A similar experiment was performed to assess the abil- high concentration (i.e., 20 mM). In the presence of 20 mM ity of metal ions to relieve EDTA mediated inhibition of io- V(III) or Hg(II) with EDTA absent from the incubations, no dide oxidase activity catalyzed by LiP. Ten mM EDTA was LiP catalyzed veratryl oxidase or iodide oxidase activity was required to obtain 95% inhibition, and a 20 mM concentra- observed. In contrast to results obtained using the veratryl tion of metal ion was used to assess the ability of each metal alcohol oxidase activity assay is the fact that Co(II), Na+,

– 30 – Inhibition of Oxidation Activity by Chelators

– + Table 3. Inhibition of Veratryl Alcohol (VA) Oxidation and Iodide (KI) I + I I2 (5) Oxidation Catalyzed by LiP. EC50 of EDTA, VA and KI

EC50 (EDTA) EC50 (VA) EC50 (KI) In either reaction sequence, the initial oxidation of io- VA oxidation 0.13 mMa N/A 0.10 mMb dide by peroxidases results in a two-electron oxidation prod- KI oxidation 5.0 mMc 50 mMd N/A uct (either IO– or I+) (Eqs. (1) and (4)) which then reacts a µ µ Reaction mixtures contained 0.05 M LiP, 1.5 mM VA, 500 M H2O2 and noneznymatically with free iodide to form iodine (I2) (Eqs. EDTA (0.01 mM – 20 mM) in 0.1 M sodium tartrate buffer, pH 3.5, moni- (2) and (5)). Iodine formed then reacts with free iodide to tored at 310 nm. – b form triiodide (I3 ) (Eq. (3)). Indeed, the absorbance of tri- Reaction mixtures contained 0.05 µM LiP, 1.5 mM VA, 500 µM H2O2 and KI (0.01 mM – 0.5 mM) in 0.1 M sodium tartrate buffer, pH 3.5, moni- iodide at 353 nm was measured in iodide oxidase assays. Thus, tored at 310 nm. when iodide concentrations were saturated, increasing the io- c µ µ – Reaction mixtures contained 0.05 M LiP, 1.5 mM KI, 250 M H2O2 and dide concentration resulted in an increased rate of I3 formation. EDTA (0.01 mM – 20 mM) in 0.1 M sodium tartrate buffer, pH 3.5, moni- This increased rate was due to a nonenzymatic reaction, as tored at 353 nm. d also mentioned by Huwlier et al. (1985). Recent investiga- Reaction mixtures contained 0.05 µM LiP, 1.5 mM KI, 250 µM H2O2 and VA (0 mM – 100 mM) in 0.1 M sodium tartrate buffer, pH 3.5, monitored tions of horseradish peroxidase suggest that peroxidases pos- at 353 nm. sess several distinct sites on the moiety where individual classes of substrates are oxidized (Ator and Ortiz de Montel- lano, 1987; Ortiz de Montellano et al., 1987, 1988). Iodide Mg(II), Ca(II) and Ni+ were able to relieve EDTA mediated and thiocyanate are thought to bind at a site equidistant from inhibition of iodide oxidase activity catalyzed by LiP. The the 1 and 8 methyl groups of the heme, approximately 6 – 10 reason for this difference is unknown. However, it is interest- Angstroms from the heme iron (Bhattacharyya et al., 1993; ing to note that Banerjee et al. (1986) showed that Mg(II) Lukart et al., 1987; Renganathan et al., 1985; Somers and and Ca(II) were able to relieve inhibition of iodide oxidation