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Optical properties and a new epr signal from type 3 copper in metal-depleted Rhus vernicifera laccase
Optical Properties and a New epr Signal from Type 3 Copper in Metal-depleted Rhus vernicif era Laccase Bengt Reinhammar Department of Biochemistry and Biophysics, University of GSteborg and Chalmers Institute of
Technology, Gothenburg, Sweden ABSTRACT Due to conflicting reports on the properties of Rhus laccase depleted in type 2 copper a further investigation of this protein derivative has been undertaken. In contrast to most other reports it is shown that the type 3 copper site retains its absorbance at 330 nm when type 2 copper is removed. The type 3 copper ions are oxidized in the resting protein and part of the type 3 Cu(ll) can be made electron paramagnetic resonance (epr) detectable on reduction by ascorbate. This new epr signal is highly rhombic and the epr parameters are comparable to those found in other metalloproteins containing Cu(ll) in binuclear sites. Certain preparations of type 2 deficient protein exhibit lower extinction coefficients at 330 nm. Since these protein derivatives have lost some type 3 copper, it is inferred that the absorbance at 330 nm is dependent on a native type 3 copper site. Also in contrast to other reports, it is found that the extinction coefficient at 614 nm of the type 1 Cu(lI) decreases from 5700 to 4700 M - l c m -I when type 2 copper is removed. The oxidized-reduced difference spectrum also shows a substantial decrease in the absorbance between 700 and 800 nm. The changes in absorbance above 600 nm are probably due to a modification of the type I Cu(ll) site on removal of type 2 copper. The present results also suggest some explanations to the apparent discrepancies among the earlier reports,
INTRODUCTION Laccase is a blue copper-containing oxidase that catalyzes the reduction of dioxygen to water. It contains four copper ions of three different types, all of which take part in the catalytic transfer of electrons from one-electron donating reductants to dioxygen. Two of the metal ions, types 1 and 2 copper, are electron paramagnetic resonance (epr) detectable in the oxidized enzyme and thus Cu(II). The two epr nondetectable ions, type 3 copper, are thought to constitute an antiferromagnetically coupled Cu(H)-Cu(II) pair, Address reprint requests to Dr. Bengt Reinhammar, Department of Biochemistry and Biophysics, Chalmers Institute of Technology, S-412 96 Gothenburg, Sweden.
JournaloflnorganicBiochemistry18,
! 13-121 (1983) (~ Elsevier Science Publishing Co., Inc. 1983 52 Vanderbilt Ave., New York, NY 10017
113 0162-O134/83/020113-0953.00
1 14
B. Reinhammar
which is associated with a 330 nm absorption band with an extinction coefficient of 2800 M ~cm ~. (For a recent review see ref. [1].) Extensive mechanistic studies have been performed in order to elucidate the role of the different metal sites in the catalytic mechanism and several models have been presented for the participation of type 2 copper in the reduction and reoxidation processes. These models are conflicting in many respects. For example, this metal was thought to be involved in the stabilization of a proposed peroxide intermediate [2] or the epr detectable oxygen intermediate [3-5] until the intermediates were further reduced to water. In another study it was inferred tha,t this metal takes part in the breaking of the O-O bond of a peroxide intermediate, which would be bound between the oxidized type 3 copper site and reduced type 2 copper [6]. However, the type 2 copper-depleted protein is reoxidized in a manner similar to that of the native enzyme, which would seem to preclude type 2 copper being involved in 02 reduction [7]. It was previously shown that it is possible to reversibly remove only the type 2 copper in both Polyporus [8] and Rhus [9] laccases. It is therefore now possible to investigate the role of this metal ion in the catalytic mechanism. Several laboratories have published the properties of their type 2-depleted tree laccase [7, 9-14]. These preparations have rather different characteristics. For example, most laboratories report that the 330 nm absorption band disappears following removal of type 2 copper [9-15]. In one case, this is suggested to depend on a reduction of the type 3 copper pair during preparation [ 14]. Other reports claim that the type 3 copper pair is still oxidized, although most of the 330 nm absorption has disappeared [10-13]. Another study shows that the 330 nm chromophore is unaffected in the type 2-depleted protein [7]. The present study is a further investigation of the properties of type 2 copper-depleted Rhus laccase. The results are in accord with an earlier report [7]. It is confirmed that the type 3 copper ions are oxidized in the resting type 2-deficient protein and that they are associated with an absorption band at 330 nm with the same extinction coefficient as the native enzyme. Samples with lower absorbancy at this wavelength contain less