chloroperoxidase (chloride : hydrogen peroxide oxidoreductase) (EC. 1.11.1.10) .... isozyme L3, from the white-rot fungus P. radium strain 79, and present kinetic ...
Eur. J. Biochem. 211,391-402 (1993) 0 FEBS 1993
Lignin peroxidase L3 from Phlebia radiata Pre-steady state and steady-state studies with veratryl alcohol and a non-phenolic lignin model compound l-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-l,3-diol Taina LUNDELL ', Ron WEVER ', Rent FLORIS z, Patricia HARVEY ', Annele HATAKKA I, Gosta BRUNOW4 and Hans SCHOEMAKERS Department of Applied Chemistry and Microbiology, University of Helsinki, Finland ' E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands ' Department of Biology, Imperial College of Science, Technology and Medicine, London, England Department of Chemistry, University of Helsinki, Finland ' DSM Research, Bio-organic Chemistry Section, Geleen, The Netherlands (Received September 15/0ctober 26, 1992) - EJB 92 1311
The catalytic cycle of lignin peroxidase (Lip, ligninase) isozyme L3 from the white-rot fungus Phlebia radiata was investigated using stopped-flow techniques. Veratryl (3,4-dimethoxybenzyl) alcohol and a lignin model compound, non-phenolic a-0-4 dimer 1-(3,4-dimethoxyphenyl)-2-(2methoxyphenoxy)propane-1,3-diol,were used as electron donors. This is the first report on the detailed kinetic analysis of a Lip-catalysed Ca-CB bond cleavage of the dimer, representing the major depolymerisation reaction in the lignin polymer. The native enzyme showed a typical heme peroxidase absorbance spectrum with a Soret maximum at 407 nm. Following the reaction with H,Oz, the Soret band decreased in absorbance, shifted to 403 nm and then to 421 nm,demonstrating the formation of compound I followed by the formation of compound 11, respectively. Similar results have been reported for the Lip from Phunerochaete chrysosporium upon reaction with H,O,. However, compound I of L3 was more stable in the absence of additional electron donors. Thesecond-order rate constant of compound I formation by H,O, was determined to be 6 X IOI M-' s-' and was the same at pH 3.0 and 6.0. Compound I was rapidly reduced to compound Il and further to native enzyme when either veratryl alcohol or the p-0-4 dimer was supplied as electron donor and in both cases veratraldehyde appeared as the major product. At pH 6.0, the second-order rate constant for compound I1 formation was similar with either veratryl alcohol or the p-0-4 dimer (6.7 X lo3 and 6.5 X 103 M-' s-', respectively). At pH 3.0 formation of compound I1 with either reductant proceeded so rapidly that determination of the respective rate constants was not possible. The results point to identical catalytic cycles of L3 with veratryl alcohol or the p-0-4 dimer involving both compounds I and I1 as intermediates and participation of the same veratryl alcohol radical as the most appropriate reductant for compound 11. Chemical evidence of such a radical, formed after the initial Lip-catalysed one-electron oxidation of p-0-4 dimeric lignin models, is presented in a separate article [Lundell, T., Schoemaker, H., Hatakka, A. & Brunow, G. (1993) Holgorschung, in the press]. The catalytic redox-cycle and the oxidation mechanism presented here reconcile seemingly contradictory results obtained in previous studies on Lip kinetics during the last decade.
40 M>a and PI 3.5, and contains one iron protoporphyrin IX as prosthetic group [3, 41. Resonance Raman spectra of native Lip indicate a high-spin ferric iron, either in the hexa[5] or penta-coordinate [6] state, depending on temperature [7]. The catalytic cycle, similar to that of horseradish peroxiCorrespondence to T. Lundell, Department of Applied Chemistry and Microbiology, Division of Microbiology, P.O. Box 27, dase (HRP), involves reaction with H,O, and both compound I and compound I1 can be detected [8-lo]. SF-00014, University of Helsinki, Finland Pre-steady-state studies have shown that the second-order F a : +358 0 708 5212. Abbreviations. Lip, lignin peroxidase, ligninase ; MnP, manga- rate constant for the oxidation of Lip from P. chrysosponum nese peroxidase ; HRP, horseradish peroxidase ; ClP, chloroperoxi- to compound I by H,O, is 4.2-6.5 X lo5 M-' s-I and this dase ; 8-0-4dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxypheno- reaction is not affected by pH [ll-131. Only at very low xy)propane-l,3-diol; compounds 1 and 11, see Fig. 1. pHcan an acid-base group (pK, about 1) be identified as Enzymes. Lignin peroxidase, Mn(II) peroxidase, horseradish per- being involved in the binding of H,O, and formation of comoxidase (donor :hydrogen peroxide oxidoreductase) (EC 1.I 1.1.7) ; chloroperoxidase (chloride :hydrogen peroxide oxidoreductase) (EC pound I [14]. The subsequent reduction of Lip compound I 1.11.1.10) ; laccase (benzenediol:oxygen oxidoreductase) (EC to I1 is strongly pH-dependent when veratryl alcohol is used as electron donor [12, 131. However, this reaction and further 1.10.3.2). Lignin peroxidase (Lip, ligninase) is an extracellular lignin-degrading enzyme, first discovered in the white-rot fungus Phanerochaete chrysosporium Burds. [I, 21. The enzyme is a glycoprotein that typically has molecular mass of
392 reduction of compound I1 back to native enzyme by veratryl alcohol are not well understood, even though several investigations have been carried out [9, 10, 12, 131 and the secondorder m e constants for separate reductions of Lip compounds I and I1 by veratryl alcohol have been determined by Wariishi et al. as 2.2 X lo6 M-' s-' and 1.6 X lo5 M-' s-', respectively (pH 3) [15]. In the presence of excess H202, compound 111 [lo, 16-18], the catalytically less active species of enzyme, is readily formed. Recently Cai and Tien reported that Lip compound I11 is converted to the resting ferric enzyme via spontaneous decay or consorted action of H,O, and a reducing substrate, such as veratryl alcohol [19]. Lip is able to oxidise a wide variety of substrates, the most important of which are aromatic compounds that are structurally related to lignin [20, 211. Model compounds of arylglycerol p-aryl ether (p-0-4 linkage) and diarylpropane (p-1 linkage) types represent the most common subunit linkages in lignin polymer [22] and in these compounds LIP from P. chrysosporium cleaves the p-0-4 and Ccx-Cp bonds leading to their fragmentation into the corresponding aromatic monomers [l-3, 23-25]. Analogous results have been obtained by using intact cultures of P. chrysosporium [26, 271 and Phlebiu rudiutu [28], implying a primary role of LiP in lignin degradation by these fungi. Depolymerisation of methylated lignins [l] and synthetic lignin [29, 301 by Lip has been observed, but apparent polymerisation of various lignins by Lip has also been reported [31], which points to a complicated mechanism involving both depolymerisation by the enzyme and uncontrolled polymerisation of radical intermediates thus formed [30, 321. Lip from P. chrysosporium Burds. is represented by a family of isozymes, and six [33] or up to fifteen [34] distinct Lips have been isolated, depending on culture conditions. Thus far, most kinetic investigations have been carried out using the Lip isozyme H8 [33]. In addition to Lip, a second heme-peroxidase is produced during ligninolysis, the manganese-dependent or manganese peroxidase (MnP) [35, 361, sonamed because of its dependence on both H,O, and manganese for catalysis. Lips have also been purified and characterised from other white-rot fungi, such as Phlebiu rudiutu [37, 381, Phlebiu (Merulius) tremellosu [38] and Coriolus (Trumetes) versicolor [391. P. rudiutu 79 is a selective and efficient lignindegrader [40] secreting ligninolytic enzymes under conditions similar to those of P. chrysosporium [41, 42). However, the ligninolytic system of P. rudiutu differs from that of P. chrysosporium since, in addition to Lip and MnP, P. rudiutu also produces laccase [42-441. Three Lip isozymes from P. rudiutu have been isolated [37] and shown to be coded by different genes [45]. Isozyme L3 is the major extracellular enzyme expressed, in particular, in growth media containing /I-0-4 dimers or lignocellulose [44] or Kraft bleach effluent [46], which demonstrates that L3 obviously is essential for the fungus to accomplish mineralisation of lignin. The H,O,-dependent oxidation of various aromatic compounds, such as veratryl alcohol and /3-0-4 dimers, has been studied [47, 481 ; these results reveal general similarities but differences in the formation of end-products by L3 from P. rudiuru 147, 481 and Lip from P. chrysosporium 13, 20, 251. In this study we report on the spectral properties of Lip isozyme L3, from the white-rot fungus P. radium strain 79, and present kinetic results obtained with two different aromatic compounds, veratryl alcohol and the more lignin-like non-phenolic /3-0-4 dimer 1-(3,4-dimethoxypheny1)-2-(2methoxyphenoxy)propane-1,3-diol. Veratryl alcohol is both a
substrate for Lip and a secondary metabolite produced de novo by several white-rot fungi such as P. chrysosporium [49] and P. rudiutu [43] ; it has been used in previous kinetic investigations of Lip [9, 12, 13, 15, 16, 181. It should be pointed out that in itself the net two-electron oxidation of veratryl alcohol to form veratraldehyde, a reaction involving the cleavage of two C-H bonds, cannot be considered as a model for lignin depolymerisation. The latter typically involves C-C bond or C - 0 ether bond cleavages. The spectral and kinetic properties of L3 show that the enzyme resembles the Lip from P. chrysosporium to a high degree. However, the results on the oxidation of veratryl alcohol by P. rudiutu Lip L3 are significantly different from those recently obtained with P. chrysosporium Lip by Wariishi et al. [15]. Moreover, this is the first study in which a real lignin model, the p-0-4 dimer, is used to elucidate the pre-steadystate kinetics of LiE? Here, we show that both veratryI alcohol and the /3-0-4 dimer are oxidised by L3 via the same mechanism probably involving, in both cases, participation of the veratryl alcohol radical as reductant for compound 11. We also describe a novel Lip-catalysed Ca-Cp cleavage of the p0 - 4 dimer, which is a model for depolymerisation of lignin. The latter is explained in more detail in a separate article [50]. In this study novel mechanisms for both the oxidation of veratryl alcohol and the oxidation and subsequent C-C bond cleavage of the /3-0-4 dimer by Lip are presented.
MATERIALS AND METHODS Chemicals Veratryl (3P-dimethoxybenzyl) alcohol was purchased from Fluka. In some experiments vacuum-distilled veratryl alcohol (originally from Aldrich) was used to exclude the possibility that some observations are due to the phenolic impurity present in trace amounts in the commercial preparations [9]. The non-phenolic /3-0-4 dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol, was synthesized from veratraldehyde (3,4-dimethoxybenzaldehyde) (Fluka) according to the method of Nakatsubo et al. [51]. The purity of veratryl alcohol and the dimer was checked by HPLC [43]. H,O, (30%, Suprapur) was from Merck and concentrations of the freshly made stock solutions were determined spectrophotometrically using E~~~ nm = 43.6 M-' cm-' 1.521. Other chemicals were of reagent grade and used as such. H,O was filtered and deionised by leading it through Elgastad (Elgagroup) and a Milli-Q (Millipore) water-purification system. This water was used throughout the study. Veratryl alcohol was dissolved in water to obtain a 10 mM stock solution. The dimer was first dissolved in 2 % by vol. dimethylformamide (Merck) and then further diluted in water to a 10 mM stock solution. Production and purification of L3 Phlebiu rudiatu Fr. strain 79 (ATCC 64658) was isolated at the Department of Microbiology, University of Helsinki [40]. The fungus was cultivated in 2-1 bioreactors at 28 "C immobilised onto a polypropylene carrier [42, 461 in lownitrogen asparaginefammonium nitrate/dimethylsuccinate medium at pH 4.5 [401 containing 56 mM glucose and 0.05 % (massfvol.) Tween 80 [42, 461. Veratric (3,4-dimethoxybenzoic) acid (1.0 mM) was added to enhance production of ligninolytic enzymes [43]. When production of LIP achieved a permanent high level, as determined by oxidation of veratryl
393 alcohol in the presence of H,O, at pH 3.0 [3], the extracellular culture liquor was harvested. Extracellular enzymes were purified from 4.7 1 culture liquor as 13-times concentrated (with a Filtron Omega casette, 10-kDa cut-off) by anion-exchange chromatography on Sepharose-Q Fast How (Pharmacia LKB Biotechnology) at pH5.5 [37] in which L3 is eluted as the last hemoprotein. The pooled L3 fractions in 25 mM sodium acetate pH 5.5 were concentrated 20-fold (with 10-kDa Filtron Microsep concentrators) to 2 ml. This preparation was characterised by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and isoelectric focusing [44] and it appeared as a homogenous 43-44-kDa protein with a PI value of 3.2. Specific activity was 890 nkat/mg protein in the H,O,-dependent oxidation of veratryl alcohol. MnP activity was not detected when using the phenol red method [43] both with and without Mn(I1). Protein concentration was determined by the method of Bradford [53] using a Bio-Rad reagence and bovine serum albumin as reference and from the absorbance at 280 nm [54]. The R, (A4n9/A28n)value was 4.5. Based on the protein concentration, an absorption coefficient at 407 nm of 129 d-' cm-' was derived for the native enzyme (see Results).
