The Electron-Accepting Sites in Rhus vernicqera Laccase as Studied by Anaerobic Oxidation-Reduction Titrations

Transcription

1 Eur. J. Biochem. 18 (1971) The ElectronAccepting Sites in Rhus vernicqera Laccase as Studied by Anaerobic OxidationReduction Titrations Bengt R. M. REINHAMMAR and Tore I. VANNGBRD Institutionen for Biokemi, Goteborgs Universitet och Chalmers Tekniska Hogskola (Received September 24/November 26,1970) 1. Laccase from Rhus vernicifera was titrated under anaerobic conditions using quinol as the reducing substrate, and changes in the optical and electron paramagnetic resonance spectra were measured. 2. Titrations of native laccase as well as enzyme in the presence of the inhibitor, fluoride, demonstrated that four electrons were required to reduce completely the enzyme as determined by the measurements noted above. 3. The titrations of native laccase revealed three different types of electronaccepting sites in the molecule. Type 1 copper is associated with the strong absorption band at 614 nm and has a potential of 420 mv. Type 2 copper has the lowest potential, 390 mv, and could not be correlated with any of the resolvable absorption bands in the visible spectrum. The absorption band at 330 nm is suggested to arise from a twoelectron accepting chromophore with an oxidationreduction standard potential of 460 mv at ph In the presence of excess fluoride, the oxidationreduction potential of some sites was changed with the result that all sites appear to get the same potential. 5. The nature of the twoelectron accepting chromophore is discussed in terms of its properties, and these are consistent with the concept that it contains the two copper ions undectable by electron paramagnetic resonance as a cupriccupric pair in the oxidized enzyme. Laccase from the lacquer tree Rhus vernicifera is a coppercontaining oxidase which catalyzes the oxidation of various compounds, including diphenols, by molecular oxygen. Recent investigations in this laboratory [ 11 have indicated that the tree enzyme is very similar to the enzyme obtained from the fungus Polyporus versicolor. Thus, both proteins contain four copper atoms per molecule, but only two of these can be detected by electron paramagnetic resonance in the oxidized enzyme [1,2]. The detectable copper ions differ in their optical absorption and electron paramagnetic resonance spectra and are designated Type 1 and Type2 copper. For fungal laccase the other two copper ions are diamagnetic [3] and it has been suggested that these ions are divalent and form a binuclear complex with the electrons totally spinpaired [4]. Results from anaerobic oxidationreduction titrations of Rhus vernicifera laccase, presented in this communication, suggest that the enzyme molecule can take up four electrons during reduction. The four electrons are accepted by three different sites with oxidationreduction potentials between 390 mv and Enzyme. Laccase or pdiphenol : 0, oxidoreductase (EC ). 460 mv at ph 7.5. Two electrons are taken up by the Type 1 and Type 2 copper ions. The other two electrons probably enter in pairs into a twoelectron accepting site which is associated with a nearultraviolet absorption band. Part of these results have been presented in a review article [15]. MATERIALS AND METHODS Protein and Chemicals Laccase was prepared as described previously [5] from acetone powder obtained from the lacquer of Rhus vernicifera. The spectroscopic properties of the enzyme were identical to those published earlier [l]. All solutions were prepared from reagent grade chemicals and deionized distilled water. The concentration of quinol (obtained from May & Baker Ltd, Dagenham, England) was calculated, using a molar extinction coefficient of 475 Mlcml at 240 nm [6]. Electron Paramagnetic Resonance and Other Xpectral Measurements Electron paramagnetic resonance spectra were recorded in a Varian E3 spectrometer at about 9 GHz and at 77" K.