Shapiro, 1989; Ortiz de Montellano et al., 1988). In con- caused by EDTA in mouse gastric mucosa mitochondrial trast, phenolic substrates bind to a site nearer the 8 methyl peroxidase. It should also be noted that control experiments group (Ator and Ortiz de Montellano, 1987; Harris et al., 1993). showed that no iodide oxidase activity occurred in the ab- Still another site for thioanisoles has been proposed to exist sence of LiP in these experiments. The results for the effects nearer the heme iron (Harris et al., 1993). Oxygen exchange of various metal chelators with regard to inhibition of LiP can occur within one of the few oxygenation reactions medi- mediated veratryl alcohol oxidase activity suggest that nitro- ated by peroxidases (Chen and Schopfer, 1999). Most reac- gen atoms of metal chelators are required to achieve inhibi- tions mediated by peroxidases are one or two electron oxi- tion (Fig. 1 and Table 2). dations. Peroxidase mediated oxygenations are relatively rare The kinetics of EDTA inhibition of iodide oxidation (Barr and Aust, 1993). are not amendable to conventional (e.g., Lineweaver-Burke, It is not known if iodide and veratryl alcohol have sepa- Eadie-Hofstee or Hanes) analysis. The reason for this is that rate binding sites on the heme moiety of lignin peroxidases. peroxidase mediated oxidation of iodide is thought to occur The fact is that they are both substrates for LiP but are in as follows (Morrison and Schonbaum, 1976; Magnusson et competition with each other. Oxidation by this LiP argues for al., 1984): at least some affects in their respective binding sites. Recently, Choinowski et al. (1999) reported that a second - H OI– 2 centered at Trp171 had been found in a crystal E + H O EO [EOI–] 2 2 structural study of LiP. Trp171 is a redox active amino acid and is involved in the oxidation of veratryl alcohol and larger E + IO– (1) aromatic substrate molecules, such as lignin. The distal heme pocket was identified as the active center for small aromatic molecules, including H O . However, even if these sites are IO– + I– + H+ I + OH– (2) 2 2 2 mutually exclusive, a pattern of competitive inhibition could be expected because both iodide and veratryl alcohol could be expected to compete for the same of the LiP. – – I + I2 I3 (3) Results showed that iodide is a better substrate for lignin per- oxidase because Vmax/Km of LiP for oxidizing potassium io- Alternatively, it may occur due to the following reac- dide (2.43 min–1) is larger than that for oxidizing veratryl al- tions (Roman and Dunford, 1972) through the triiodide equi- cohol (0.033 min–1). Also, it needs only 0.1 mM potassium librium (Schwarz and Bielski, 1986): iodide to inhibit 50% of LiP mediated veratryl alcohol oxida- tion while for iodide oxidation, it needs 50 mM veratryl alco- – H2OI hol to inhibit 50% of the enzyme activity. Mediated inhibi- + E + H2O2 EO E + I (4) tion of iodide oxidation due to the addition of potassium io-