that two type 3 copper ions per protein molecule. The disappearance of this chromophore is theretore not due to the loss of type 2 copper per se, although the procedure to remove t h i s m e t a l ion might cause a loss of or a modification of the type 3 copper site. It is also shown that one of the type 3 copper ions can be selectively reduced and that the other Cu(II) then becomes epr detectable in certain preparations of type 2 copper-deficient enzyme. This metal shows epr parameters similar to those in other bimetallic sites where the Cu(ll) is coordinated in a rhombically distorted site. The present study also confirms an earlier result [7] that the 614 nm absorption band of type 1 Cu(ll) decreases by about 1000 M ~cm ~when type 2 copper is taken away. There is also a considerable change in the absorbance at 700 to 800 nm. These changes are probably due to a perturbation of the type 1 Cu(ll) site as an effect of a conformational change when type 2 copper is removed.
MATERIALS AND METHODS
Rhus vernicifera laccase was prepared according to previously published methods [7, 17]. Removal of type 2 copper was performed by a recently published method [7] or with such modifications as are mentioned in the results. Optical absorption spectra were recorded on a Beckman ACTA M IV spectrophotometer. The anaerobic reduction experiments were made in an optical quartz cell with a Thunberg top containing the
Spectroscopic Properties of Metal-Depleted Tree Laccase
1 15
reductant. The cell was made anaerobic by several cycles of evacuating and flushing with O2-free nitrogen gas. Electron paramagnetic resonance (epr) spectra were recorded on Varian E-3 or E-9 instruments at about liquid nitrogen temperature. The concentration of paramagnetic copper was determined by double integration of epr spectra. Total copper in the protein samples was determined by the biquinoline method [18]. Protein concentration was determined from the absorption at 280 nm, using the molar extinction coefficient value previously published [ 19].
RESULTS
epr Properties of Type 2 Copper-depleted Laccase Several prepartions of type 2-depleted laccase were examined, epr spectral measurements were performed with the same protein samples that were subjected to optical absorption measurements. The epr samples were frozen in liquid nitrogen at the same time as the optical absorption spectra were recorded. Figure I shows the epr spectra of native (A) and type 2 copper-depleted samples (B and C). Sample B represents a normal type 2-depleted protein with properties as reported earlier [7]. Sample C originates from a preparation of type 2-depleted protein that showed the presence of a low-field line at about 0.265 T similar to the line at this field in C, but smaller. Several laboratories have reported the presence of this line in their preparations of type 2-depleted laccase and it has been suggested to have come from residual type 2 copper [ 12, 15]. Since there are no other copper hyperfine lines characteristic for this Cu(ll), it cannot come from this metal. In an order to remove this impurity, the sample was dialyzed for 24 h against anaerobic potassium phosphate buffer, pH 7.4, ionic strength 0.25 M, which also contained I mM sodium ascorbate and l mM ethylenediaminetetraacetate (EDTA). The protein sample was thereafter dialyzed against several changes of the same buffer in the presence of air. Sample C contains 1.0 type l Cu(II) but only 1.6 type 3 copper. The low-field line in C consists of at least two signals. One of the signals belongs to the type 3 Cu(II) shown in sample D. It is estimated that sample C contains about 0. I mol of signal D per mol of protein. The other contribution is probably from Fe(CN)63, since metal analyses show that there is 0.4 mol of iron per mol of protein in this sample. Since iron is still present after the extensive dialysis, it must be very strongly bound to some site in the protein. There is also a weak band at about 420 nm in the absorption spectrum (not shown) that indicates the presence of Fe(CN)63. Preparations of type 2 copper-depleted tree laccase from other laboratories show similar spectral properties [10, 12, 13, 15] and the low-field line was inferred to originate from type 2 Cu(Il). The reported values of this copper in several preparations of type 2-depleted laccase [10, 12] are therefore presumably too high. Sample D is the same as sample C but after reduction for about l min with 5mM ascorbate at 20°C. The integrated epr intensity corresponds to 0.4 Cu(II) per protein molecule. This signal must originate from the oxidized type 3 copper pair, since it can be made epr detectable on reduction. It is very similar to the new Cu(ll) epr signal that is obtained on reduction of type 2-depleted Polyporus laccase [20] and shows a Cu(II) that is coordinated in a rhombically distorted site. The epr spectrum can be simulated with the parameters given in Table 1; the parameters from Cu(II) in bimetallic centers found in some other proteins are also presented.