Spectra of L3 oxidation states A Hewlett-Packard 8452A photodiode-array spectrophotometer was used to record optical absorption spectra of the oxidation states of L3, and from these spectra the most suitable wavelengths for stopped-flow experiments were selected. The experiments were carried out at room temperature in 20 mM potassium phosphate pH 6.0 with 4 pM L3.
compound I generated by H,O, since, after several trials, we noticed that compound I1 was formed rapidly prior to mixing with the electron donor. This was most probably due to the contact of compound I with remaining electron donor adsorbed on the walls of the stopped-flow apparatus. Reduction of compound I1 to native enzyme at pH3.0 was determined from the decrease of absorbance at 426 nm. This wavelength was selected since at 426 nm the difference of absorbance is most distinct between compound I1 and native enzyme (see Results, Figs 1 and 5). Veratryl alcohol was used as reductant in concentrations ranging over 0.025 4 mM. L3 compound I1 was generated in one stopped-flow syringe by adding 3 mol H,O,/mol native L3 (4 pM) in water and after a 5-min incubation time, its contents were mixed with that of the other syringe containing veratryl alcohol in buffer. Formation of compound 11in water by H,O, was carefully checked by scanning the absorption spectrum of L3 (350-650 nm) with the Hewlett-Packard photodiode-array spectrophotometer prior to the stopped-flow analysis. L3 was completely converted to compound 11 5 min after addition of 3 mol H,O,/mol and it was stable for up to 10 min in the absence of additional reductants (here veratryl alcohol).
Rapid scans of the catalytic cycle of L3 To detect the oxidation states of L3 during catalysis at pH 3.0, the optical absorption spectra (380-480 nm, 10-ms or 110-ms intervals) were rapidly recorded by using stoppedflow techniques and the Union-Giken rapid-scan spectrophotometer. In addition, the formation of oxidation products from veratryl alcohol and the dimer were monitored by scanning the absorption spectra (250-650 nm, 10-s intervals) using the photodiode-array spectrophotometer.
Stopped-flow traces at single wavelength A Union-Giken RA-401 stopped-flow, rapid-scan spectrophotometer (optical path length 10 mm, wavelength accuracy better than 1 nm) was used to determine the rates of formation of compounds I and I1 at 20°C. The pseudo-firstorder reaction rate, k(ohs),was calculated from three averaged traces after first-order curve fitting by the standard software of the apparatus. The k(oba)was plotted against substrate concentration and, from the slope of the line, the second-order rate constant k, was determined. The second-order rate constant for compound I formation by H,O, was determined at pH 3.0 and pH 6.0 in a 10 mM potassium phosphate at 399 nm, which is the isosbestic point between compounds I and I1 and where the difference of absorbance between the native enzyme and compound I is most distinct (see Results). Four different concentrations (10, 15,20,25 pM) of H,O, were used and 1 pM L3. All concentrations mentioned in the text are those after mixing and the ionic strength of the buffer was kept constant in all stoppedflow experiments. One syringe contained H,O, in water and the other contained enzyme in buffer. Formation of compound I1 in the presence of H,O, was monitored by using two different electron donors, that is veratryl alcohol and the p-0-4 dimer. The second-order rate constants were determined at 420nm, which is near the maximum of absorbance of compound I1 (see Results) and has been used previously to determine Lip compound I1 formation [13], by using concentrations of 15, 25, 50, 75, 100 and 150 yM of the electron donor, 100 yM H,O, and 2 yM L3. One syringe contained enzyme in buffer, the other the reductant and H,O, in water. It was not possible to start with
Determination of steady-state kinetic constants Steady-state kinetic experiments were carried out using Michaelis-Menten assumptions, i.e. at a low concentration of enzyme and the concentration of one substrate was kept constant whereas the concentration of the other was varied. Under these conditions the Lip reactions show saturation with the respective substrate (see for instance [9, 331). The K , and k,,, for H,O, were determined with 40 nM L3, 1 mM veratryl alcohol and 12 different concentrations (1 -50 pM) of H,O,. The kinetic constants for veratryl alcohol and the dimer were obtained with 40 nM L3, 200 pM HZ02,and concentrations of the electron donors varied from 2-500 pM. Duplicate experiments with parallel samples were carried out. The reactions were initiated by addition of H,O,. Catalytic activity was monitored as formation of veratraldehyde at 310 nm [3] (for the dimer, see Results) at 25 "C using a Shimadzu 160A spectrophotometer. The second-order rate constant for each substrate was determined from the slope of double-reciprocal plots and the true k,, and K,,, values were calculated [55].