– 31 – H.C. Chang and J.A. Bumpus dide released veratryl alcohol also supports the idea that po- peroxidase-catalyzed conversion of iodine to iodide in the presence of tassium iodide is a better substrate for lignin peroxidase (also EDTA and H2O2. J. Biol. Chem., 261:10592-10597. see Fig. 7). Banerjee, A.K. (1989) Mechanism of horseradish peroxidase-catalyzed con- version of iodine to iodide in the presence of EDTA and H2O2. J. Biol. The fact that EDTA functions as a non-competitive in- Chem., 264:9188-9194. hibitor of veratryl alcohol oxidase activity can be explained Barr, D.P. and Aust, S.D. (1993) On the mechanism of peroxidase-mediated by the classic interpretation that it binds to an inhibitory site, oxygen production. Arch. Biochem. Biophys., 303:377-382. which could be the primary substrate binding site, the heme Barr, D.P. and Aust, S.D. (1994) Effect of superoxide and superoxide pocket on the enzyme rather than a second substrate-binding dismutase on lignin peroxidase-catalyzed veratryl alcohol oxidation. Arch. Biochem. Biophys., 311:378-382. site (Choinowski et al., 1999), and that it cannot be totally Bhattacharyya, D.K., Bandyopadhyay, U. and Banerjee, R.K. (1993) EDTA displaced by substrate (veratryl alcohol). It has been shown inhibits -catalyzed iodide oxidation by acting as an elec- that EDTA is decarboxylated by LiP (Shah et al., 1992). There tron-donor and interacting near the iodide binding site. Mole. Cell. might be several other differences in the ways EDTA inhibits Biochem., 162:105-111. veratryl alcohol oxidation and iodide oxidation by means of Bumpus, J.A. and Aust, S.D. (1987) Biodegradation of DDT [1,1,1-trichloro- 2,2-bis(4-chlorophenyl)ethane] by the white rot fungus Phanerochaete lignin peroxidase. For example, substantially greater concen- chrysosporium. Appl. Environ Microbiol., 53:2001-2008. trations of EDTA are required for complete inhibition of io- Bumpus, J.A. and Brock, B.J. (1988) Biodegradation of crystal violet by the dide oxidation compared to that required for complete inhibi- white rot fungus Phanerochaete chrysosporium. Appl. Environ. tion of veratryl alcohol oxidation. Similarly, there are several Microbiol., 54:1143-1150. relatively subtle differences in the patterns and types of met- Chang, C.W. (1994) Inhibition of Mammalian, Plant and Fungal Peroxi- dases by Selected Nitrogen-containing Compounds: a Comparative Study. als that relieve EDTA mediated inhibition of these two sub- Ph.D. Dissertation. University of Notre Dame, South Bend, IN, U.S.A. strates. It is also important that superoxide is involved in the Chen S.X. and Schopfer, P. (1999) Hydroxyl-radical production in physi- oxidation of LiP catalyzed veratryl alcohol oxidation (Barr ological reactions. A novel function of peroxidase. Eur. J. Biochem., and Aust, 1994). In a peroxidase system, superoxide is gener- 260:726-735. ated in the presence of EDTA and is responsible for nonenzy- Choinowski, T., Blodig, W., Winterhalter, K.H. and Piotek, K. (1999) The crystal structure of lignin peroxidase at 1.70 A resolution reveals a hy- matic iodide oxidation (Chang and Bumpus, unpublished). droxyl group on the Cbeta of tryptophan 171: a novel radical site formed This phenomenon may explain why the kinetics of EDTA in- during redox cycle. J. Mole. Biol., 286:809-827. hibition of iodide oxidation are not amendable to conventional Crawford, R. (1981). Lignin Degradation and Transformation, pp. 154, John analysis. Wiley, New York, NY, U.S.A. In summary, EDTA inhibits LiP mediated iodide oxi- Fernando, T., Aust, S.D. and Bumpus, J.A. (1989) Effects of culture param- eters on DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] biodegra- dation and veratryl alcohol oxidation. The mechanisms by dation by Phanerochaete chrysosporium. Chemosphere, 19:1387-1398. means of which EDTA mediated inhibition occurs are differ- Glenn, J.K., Morgan, M.A., Mayfield, M.B., Kuwahara, M. and Gold, M.H. ent for different substrates. A greater concentration of EDTA (1993) An extracellular H2O2-requiring enzyme preparation involved in is required for complete inhibition of iodide oxidation than is lignin biodegradation by the white rot Basidiomycete Phanerochaete required for complete inhibition of veratryl alcohol oxidation. chrysosporium. Biochem. Biophys. Res. Comm., 114:1077-1083. Haemmerli, S.D., Leisola, M.S.A., Sanglard, D. and Fiechter, A. (1986) Inhibition of both reactions is relieved by metal ions in differ- Oxidation of benzo(a)pyrene by extracellular ligninases of Phanerochaete ent patterns. This conclusion is supported by the fact that LiP chrysosporium: veratryl alcohol and stability of ligninases. J. Biol. 