1 16
B. Reinhammar
g-VALUE 26
24
I
I
2.2
20
I
D
I
l
0.25
I
I
I
I
1
I
1
030
1
0.35
MAGNETIC FLUX DENSITY (T) FIGURE 1. epr spectra of native (A) and type 2 copper-depleted (B and C) Rhus laccase, (D) The same as C but after reduction with ascorbate. All samples in potassium phosphate buffer, pit 7.4, ionic strength 0.25 M. Protein concentration about 0.5 raM. Spectra were recorded at 77°K and 9.1 GHz with a microwave power of 20 mW and a modulation amplitude of 2 roT.
Optical Absorption Properties of Type 2 Copper-depleted Laccase The optical absorption difference spectra from 300 to 800 nm of the oxidized-reduced protein samples are shown in Figure 2. The type 3 copper site is generally very slowly reduced by ascorbate in the type 2-deficient protein, but it can be reduced within a few hours w i t h excess sodium dithionite. However, about 40% of the 330 nm band is sample C can be reduced within 1 rain with ascorbate, as shown in D. Further reduction with this reductant was extremely slow and no change in the 330 nm absorption was observed even after 30 rain of reaction. Full reduction was, however, possible within 15 rain with dithionite. In agreement with the native enzyme (A), the type 2-depleted samples (B and C) show the presence of an absorption band at 330 nm. The extinction coefficient of this band in sample B is almost comparable to the native protein but is only about 805 of the native in
Spectroscopic Properties of Metal-Depleted Tree Laccase
1 17
T A B L E 1. epr Parameters for Cu(II) Signals from Bimetallic Centers in
Various Proteins gx
gy
gz
2.03
2.15
2.277
13.2
This study
2.025 2.04
2.148 2.09
2.268 2.30
13.2 13.2
20 24
2.025
2.103
2.257
13.9
16
2.052
2.109
2.278
10.8
20
2.05
2.15
2.305
8.4
23
Protein
Az × ( 103 c m - l )
References
Rhus laccase, type 2-depleted
Polyporus laccase, type 2-depleted Half-met-hemocyanin Bovine superoxide dismutase Cytochrome c oxidase Native Rhus laccase
sample C. Since this latter sample contains only 1.6 type 3 copper per protein molecule, it seems likely that about 20% of the molecules have lost the type 3 copper pair and thus the absorption at 330 nm. All samples display similar extinction coefficients at 450 nm where the blue proteins have a weak absorption band [l]. The extinction coefficient at 614 nm, calculated from the concentration of epr detectable type 1 Cu(II) and the optical absorbance, is 5500 M-~cm t in the native enzyme, in accordance with earlier results [19]. The type 2-depleted samples show lower values, 4400 M ~cm -~, and also a significantly lower absorbance at higher wavelengths, as first reported by Morpurgo et al. [10]. These
FIGURE 2. Oxidized-reduced difference spectra of native (A) and type 2-depLeted samples (B-D). The same samples as in Figure 1A-D but diluted to about 0.1 mM protein. Samples A-C were reduced at 20°C for 15 min to 5 hr with a few crystals of sodium dithionite under anaerobic conditions. Curve D shows sample C reduced for 30 min with 1.5 mM sodium ascorbate.