HPLC analysis Formation of veratraldehyde and other products from veratryl alcohol or the dimer by L3 were analysed by using a Nova-Pak C,, column (WatersMillipore) and a HewlettPackard 1090 M high-performance liquid chromatograph employing both a photodiode array and a fluorescence detector [43]. The compounds eluted were identified based on both the retention times and ultraviolet spectra of authentic refer-
394 0.06
3
o.6
0.04
0.02 7
350
I
I
I
400 Wavelength (nm)
450
I
0
Wavelength 550 (nm)
650
Fig. 1. Absorption spectra of the oxidation states of L3. (a) Native enzyme (4 pM) in 20 mM potassium phosphate pH 6.0; (b) compound I, 2 s after addition of 10 pM H,O,; (c) compound 11, 7 min after addition of H,O,. Table 1. Oxidation states of the lignin peroxidase L3 from Phlebia radiata and of other peroxidases characterisedby their absorption spectral maxima. Peroxidase
, ,a
of compound I
compound I1
407,503, 635
403, 550,607, 650
421, 527, 554
407.6, 496, 630 408 406, 502,632 403,498,640 403, 515,542, 650
408, 550, 608, 650 401 407, 558, 617, 650 400, 577,622, 651 367, 545,610, 688
420, 525,556 420, 529,553 420, 528, 555 420, 527,554 438, 542,571
native enzyme nm Lip L3 Lip from P. chrysosporium: [I01 [I31 MnP from P. chrysosporium [65] HRP [56] C1P [57, 66, 671
ence compounds ; the end products were quantified using the external standard method [43].
RESULTS
Table 2. Molar absorption coefficients ( 8 ) at absorbance maxima of the oxidation states of L3. Absorption coefficients were determined from the photodiode-array scans and are based on the absorbance of native L3 at 407 nm. Oxidation state
Native enzyme The absorption spectrum (Fig. 1) of lignin peroxidase isozyme L3 from Phlebia radiata at resting state had a Soret maximum at 407nm and other maxima at 503nm and 635 nm, very similar to those of native Lip and MnP from Phanerochaete chrysosporium (Table 1). The absorbance maxima in the Soret region of native L3, P. chrysosporium Lip and MnP are the same within 1 nm. In the visible region the two other maxima of L3 differ by 5-7 nm from those of P. chrysosporium Lip and are nearer to those of MnP. An absorption coefficient for native L3 at 407 nm of 129 mM-' cm-' was calculated from this spectrum as based on the protein concentration (Table 2).
Formation of compounds I and I1 After 2 s following the addition of 2.5 mol H,O,/mol to L3, a rapid decrease of absorbance in the Soret region and shift of the maximum to 403 nm was observed (Fig. l), to-
Native enzyme
Compound I
Compound I1
An,
E
nm
mM-' cm-'
407 503 635 403 550 650 421 527 554
129 8.0 3.0 37 5.5 5.1 65 6.1 6.5
gether with the formation of a broad band around 550 nm and two weak shoulders at 607 nm and 650 nm. The spectrum is ascribed to that of compound I of L3 and resembles most the spectrum of compound I of P. chrysosporium Lip described by Harvey et al. [13] (Table 1). An absorption coefficient for
395 Table 3. Pre-steady-state kinetic constants of L3. The secondorder rate constants were determined by using stopped-flow techniques. Compound I formation by H,O, was monitored at 399 nm, and compound I1 formation by veratryl alcohol or the 8-0-4 dimer was monitored at 420 nm. For more details, see Materials and Methods. Substrate
PH
Second-order rate constant M-I
HZ02 Veratryl alcohol 8-0-4 dimer
300 ms
s-l
6.30 2 0.33 X lo5 6.14 2 0.07 X lo5 fast 6.68 2 0.76 X 103 fast 6.55 2 0.25 X lo3
3.0 6.0 3.0 6.0 3.0 6.0
Table 4. Steady-statekinetic constants of L3. The constants were
10 ms
Time (ms)
Fig. 2. Formation of L3 compounds I and I1 followed at 408 nm and 420 nm, respectively, by stopped-flow techniques. 2 pM L3 in 10 mM (final concentration) potassium phosphate pH 6.0 was mixed with 100 pM veratryl alcohol and 100 pM H,O, in water.
determined by measuring the rate of formation of veratraldehyde at 310 nm [3] at pH 3.0. For more details, see Materials and Methods. Substrate
Km
HzOz Veratryl alcohol
VM 11 2 0.2 149 2 1.5
8-0-4 dimer
192 2 1.9
k,,
kc.&
s-'
M-l
3.7 ? 0.06 7.1 2 0.07 4.7 2 0.05
3.56 2 0.07 X lo5 4.76 2 0.05 X 104 2.48 2 0.04 X 104
s-l
the Soret band, of 37 mh-' cm-' (Table 2), was calculated based on the estimated absorption coefficient of the native enzyme. An isosbestic point between native enzyme and compound I appeared at 430 nm (Fig. 1). Conversion of compound I to I1 in the next 7 min was associated with formation of a peak at 421 nm and an isosbestic point at 399 nm (Fig. 1). An isosbestic point between native enzyme and compound I1 was observed at 418nm. The absorbance maxima of L3 compound I1 were very similar to those of P. chrysosporium Lip, MnP and HRP (Table l), differing by only a few nanometers. However, compound I of L3 was stable for several minutes (estimated tln 3.5 min) under these conditions and only slowly converted to compound II.