2 464 ElectronAccepting Sites in Tree Laccase Eur. J. Biochem. Optical spectra were recorded at 25" with either a Zeiss M4Q I1 or in a Cary model 15 recording spectrophotometer. Anaerobic Titration Technique For the anaerobic oxidationreduction titrations, optical spectra were measured in a cell assembly similar to the one shown in Fig. 1 of [4] but with no electron paramagnetic resonance tube attached. Quinol was added in portions corresponding to about half an electron equivalent, and after each addition the optical spectrum in the range 300 to 800 nm was recorded. When the enzyme had been completely reduced by four equivalents, it wa0 reoxidized by exposure to air and the optical spectrum was recorded again. Concomitant measurements of optical and electron paramagnetic resonance properties were made with an assembly consisting of an optical cell and an electron paramagnetic resonance tube and provided with a small Thunberg top instead of the gas chromatography injection gasket. The total volume of this cell was about 8 ml. In these titrations between one and four electron equivalents of quinol was added to the bulb of the Thunberg top and the system was made anaerobic. The titrant and enzyme solutions were mixed and when the absorbance at 614nm became constant the optical spectrum was recorded. The solution was transferred to the electron paramagnetic resonance tube, frozen in liquid nitrogen, and the electron paramagnetic resonance spectrum was recorded. The sample was thawed and mixed with air, refrozen, and the electron paramagnetic resonance spectrum was recorded in the same tube. Finally, optical measurements were made on the thawed enzyme. The increases in concentrations of the enzyme and substrate solutions due to evaporation during evacuation were 5 10 Ole. OxidationReduction Potential of Type 1 Copper The oxidationreduction potential at 25" of the 614 nm chromophore was determined by an anaerobic spectrophotometric titration method [7] using the oxidationreduction buffer ferroferricyanide. The concentration of laccase, with the 614 nm chromophore in the reduced and oxidized states, was determined spectrophotometrically at each point during the titration. An optical cell provided with a gas chromatography injection gasket was employed and anaerobic ferrocyanide solution was added to the anaerobic enzyme ferricyanide mixture with a Hamilton syringe. Appropriate corrections were made for the change in concentrations of the solutions during evacuation. Titrations were performed with native laccase in 88mM sodium phosphate buffer, ph 6.8, and with enzyme solutions containing 1OOmM sodium phosphate buffer, ph 7.5, and 10 mm sodium fluoride. The ionic strengths of the reaction mixtures containing equal amounts of ferrocyanide and ferricyanide were and 0.408, respectively. These ionic strengths correspond to oxidation reduction potentials for the ferroferricyanide system of 428 and 430 mv, respectively [8]. RESULTS OxidationReduction Potential of Type I Copper The results from an anaerobic oxidationreduction titration of native laccase with the ferroferricyanide system is shown in Fig. 1, upper line. In agreement with Nakamura's earlier report [7] there is a Linear relation between log ([ferricyanide]/[ferrocyanide]) and log ([blue laccase]/[leuco laccase]) with a slope close to unity. Most of the observed deviation from unity originates from the dependence of the ferroferricyanide potential on the ionic strength which varies during the titration. 0.6 r 'f oz T. 0 6 T I B 0.2 t 0.6' I ' I I ' I ' I I log [Fe(CN)g]/[Fe(CN)g] Fig. 1. Oxidationreduction potential of Type I wpper. Plot of log ([Type 1 Cu2+]/[Type 1 Cu+]) against log ([ferricyanide]/ [ferrocyanide]). The lines were obtained by the method of least squares. The slopes are 0.82 (upper line: native laccase) and 0.75 (lower line: fluoridetreated laccase). The anaerobic cell contained about 0.7 ml approximately 0.1 mm laccase and 0.1 ml 0.1 M ferricyanide in sodium phosphate buffer, ph 6.8 (upper line) or ph 7.5 plus 10 mm sodium fluoride (lower line). The cell was made anaerobic as described earlier [a]. An anaerobic solution of 0.1 M ferrocyanide in the same buffer was added in 25 pl portions up to 200 p1 with a Hamilton micro syringe. Corrections for the dilution of laccase during titrations were made. The amount of Type 1 Cu2+ was determined by measuring the optical absorption at 614 nm. A:l4 represents the corrected optical absorption at 614nm of the fully oxidized enzyme at the beginning of the titrations and A,,, stands for the corrected absorption at this wavelength during the titrations