6000
A
4000 A
2000
300
400
I
l
I
I
500
600
700
800
WAVELENGTH ( n m )
l 18
B. Reinharnmar
6000 A
/..000
2000
....... 300
I 400
t 500
t 600
I 700
t 800
WAVELENGTH ( n m ) FIGURE 3. Optical absorption spectrum (A) of type 2-depleted laccase in 0.1 M sodium acetate buffer, pH 5.0. (B) Oxidized dithionite-reduced difference spectrum recorded after 5 hr of reduction. (C) Difference spectrum (reoxidized-reduced) after reoxidation with air for 5 min. authors report a decrease in absorbance at 750 nm of 300 M - tcm- t. The results in Figure 2 show that it is even higher, or 500 M-~cm -~ . Figure 3 shows the optical absorption spectra of a sample that was dialyzed for 50 h to remove the type 2 copper. In addition to the loss of this metal, type 3 copper was also partially removed. The sample contains 1.0 type 1 Cu(II) and 1.3 type 3 copper per protein molecule. The oxidized-reduced difference spectrum (B) shows that the 330 nm band retains only about 45% of the absorbance of the native protein. Similar results were obtained from the same sample in 0.1 M sodium acetate buffer, pH 5.0, or in 0.1 M potassium phosphate buffer, pH 7.4. On reoxidation by air for 5 min, this band reappears but the absorbance at 614 nm returns by only 45%. This result indicates that the loss of absorbance at 330 nm is due to the removal of some type 3 copper and that the type 1 Cu(I) is only reoxidized in molecules having a functioning type 3 copper pair. Since only 45% of the molecules appear to contain a native copper pair, the remaining type 3 copper is either in a modified oxidized or reduced, metal pair that is unreactive or as a single metal site where the copper would be reduced, since it is not detected by epr. DISCUSSION
The results in this study demonstrate that the type 3 copper ions are oxidized and associated with a 330 nm absorption band with the same molar extinction coefficient as in the native protein and also in the present preparation of type 2 copper-depleted tree laccase. In samples containing less than two type 3 copper ions per protein molecule (Figs. 2C and 3), this absorption band shows a diminished value that is not unexpected. The reported disappearance of this absorption band in other preparations of type 2-depleted laccase is therefore not likely due to the removal of the type 2 copper as suggested [10, 13], but to some other reason. It is notable that several investigators
Spectroscopic Properties of Metal-Depleted Tree Laccase
1 19
report that their type 2-depleted laccase has lost some type 3 copper (and also part of the type 1 copper). Thus, Morpurgo et al. [10, 12] report between 1.25 and 1.79 type 3 copper in nine samples of their type 2-depleted protein. If the 330 nm chromophore depends on the presence of both type 3 Cu(II) ions, which seems reaonable, the 330 nm absorbance would be only 25-90% compared to the native protein. Since they report that the samples have only 20-45% residual absorbance at this wavelength, it seems likely that the loss of type 3 copper is responsible for the disappearance of the 330 nm absorption band, although this idea was rejected [10]. The results in this investigation show, however, that the extinction coefficient of the 330 nm absorption band is apparently unaffected following removal of type 2 copper. Therefore, this metal does not seem to contribute significantly to the absorbance of this chromophore. The type 2-depleted laccase prepared by Lu Bien et al. [14] lacks the 330 nm absorption band, and is shown that the type 3 copper ions are in the Cu(I) oxidation state even in the presence of dioxygen. This result indicates that the copper pair contains modified protein, since other preparations can be rapidly reoxidized by dioxygen [7, 12]. Since the 330 nm band reappears on reoxidation of the type 3 copper ions with hydrogen peroxide, it again shows that this chromophore depends on the type 3 Cu(II) pair and not on the type 2 Cu(II). The present study also confirms the earlier results which show that the molar extinction coefficient