observed, followed by a slower increase (Fig. 2b). The rapid first phase corresponds to initial oxidation of the native enzyme by H,O, to compound I as also can be seen from the fast decrease of absorbance at 408 nm (Fig. 2a). The amount of compound I1 seemed to be stable for several seconds under these conditions. Similar changes in absorbance at 420nm were recorded when the 8-0-4 dimer was used as an electron donor (data not shown). The pseudo-first-order rate constants kobs for compound I1 formation were determined from the increase of absorbance at 420 nm. From a plot of these rate constants versus the concentration of veratryl alcohol or the 8-0-4 dimer the second-order rate constants were calculated and these are listed in Table 3. In both cases the kobswas linearly dependent on the concentration of the reductant. The second-order rate constants for veratryl alcohol and the 8-0-4 dimer were similar at pH 6.0. These rates are 10, times slower than that of compound I formation by H,O,. At pH 3.0 it was not possible to evaluate the reaction rates for compound I1 formation at 420 nrn for either of the two electron donors. This was due to the lack of kinetic separation between formation of compounds I and I1 (see Discussion) and a significant decrease in the amount of compound I1 (Fig. 4a). Reduction of L3 compound I1 back to native enzyme, when monitored as the decrease of absorbance at 426nm, was not clearly dependent on the concentration of veratryl alcohol. Although the variance in the data was considerable, the k,,, showed a slight linear dependence on the veratryl alcohol concentration (data not shown). However, the fistorder rates were slow, 3-6 s-', and with the concentrations of veratryl alcohol that were used in the other experiments (under 300 pM), the rate was about 3 s P .
Kinetic constants for formation of compounds I and I1 The second-order rate constants for the H,O,-dependent formation of compound I were determined at the isosbestic point for conversion of compound I to I1 at 399 nm. Formation of compound I was linearly dependent on the concentration of H,O,. However, the second-order rate constant for compound I formation was pH-independent in the pH range used in this work, with values of 6.3 X lo5M-ls-' and L3 intermediates formed during catalysis 6.1 X 105 M-' s-' at pH 3.0 and 6.0, respectively (Table 3). These values are in good agreement with the second-order Fig. 3 shows time courses of the events after mixing the rate constant of H,O, binding as determined from the steady- enzyme with 100 pM veratryl alcohol or the dimer and sevstate kinetic data at pH 3.0 (Table 4). eral low concentrations of H,O, at pH 3.0. These turn-over In the next set of experiments the absorbance changes experiments were monitored at 408 nm to investigate comwere monitored at 420 nm to determine the second-order rate pounds I, I1 and native enzyme. At first there was a rapid constants for compound I1 formation by the aromatic com- decrease in absorbance corresponding to the formation of pounds veratryl alcohol and the 8-0-4 dimer. Upon mixing compounds I and 11, followed by a sigmoidal increase in absorbance indicating reduction of compound I1 to native enthe enzyme with 100 pM veratryl alcohol and 100 pM H,O, at pH 6.0, a very rapid decrease of absorbance at 420 nm was zyme. When the concentration of H,O, was raised, the de-
396
1
I
I
I
I
0
10
20
30
40
I
50
60
p -0-4
0
I
70 380
Time (s)
400
420
440
460
10
Wavelength (nm)
dimer
I
I
10
20
I
I
30 40 Time (s)
I
I
I
50
60
70
Fig. 3. Effect of different H,O, concentrations on the conversion of L3 to resting state by reductants through formation of compounds I and 11. The reactions were followed at 408 nm and 2 pM L3 in 10 mM (final concentration) potassium phosphate pH 3.0 was mixed with (a) 100 pM veratryl alcohol or (b) 100 pM 8-0-4 dimer in water containing 5, 10 or 25 pM H,O,.
crease in absorbance was more prolonged with either aromatic compound present as reductant, but the subsequent rate of increase in absorbance was independent of the initial concentration of H202.This may simply reflect turn-over of the enzyme and, after consumption of H202, regeneration of native enzyme. The p-0-4 dimer yielded traces of a similar pattern (Fig. 3b) as veratryl alcohol (Fig. 3a) although the decrease of absorbance lasted longer, indicative of a slower turn-over for the dimer. The steady-state results are consistent with this observation: the k,,, for the p-0-4dimer is 66 % of that of veratryl alcohol at pH 3.0 and the second-order rate constants differ by a factor of two (Table 4). Moreover, with both veratryl alcohol and the dimer slow changes, first a small decrease and then an increase, in absorbance at 408 nm were detected in the late phase of the reactions, that is 50 s after mixing (Figs 3a,b). Fig. 4 shows rapid spectral scans of absorbance changes that occurred when the enzyme was mixed with H,O, and either veratryl alcohol or the dimer. In the first rapid scans at 10-ms intervals a shoulder was formed at 421 nm with an isosbestic point at 417 nm, which is very near to the isosbestic point between native enzyme and compound I1 at pH 6.0 (418 nm, Fig. 1). These changes correspond to the partial conversion of native enzyme to compound 11. On the subsequent slower time scale (110-ms scans), the absorbance of
I
380
I
I
400
420
I
440 Wavelength (nm)
I
460
4t
Fig. 4. Conversion of native L3 to compound I1 (a) with 100 pM veratryl alcohol or (b) with 100 pM p-0-4 dimer. In both cases 2 pM L3 in 10 mM (final concentration) potassium phosphate pH 3.0 was mixed with 15 pM H,O, and the reductant in water. Optical absorption spectra were recorded at 10-ms or 110-ms intervals.
the Soret band decreased more and the maximum changed to 421 nm (Fig. 4). These changes are consistent with the disappearance of native enzyme and generation of compound 11. However, in these traces a single isosbestic point is not observed, showing that more than two enzyme species were present. With the p-0-4 dimer similar changes were observed (Fig. 4b) as with veratryl alcohol (Fig. 4a), although the rates of the decrease of absorbance were slightly slower. Since compound I1 was detected as an intermediate in all spectra, we tried to elucidate its role in catalysis at pH 3.0 by using veratryl alcohol or the p-0-4 dimer in different concentrations with respect to the oxidant, H,O, . The reactions were followed by scanning the absorption spectrum from 250 nm to 650 nm at 1-10-s intervals. In the presence of H,O, L3 oxidises veratryl alcohol primarily to veratraldehyde at pH 3.0 [47] and under similar conditions, it cleaves the /3-04 dimer to veratraldehyde and other products [47, 481. Veratraldehyde has a distinct absorbance maximum at 310 nm (absorption coefficient 9300 M-' cm-I) [9] that is well separated from the Soret region of the enzyme, so it is possible to monitor changes both in the enzyme and in product formation simultaneously. When veratryl alcohol was used as electron donor the spectra showed formation of veratraldehyde (310 nm) and
397 a 0.20 310 nrn
0.15 W
0
z a
5m
0.10
v)
m
a
0.05
0
WAVELENGTH (nm)
b 0.20
p,;:
8-0-4 dirner
0.15 W
sa
m n 0 v)
m
0.10
a
0.05
0
300
400
500
600
WAVELENGTH (nrn)
Fig. 5. Intermediates and oxidation products detected upon turn-over of L3 in the presence of H,O, and (a) veratryl alcohol or (b) p-0-4 dimer. Optical absorption spectra were recorded at 1, 2, 12, 20, 30 and 75 s by using the photodiode-array spectrophotometer. In both cases, 2 pM L3 was mixed with 100 pM of the respective reductant and 10 pM H,O, in 10 mM potassium phosphate pH 3.0.