3 Vol.18, N B. R. M. RE~HAMMAR and T. I. VANNQARD 466 The blue color has earlier been associated with the Type 1 Cu2+ [l] and thus the results show that this ion is reduced by one electron with no evidence for cooperativity involving other possible electron acceptors in the protein. Therefore, the oxidationreduction potential measured in this experiment can be ascribed to the Type 1 copper alone. When log ([Type 1 Cu2+]/ [Type 1 &+I) is equal to zero, the value of log ([ferricyanidel [ferrocyanide]) is With an oxidationreduction potential of 428 mv for the ferroferricyanide system [S], the potential for the Type 1 copper becomes 432 mv. This value is somewhat higher than the one reported by Nakamura [7]. However, his calculations were based on an oxidationreduction potential of 409 mv for the ferroferricyanide system. The potential of this system is very sensitive to the ionic strength [8] and for his determination a value of about 440 mv seems more realistic. Recalculation of his data gave an oxidationreduction potential of 432 mv for Type 1 copper, in agreement with our value. Nakamura [7] also found that the oxidationreduction potential of the blue copper in native laccase decreased by about 15 mv per ph unit between ph 3.4 end 9.5. From this phdependence an oxidationreduction potential for Type 1 copper of about 420 mv at ph 7.5 was calculated. Fig. 1, lower line, also shows the results from titrations of fluoridetreated laccase with the ferroferricyanide system. Apparently, the titration behavior of the Type 1 copper does not change significantly when fluoride is added, as compared to the results obtained with native laccase. Anaerobic Titration of Laccase at ph 7.5 In this series of experiments, enzyme was titrated with increasing amounts of quinol in the two types of ceu assemblies described above. In particular, the strong optical absorption bands at 614nm and 330nm and the electron paramagnetic resonance signal [l] were observed. On deoxygenation of the enzyme solutions, there was no change in these properties. The reaction between substrate and enzyme was completed within 5 min, except in the final atages of titration, when the enzyme had been reduced with three electron equivalents of quinol. When another half equivalent of quinol was added, the blue color disappeared slowly and became constant only after 15 to 20 min. A complete stepwise titration of laccase in the cell with the rubber gasket took about 1 h, while in the onepoint titrations in the Thunberg cell the reaction was completed within a few minutes. The results of these experiments are summarized in Fig. 2 where the symbols respresent the experimental values. Neither the absorption band at 614nm (Type 1 CU~+) nor Type 2 Cu2+ were reduced completely when two electron equivalents of quinol were I I ELectron equivalents added Pig. 2. Anaerobic optical and electron paramagnetic resonance titration of laccase at ph 7.5. The reaction mixture contained about 1 ml of approximately 0.1 mm laccase in 0.1 M sodium phosphate buffer, ph 7.5. Quinol in water was added in a concentration of about 1 mm. Spectra were recorded after each addition of titrant, using the buffer as the reference. Corrections have been made for the absorbance of the reduced enzyme and the quinone formed. Type 1 Cu2+ was estimated by reading the absorption at 614 nm. Type 2 Cu2+ was obtained by subtracting the amount of Type 1 Cu2+ from the total electron paramagnetic resonance intensities which was obtained by double integrations of electron paramagnetic resonance spectra. The presented values are mean values of results obtained from three stepbystep titrations in the cell with the rubber gasket and from ten onepoint titrations in the cell with the Thunberg t,op. 0, absorbance at 614 nm. 0, absorbance at 330 nm. A, Type 2 copper electron paramagnetic resonance intensity. Full lines represent the computed titration pattern of the three electron acceptors with the oxidationreduction potentials given in the Results added. Instead, the chromophore with an absorption band at 330 nm was almost fully reduced. The third equivalent of titrant reduced the 330 nm absorption band completely, while 30 /, of the Type 1 copper was still oxidized and Type 2 Cu2+ intensity was 55 to 65O/, of the original value. With four equivalents of quinol, Type 1 copper was completely reduced and the electron paramagnetic resonance intensity was almost zero. It was observed that the absorption band at 614 nm and the shoulder at 800 nm disappeared concomitantly. During the course ofthe titrations no new electron paramagnetic resonance signals or optical absorption bands appeared. Only a decrease in the intensities of the signals and of the optical bands was observed. When the cells were opened to air, the enzyme reoxidized within seconds. The absorption at 614 nm of the reoxidized enzyme was in most cases about 9501, of the original value. This decrease probably is due to slight denaturation of laccase during the titration procedure.