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spectral evidence for a heme active site similar to those of peroxidases. 314. Biochemistry, 24:3394-3397. Roman, R. and Dunford, H.B. (1972) pH Dependence of the oxidation of Kuwahara, M., Glenn, J.K., Morgan, M.A. and Gold, M.H. (1984) Separa- iodide by compound I of horseradish peroxidase. Biochemistry, 11:2076- tion and characterization of two extracellular H2O2-dependent oxidases 2082. – from ligninolytic cultures of Phanerochaete chrysosporium. FEMS 169: Schwarz, H.A. and B.H.J. Bielski. (1986) Reaction of HO2 and O2 with – 247-250. iodine and bromine and the I2 and I atom reduction potentials. J. Phys. Lukart, G.S., Rodgers, K.R. and Goff, H.M. (1987) Electron paramagnetic Chem., 90:1455-1448. resonance spectroscopy of lactoperoxidase complexes: characterization Schreiner, R.P., Stevens, S.E., Jr. and Tien, M. (1988) Oxidation of thian- of hyperfine splitting for the NO adduct of lactoperoxidase. Biochemistry, threne by the ligninases of Phanerochaete chrysosporium. Appl. Environ. 26:6927-6932. Microbiol., 54:1858-1860. Magnusson, R.P., Taurog, A. and Dorris, M.L. (1984) Mechanism of io- Shah, M.M., Grover, T.A., Barr, D.P. and Aust, S.D. (1992) On the mecha- dide-dependent catalytic activity of and lactoper- nism of inhibition of veratryl alcohol oxidase activity of lignin peroxi- oxidase. J. Biol. Chem. 259:197-205. dase H2 by EDTA. J. Biol. Chem., 267:21564-21569. Mileski, G. J., Bumpus, J.A., Jurek, M.A. and Aust, S.D. (1988) Biodegra- Shah, M.M. and Aust, S.D. (1993) Iodide as the mediator for the reductive dation of pentachlorophenol by the white rot fungus Phanerochaete reactions of peroxodases. J. Biol. Chem., 268:8503-8506. chrysosporium. Appl. Environ. Microbiol., 54:2885-2889. Somers, C.E. and Shapiro, S.M. (1989) The heme environment of ovoper- Morrison, M. and Schonbaum, G.R. (1976) Peroxidase-catalyzed oxidase as determined by optical spectroscopy. J. Biol. Chem., 264:17231- halogenation. Ann. Rev. Biochem., 45:861-888. 17235. Ortiz de Montellano, P.R., Choe, Y.S., DePillis, G. and Catalano, C.E. (1987) Tien, M. and Kirk, T.K. (1983) Lignin-degrading enzyme from the Hymeno- Structure-mechanism relationships in hemoprotein. Oxygenations cata- mycete Phanerochaete chrysosporium Burd. Science, 221:661-663. lyzed by chloroperoxidase and horseradish peroxidase. J. Biol. Chem., Tien, M. and Kirk, T.K. (1984) Lignin-degrading enzyme from the hy- 262:11641-11646. menomycete Phanerochaete chrysosporium: purification, characteri- Ortiz de Montellano, P.R., David, S.K., Ator, M. and Tew, D. (1988) Mecha- zation, and catalytic properties of a unique H2O2-requiring oxygenase. nism- based inactivation of horseradish peroxidase by sodium azide. Proc. Nat. Acad. Sci. U.S.A., 81:2280-2284. Formation of mesoazidoprotoporphyrin IX. Biochemistry, 27:5470-5476. Tien, M. (1987) Properties of ligninases from Phanerochaete chrysosporium Renganathan, V. and Gold, M.H. (1986) Spectral characterization of the and their possible application. CRC Crit. Rev. J. Microbiol., 15:141- oxidized states of lignin peroxidase, an extracellular heme enzyme from 168. the white rot basidiomycete Phanerochaete chrysosporium. Biochemistry, Tuisel, H., Sinclair, R., Bumpus, J.A. and Ashbaugh, W. (1990) Lignin per- 25:1626-1631. oxidase H2 from Phanerochaete chrysosporium: purification, charac- Renganathan, V., Miki, K. and Gold, M.H. (1985) Role of molecular oxy- terization and stability to temperature and pH. Arch. Biochem. Biophys., gen in lignin peroxidase reactions. Arch. Biochem. Biophys., 246:304- 279:158-166.

EDT TEMED

* JOHN A. BUMPUS**

* **Department of Chemistry University of Northern Iowa Cedar Fall, IA, U.S.A.

DDT 8 mM EDTA 90% 8 mM TEMED 80% 2 mM 10 mM EDTA veratryl alcohol (95%) (97%) TEMED veratryl alcohol: 79% 92% EDTA veratryl alcohol veratryl alcohol

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