compound I1 (shoulder at 421 nm) (Fig. 5a). After a turnover period of 20 s, native enzyme was reformed as judged by the absorbance maximum at 407 nm. Table 5 shows that one equivalent of H,02 yielded one equivalent of veratraldehyde from veratryl alcohol. This was also supported by experiments carried out by using HPLC to quantify product formation and when 1OpM or 100 pM H,O, were used, 10 pM and 95 pM of veratraldehyde, respectively, were detected (Table 5). However, when an excess of 200 pM H20, was used, i.e. twice the concentration of veratryl alcohol, the absorption spectrum of L3 in the Soret region remained the same for 75 s (data not shown). The spectrum had a maximum at 41 8 nm and was different from that of L3 compound I1 or P. chrysosporium Lip compound 111 [lo, 16-19]. When the a-0-4 dimer was used as electron donor and mixed with H20z, the changes in absorbance in the Soret region were similar to those observed with veratryl alcohol. During turn-over, compound I1 was detected first and after
30 s the enzyme was reformed to the native state (Fig. 5b). However, significant differences in the spectra of the products originating from the p-0-4 dimer were observed when compared to the spectra that were recorded with veratryl alcohol even though, in both cases, the same unexpected peak appeared at 290 nm (Fig. 5a,b). In addition, the increase of absorbance at 310 nm was much less with the dimer (Fig. 5b) than with veratryl alcohol (Fig. 5a), indicating a lower yield of veratraldehyde formation (Table 5). HPLC analysis showed that formation of veratraldehyde did not occur stoichiometrically with respect to H,Oz, and when 10pM and 100 pM H,O, were used, 5 pM and 33 pM of veratraldehyde, respectively, were detected (Table 5). The amounts of veratraldehyde were less than 50 5% of those when veratryl alcohol was used as electron donor. When a twofold excess of H,O, compared to that of the dimer was used, a similar absorption spectrum of L3 was observed as with veratryl alcohol having a maximum at 418 nm (data not shown).
398 Table 5. Effect of various concentrations of H,O, on the turnover of veratryl alcohol or the /?-0-4 dimer by L3. The concentration of L3 was 2 pM in 10 mM potassium phosphate pH 3.0 and formation of veratraldehyde was calculated from the increase of absorbance at 310 nm observed after 75 s. To confirm that product formation was quantitative, 0.1 pM L3, the electron donor and H,O, were mixed in 20 mM sodium tartrate pH 3.0 and the end-products were analysed by using HPLC [43] after a 30-min reaction time (values with an asterisk). In both cases the experiments were carried out at 20 "C ; n.d., not determined.
Electron
Concentration of
donor
electron
H,O,
donor
added
veratraldehyde formed
100 100 1000 100 100
5 10 10 100 10
5 10 8 95 * 10*
100 1000 100 100
10 10 100 10
4 1 33 * 5*
L3 oxidation state after 75-s
PM
Veratryl alcohol
p-0-4
dimer
native enzyme native enzyme native enzyme
n.d. n.d. native enzyme native enzyme n.d. n.d.
DISCUSSION
isation of the cation radical in compound I from the porphyrin ring to the proximal histidine [59]. The similarity in the bleaching of the Soret band in L3, P. chrysosporium Lip and HRP upon oxidation by H,02 points to a similar cation radical arrangement in compound I of the enzymes. Furthermore, the absorption spectrum of compound I is similar in L3 and P. chrysosporium Lip, and a maximum is observed at 650nm in L3, P. chrysosporium Lip and MnP, and in HRP. Conversion of compound I to compound I1 leads to a shift of the Soret maximum to 421 nm, which is 1 nm higher than in P. chrysosporium Lip, MnP and HRP (Table 1).The position of the two additional maxima of compound I1 in the visible region (527 nm and 554 nm) are identical to those of HRP and within 2 nm of those of P. chrysosporium Lip. However, compound I of L3 was exceptionally stable at pH 6.0: the t1,2of the conversion of L3 compound I to I1 was estimated to be several minutes, whereas in P. chrysosporium Lip it is 1.0 min at the same pH [lo]. The shorter lifetime of compound I of P. chrysosporium Lip may be explained by the presence of an endogenous electron donor, which generates some compound I1 [13]. The reduction of compound I to I1 occurs in LIP either spontaneously [lo] or by H202[9]. The higher stability of L3 compound I may be due to the methods applied in the purification of the enzyme and the use of highly purified water for buffers and solutions, which obviously decreased the amount of exogenous reducing agents.