4 466 ElectronAccepting Sites in Tree Laccase Eur. J. Biochem. Although one of the cells used had a larger volume than the other cell and had a rubber gasket which might allow some leakage of oxygen, the titration data with the two cells were not significantly different. However, some leakage of air through the gasket was detected when the much slower substrate, ascorbate, was used. Complete stepwise reduction of laccase by this substrate usually required more than 2 h. Simulation of Titration of Laccase at ph 7.5 In an attempt to interpret the experimental data, a computer program was designed for simulating the anaerobic reductive titration curves. The program can be used for systems with up to five different electron acceptors which can take an arbitrary number of electrons each. All electron acceptors were assumed to be fully oxidized at the beginning of the titration and to occur at equal concentrations. The titrant can have any standard oxidationreduction potential and donate any number of electrons per molecule. For the simulation it was assumed that the Type 1 copper is a oneelectron acceptor associated with the absorption band at 614nm and with a potential of420 mv (cf. above). The best fit between computed and experimental values was achieved (see Fig. 2) with the following assumptions about the other electron acceptors : a) The chromophore with the absorption band at 330 nm is a twoelectron acceptor with an oxidationreduction potential of 480 mv (cf. below). b) Type 2 copper is a oneelectron acceptor with a potential of 390 mv. An even better demonstration that the 330nm chromophore is associated with a twoelectron acceptor rather than two oneelectron acceptors with similar potentials is given in Fig.3. This contains data from three titrations performed as in Fig. 2 but presented in a different fashion. The 330 nm absorption is plotted against the 610nm absorption in the form of a Nernst plot. Clearly most of the experimental points in this figure fall close to the full line, which is drawn with a slope of exactly2. As the blue color is due to a oneelectron acceptor (see above) it follows that the 330 nm chromophor takes electrons in pairs. This conclusion is independent of the way the reduction has occurred and is not impaired by the presence of oxygen leaks or other experimental difficulties. Titration of Laccase Treated by Excess Pluoride If laccase was incubated with excess of the inhibitor fluoride [9], the protein did not change the visible absorption spectrum but the electron para c OR T a.f" Y, I I I I Fig.3. Plot of log ([oxidized 330 nm chromophore]l[reduced 330 nm chromophore]) against log ([Type 1 Cu2+]/[Type 1 Cuf]). A&, and A:,* represent the corrected optical absorptions of the fully oxidized enzyme at the beginning of the titrations. A,,, and A,,, stand for the absorptions, corrected for the background absorption, at these bands during titrations. The values are taken from three titrations performed as in Fig.2. The full line is drawn with a slope of 2 magnetic resonance signal of the Type 2 copper was altered as described previously [I].Also, the anaerobic titration behavior of the enzyme was markedly changed. The results of such experiments are shown in Fig. 4. Contrary to the titration behavior of native laccase, all electron acceptors now titrate concomitantly and in an apparently linear fashion. However, as in the case of native laccase, four electron equivalents of titrant are required to reduce the enzyme as determined by the intensities of the visibie optical spectrum and the electron paramagnetic resonance signals. Also similar to native laccase, no new absorption bands appear in the optical and electron paramagnetic resonance spectra on deoxygenation of the enzyme solutions or during the titrations. Simulations of the titration curves obtained in the presence of fluoride were performed with the same program as above, and the results are shorn in Fig.4 as lines, while the symbols represent the experimental values. The following assumptions were used in the simulations : a) AU electron acceptor sites have the same oxidationreduction potential, much higher than that of the reductant.

5 Vol.18, N0.4, 1971 B. R. M. REINHAMMAR and T. I. VANNGARD 467 Electron equivalents added Fig.4. Anaerobic optical and electron paramagnetic resonance titrations of laccase in the presence of excess fluoride. The reaction mixture contained about 0.1 mm laccase, 0.1 M sodium phosphate buffer, ph7.5, and 10mM sodium fluoride in a final volume of about 1 or 3ml. The system was titrated with approximately 1 mm quinol in water. Spectra were recorded about 5 min after addition of titrant. Corrections were made as in Fig.2.0, absorbance at 614 nm. 0, absorbance at 330 nm. +, total electron paramagnetic resonance intensity. The lines represent the computed titration pattern where all three electron acceptors ere given the same oxidationreduction potential. Full line : the oneelectron acceptors. Broken line : the twoelectron acceptor b) Type 1 and Type 2 copper are oneelectron acceptors. The chromophore with the optical maximum at 330 nm is a twoelectron acceptor. DISCUSSION The anaerobic oxidationreduction titrations of both native and fluoridetreated laccase conclusively show that the enzyme molecule requires four electrons for reduction, as determined by changes in the visible optical spectrum