Oxidation states of L3
Kinetic constants
Absorbance spectra of L3 from P. rudiutu at different oxidation states indicate that the enzyme resembles the Lip (H8) from P. chrysosporium (see Table 1). At resting state, the Soret maxima of these two Lips differ by only 1nm (L3 407 nm; P. chrysosporium Lip 407.6 or 408 nm) and the absorption coefficient of L3, 129 mW' cm-I at 407 nm, is nearly the same as for P. chrysosporium Lip, 133mM-'cm-' at 407.6 nm [lo]. However, the two additional absorbance maxima of native L3 are red shifted 5-7 nm in comparison to those of P. chrysosporium Lip. The absorbance maxima of native L3 and P. chrysosporium MnP are very similar. The similarity of the absorption spectra of L3 and P. chrysosporium Lip and MnP, bearing only minor differences, suggests that the prosthetic group, which in P. chrysosporium Lip is iron protoporphyrin IX (heme) in the high-spin ferric state [S, 61, is the same in all these enzymes. Upon oxidation by H20, and formation of compound I, L3 shows a similar rapid bleaching of Soret band as occurs in P. chrysosporium Lip [9-11, 131. Compound I of L3 showed a broad, flat band in the Soret region absorbing around 403nm and having an absorption coefficient of 37 mM-' cm-', which is lower than that of P. chrysosporium Lip (55.0 mM-' cm-') at 408 nm [lo]. Compound I of P. chrysosporium has been reported to have an absorbance maximum at 408 nm [9, 101, though Harvey et al. [13] showed that the maximum is positioned at 401 nm (see Table 1) and the absorbance is low in the Soret region. In HRP the absorbance maximum shifts to 400 nm upon compound I formation [56] and in C1P (chloroperoxidase), down to 367 nm [57]. Dolphin et al. [S8] have demonstrated a Fe(IV)=O porphyrin n-cation radical in HRP compound I after two-electron oxidation, which has also been proposed for the compound I of P. chrysosporium Lip [lo]. However, 'H-NMR studies of HRP have provided evidence for delocal-
The lack of pH dependence in the formation of L3 compound I is consistent with the results obtained with P. chrysosporium Lip [ll-131. In HRP and other histidine peroxidases formation of compound I is affected by pH [56] ; the acid-base group that controls their catalytic activity may be the distal histidine [60]. Recently, Cai and Tien [14] showed that, in P. chrysosporium Lip, an acidic ionisable group (pK, about 1) participates in catalysis. This group is deprotonated in Lip and it accounts for the low pH optimum of the enzyme [14]. The second-order rate constant for L3 compound I formation by Hz02,6.1 -6.3 X lo5M-' s-', is in the same range as the values reported with P. chrysosporium LIP, 5.4 X lo5 M-' s-' [ l l ] and 4.2 X lo5 M-' s-' [13]. These rates are several orders of magnitude lower than those of other peroxidases, which may, as suggested by Andrawis et al. [ l l ] , be a reflection of either steric constraints in the active center or of a two-step reaction mechanism upon binding of H202. The rate of generation of L3 compound I1 by subsequent action of H,02 and a reducing compound, either veratryl alcohol or the p-0-4 dimer, was clearly enhanced when the pH was decreased. This is analogous to formation of P. chrysosporzum Lip compound I1 by veratryl alcohol [9, 12, 131. A biphasic curve was observed at 420 nm, in which a rapid decrease of absorbance corresponding to compound I formation, was followed by an exponential increase that, at pH 6.0, reached a steady level corresponding to compound 11 formation. The second-order rate constants of formation of L3 compound I1 at pH 6.0 by either veratryl alcohol or the p-04 dimer, 6.7 and 6.5 X lo3 M-' s-', respectively, are similar to the values reported for reduction of P. chrysosporium Lip compound I by veratryl alcohol (7.0 X lo3M-' s-' [13], =lo4 M-' s-' [12]) at the same pH. At low pH, pH 3.0, formation and disappearance of L3 compound I and formation
399 of compound I1 proceeded so rapidly at 420nm with both veratryl alcohol and the dimer that it was not possible to separate kinetically these processes or identify compound I1 as a steady-state intermediate. Analysis of such a system of consecutive reactions with similar rate constants is, in general, not possible. A similar phenomenon has been observed by Harvey et al. [13] with P. chrysosporium LIP H8 and veratryl alcohol. However, a second-order rate constant of 2.5 X lo6M-' s-' for formation of P. chrysosporium Lip compound I1 by veratryl alcohol at pH 3.0 has been estimated [12] and recently, starting from compound I, Wariishi et al. [15] found two enzyme-derived acid dissociations for this reaction and calculated the respective pH-independent second-order rate constants (1.29 and 4.6 X lo6M-' s-I).
Catalytic cycle with veratryl alcohol Even though the pre-steady-state and steady-state kinetics of P. chrysosporium Lip with veratryl alcohol have been intensively studied, it is not clear whether the oxidation of veratryl alcohol occurs via one-electron oxidation yielding veratryl alcohol cation radicals, as suggested by Schoemaker et al. [61] and Renganathan and Gold [lo], or via one direct two-electron oxidation yielding veratraldehyde, as suggested by Tien et al. [9]. In the first case two veratryl alcohol cation radicals would be produced in each catalytic cycle and after diffusing from the active center of Lip, they could undergo non-enzymic reactions yielding veratraldehyde and other products [21]. It has been proposed that veratryl alcohol cation radical may also act as a charge-transfer mediator in lignin breakdown [62]. The reduction of Lip compound I to I1 by veratryl alcohol occurs readily at low pH, as was also observed in this study. Steady-state kinetics may provide estimates for overall turnover; in this study, a k,,, of 7.1 s-' and a second-order rate constant of 4.8 X lo4M-' s-' for veratraldehyde formation at pH 3 .O were determined. Veratraldehyde accumulated in a stoichiometric relationship with the amount of H,O, supplied. Upon turn-over and in the presence of H,O, and excess concentrations of veratryl alcohol, compound 11 was only present as an intermediate, and the end-products were the native enzyme and veratraldehyde. The observation that reductions of compounds I and I1 by veratryl alcohol at low pH cannot be separated kinetically has been proposed as an indication of the formation of an intermediate veratryl alcohol cation radical-compound I1 complex [13], which would then enhance the reduction by another veratryl alcohol molecule leading to regeneration of native enzyme. Our results also point to oxidation pathways of veratryl alcohol consistent with this explanation. We propose that L3 compound I oxidises veratryl alcohol to veratryl alcohol cation radical which (a) either stays in the active center or (b) following proton abstraction from the Ca, the veratryl alcohol radical thus formed acts as electron donor for compound II. The possibility that veratryl alcohol radical reduces Lip compound I1 has been previously suggested by Schoemaker [21]. Thus, in (b) one veratraldehyde molecule is producedcatalytic cycle involving two separate one-electron oxidation steps. This mechanism is supported by the stoichiometric relationship of veratraldehyde formation by Lip with H,O, supplied which was observed in this work. Even though the spectral changes of L3 at steady-state and the kinetic constants calculated here for veratryl alcohol do not allow us to determine between the two mechanisms, (a) or (b), the analogy in the oxidation of veratryl alcohol and the
p-0-4 dimer by L3, as discussed later, suggests that (b) is more likely to operate. Previously Marquez et al. [12] reported pH-dependent rate constants for reduction of P. chrysosporium Lip compound I1 to native state (1.6 X lo5M-' s-' at pH 3.06) by veratryl alcohol but these values were calculated indirectly from the second part of biphasic curves observed at 426 nm. Recently, using P. chrysosporium Lip compound 11, which was made by oxidising native enzyme with m-chloroperoxybenzoic acid and ferrocyanide, Wariishi et al. [15] observed saturation kinetics in the reduction of compound I1 by veratry1 alcohol. They obtained first-order reaction rates up to 30 s-' (1mM veratryl alcohol) and estimated second-order rate constants (1.6 X lo5M-' s-' at pH 3.05) for the conversion of compound I1 to native enzyme. However, because the experimental conditions are different, their results may describe only a special case of Lip catalysis. We measured much slower first-order rates (kObs= 3 s-') when compound I1 of L3 was generated by adding H,O, to the native enzyme and observed a slight, more linear than saturating, dependence on veratryl alcohol concentration in the reduction of compound I1 back to native enzyme, indicating the improbability of this reaction to occur in the fast redox-cycle of Lip to L3.
Degradation mechanism of the p-0-4 dimer We have recently shown that the true mechanism for the Lip-catalysed Ca-Cp cleavage in p-0-4 dimers [50] is different from the, thus far, generally accepted mechanism which was based on labelling studies with p-1 lignin model compounds [24, 50, 611. One-electron oxidation of the p-1 dimer results in Ca-Cp cleavage to form a Ca-protonated benzaldehyde (or protonated veratraldehyde when 3,4-dimethoxysubstituted) and a benzylic radical at the P-position. However, different results have been obtained in labelling studies with p-0-4 dimers (see [50]). We demonstrate that the Lip-catalysed Ca-Cp cleavage in the p-0-4 dimeric lignin model compound proceeds via a cation radical center localised at the Cp-ether oxygen [63] and, after electron rearrangement, a fragmentation of the dimer occurs in which the Ca-Cp bond is cleaved to form a veratryl alcohol radical and a positively charged Cp-moiety [50].The same veratryl alcohol radical would also be an intermediate in the Lip-catalysed oxidation of veratryl alcohol (see before). The formation of the identical radical intermediate from both veratryl alcohol and the p-0-4dimer is supported by the corresponding values of the kinetic constants and by the formation of the same spectral intermediates of the enzyme during catalysis. We therefore propose that, in the catalytic cycle of L3, veratryl alcohol radical will act as one-electron donor and reduce compound II to native enzyme (see Fig. 6). The second part of the dimer, after Ca-Cp cleavage, is left as a Cp cation which would easily be attacked by a nucleophile, such as HzO, and undergo further fragmentation to guaiacol (2-methoxyphenol) and glycolaldehyde [3]. Guaiacol has been detected as a product from this dimer after oxidation by L3 [48] and by P. chrysosporium Lip H8 [25]. Furthermore, LIP oxidises guaiacol in the presence of H,O, [64] apparently to phenoxy radicals which is a one-electron oxidation. However, in this process the catalytic cycle of LiP is inhibited [64]. The formation of guaiacol from the p-0-4 dimer may still explain why less veratraldehyde is generated from it than from veratryl alcohol, even though the kinetic
400 REFERENCES
/
Compound II
I
FHOH
OMe
Fig. 6. Proposed catalytic cycle of Lip L3 with the p-0-4 dimer.
constants and the reduction of L3 back to native enzyme are so similar with both compounds. If the /3-0-4 dimer could be considered as a two-electron donor and if, following Cu-Cp cleavage, an additional one-electron donor, guaiacol, were formed, less of the dimer would be oxidised compared to veratryl alcohol by L3 upon turn-over. This is consistent with the lower second-order rate constant of the dimer obtained from steady-state experiments (Table 4). Our results show a high degree of similarity in the catalysis of L3 using veratryl alcohol or the /3-0-4 dimer as electron donors, which indicates that the binding site and mechanism in the enzyme is identical for both compounds. This is supported by the turn-over experiments, in which the same sequence of events was observed with both reductants, and by the corresponding reaction rates obtained under presteady-state or steady-state conditions. Because veratryl alcohol and the p-0-4 dimer are substantially different in size and the structure of the heme active site of L3 is apparently closed as well as in P. chrysosporium Lip [45], the binding site may be on the surface of the protein. However, the yield and nature of oxidation products are not completely identical, pointing also to differences in the reducing capacities of veratryl alcohol and the dimer. The novel Lip-catalysed C-C bond cleavage of the 8-0-4 dimer [50], which obviously operated under these experimental conditions, will certainly enable us to probe the real mechanism of depolymerisation of the lignin polymer by Lip. This study was partly presented at The 5th International Conference on Biotechnology in the Pulp and Paper Industry, May 27-30, 1992, in Kyoto, Japan. The skilful technical assistance of Mika Kalsi in the purification of L3 is kindly acknowledged. The study was supported by the Academy of Finland and by the Netherlands Foundation for Chemical Research (SON). We thank DSM (Geleen, The Netherlands) for their support; we gratefully acknowledge the gift by Hewlett-Packard, The Netherlands, of the 8452A photodiodearray spectrophotometer.
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