and the intensity of the electron paramagnetic resonance signals. The total number of electrons that the protein can accept has not been determined (cf. titrations on fungal laccase [4]) due to the lack of a suitable titrant. For fungal laccase it was found that no more than four electrons were accepted and we suggest that this is true also for the Rhus vernicifera laccase in view of the close similarity between the two proteins (see below). In native laccase two electrons are accepted by the Type 1 and Type 2 Cu2+ ions and the other electrons by a twoelectron acceptor. Its nature has not yet been established but in view of its known properties some suggestions can be made. As for fungal laccase [4], the high oxidationreduction potential of the acceptor compared to that of known organic 32 Eur. J. Biochem., Vo1.18 groups in this protein and the relatively long wavelength of the associated optical band, leads to the proposal that the two copper ions undetecable by electron paramagnetic resonance are involved. It has been suggested that these two ions might exist as a Cu2+Cu2+ pair in the oxidized protein [4], but the actual valence state of these ions may be undeterminable. The titration [lo] and magnetic susceptibility [ll] experiments performed by Nakamura on Rhus vernicifera laccase from trees grown in the Ken Shii district probably have no direct relevance for the conclusions drawn in this paper, as he works with a preparation with rather different properties. For example, he finds almost all copper to be detectable by electron paramagnetic resonance [12] whereas we find only GO0/, PI. With fluoridetreated laccase the titration experiments (Fig. 4) suggest that the oxidationreduction potentials are altered in such a way as to approach a common value. However, the 614 nm chromophore still is a oneelectron acceptor (Fig. 1) and if we wish to retain the view that the band at 330 nm is associated with a twoelectron acceptor the titration curves must depart from linearity. This is ahown by the computed lines in Fig.4. Our determinations are not sufficiently precise to show whether or not these small predicted deviation from linearity occur. It is interesting to note that for fungal laccase titration curves similar to those in Fig.4 are observed in the absence of fluoride and that addition of fluoride leads to a differentiation between the acceptors [13]. Apparently the two laccases behave quite differently but for both proteins the effect of F can be explained as a lowering of the standard potential of the twoelectron acceptor relative to that of the Type 1 copper. For the Type 2 copper ion a similar generalization is not so easy to make as its valence state is difficult to measure accurately. In the present model, the cooperativity suggested earlier [4] between the Type 1 copper ion of fungal laccase and the two acceptors undetectable by electron paramagnetic resonance is no longer included; there is cooperativity only within the twoelectron acceptor. As a consequence, the finding that in determinations of standard potential by Nernst plots, the blue color is associated with a oneelectron acceptor in both laccases (see above and [14]) is quite reasonable and presents no discrepancy [4] when compared to the linear titration curves discussed above. The electronaccepting sites in native Rhus vernicifera laccase have standard potentials which are considerably lower than those of the related sites in native fungal laccase [4]. Yet, they are probably reduced by substrate and reoxidized by oxygen by similar mechanisms. The differences in potential might be advantageous in investigating the mechanism of the catalytic process. As with fungal laccase

6 468 B. R. M. REINHAMMAR and T. I. VLNNQHRD: ElectronAccepting Sites in Tree Laccase Eur. J. Biochem. [13], the 330 nm chromophore in Rhus vernicifera 4. Fee, J. A., Malkin, R., Malmstrom, B. G., and Vlinnseems to be in the ghd, T., J. Biol. Chem. 244 (1969) Reinhammar, B., Biochim. Biophys. A&, 205 (1970) 35. nism* Preliminary aerobic stoppedflow studies, 6. Pecht, I., Levitzki, A., and Anbar, M., J. Amer. Chem. excess ascorbate, indicate that this absorption band Soc. 89 (1967) undergoes changes at rates similar to those of the 7. Nakamura, T., Biochim. Biqhys. Acts, 30 (1958) nm band. The electron acceptors related to these 8. Kolthoff, 1 and Tomsicek, W. J., J. Phys. Chem. 39 (1935) 945. absorption bands behave in both laccases. 9. Peisach, J., and Levine, W. E., J. Bid. Chem. 240 (1965) The authors like to Dr' Bo G' MalmstrOm 10. Nakamura, T., Biochim. Biophys. Ada, 30 (1958) 945. for discussions and s. O* and Mr' A' 11. Nakamura, T., Biochim. Biophys. A&, 30 (1958) 640. Magnusson for very valuable technical assistance. This work 12. Nakamura, T., Ikai, and Ogura, y., J. Biochem. was supported by grants from the Swedish Natural Science (Tokyo), 57 (1965) 808. Research Council and the U. S. Public Health Service (GM 13. Malkin, R,, Malmstrom, G., and Viinngkd, T., ). J. Biochem. 10 (1969) Fee, J. A., and Malmstrom, B. G.. Biochim. Biwhvs. REFERENCES &a, 153 ( Malmstrom, B.*G.: Reinhamma'r, B.,' and Viinngkd, T., Biochim. Biophys. Acta, 156 (1968) 67. B. R. M. Reinhammar and T. I. Viinngird 3. Ehrenberg, A., Malmstrom, B. G., Broman, L., and Institutionen for Biokemi, Chalmers Tekniska Hogskola Mosbach, R., J. Mol. Biol. 5 (1962) 450. Fack, S40220 Goteborg 5, Sweden