of the 614 nm chromophore decreases from 5500 to 4400 (oxidized-reduced protein) when type 2 copper is removed. It should be noted that this extinction coefficient is calculated from the optical absorbance and the integrated epr intensity of type 1 Cu(II) in the same samples. Since the optical spectra were recorded at the same time as the epr samples were frozen in liquid nitrogen, the suggestion [12, 13] that this lower value is due to denaturation or slow autoreduction of the type 1 Cu(II) above pH 7 is not sound. Moreover, the same preparations have been analyzed at pH 5.0 and 7.4. No difference in the molar extinction of the type 1 chromophore was detected at the two pH values. In an earlier report [7] no significant changes of the optical absorbance were observed above 700 nm following removal of type 2 copper. Two other laboratories have obtained similar results. In the article by Graziani et al. [9], the e750nm decreases by only about 10% and the type 2-depleted laccase prepared by Lu Bien et al. [14] exhibits almost the same extinction as the native enzyme at this wavelength. However, a later report by the Italian group [10] shows that the e decreases by 300 at 750 nm. The oxidized-reduced difference spectra in Figures 2 and 3 confirm that there are substantial absorbance decreases at 700-800 nm in the type.2-deficient protein. It was inferred that the decrease in the extinction coefficient at 750 nm is due to type 2 Cu(II) contribution [10]. It could also depend on a change in the type 1 Cu(II) coordination when type 2 is removed. This is likely to occur, since the extinction of the 614 nm absorption band is altered and the epr parameters are somewhat changed following removal of type 2 copper [7]. A resonance Raman band at about 390 cm- ~also loses about half of its intensity, which indicates that a small angular perturbation of the type 1 Cu(II) site occurs when type 2 copper is removed [21]. The new epr signal that appears on reduction of the modified type 2-deficient laccase (sample D in Fig. 1) originates from one of the type 3 Cu(II). The signal shows strong rhombic character and appears to be almost identical to the type 3 Cu(II) signal appearing on reduction of type 2-depleted Polyporus laccase [20]. This indicates that the metal is possibly bound in similar sites in both laccases. Very similar rhombic Cu(II) epr signals
120
B. Reinhammar
have also been observed in other proteins containing bimetallic centers, such as superoxide dismutase, half-met-hemocyanin, and cytochrome c oxidase (see Table 1). There is also a pronounced amino acid sequence homology between a blue oxidase (ceruloplasmin) and cytochrome c oxidase with the copper-binding site in superoxide dismutase [22]. This similarity of Cu(II) epr signals and amino acid sequences indicate that the metals might be co-ordinated in a similar way in the different proteins. Since it is possible to obtain this new Cu(II) signal on reduction of type 2-depleted laccase the type 3 copper ions must be oxidized in the resting protein. This result is in accord with earlier suggestions that these metal ions consist of a pair of contiguous Cu(II) ions that are strongly antiferromagnetically coupled [ 1]. The physical demonstration of such a pair has, however, not been reported for any laccase. The treatment leading to the modified type 2-depleted sample in Figure 1C apparently changes the reactivity of the type 3 copper pair in some molecules, since one of the Cu(II) ions can be rapidly reduced in about 40% of the molecules with ascorbate. This is not possible with the usual preparation of laccase [7]. The modified type 3 site seems to retain its absorbance at 330 nm, since the absorption decrease occurs simultaneously with the development of the new epr signal in Figure 1D. Since the decrease in 330 nm absorbance matches the reduction of the type 3 Cu(II) ions, the chromophore might depend on this metal ion, with the other Cu(II) then making only a negligible contribution to the 330 nm absorbance. On the other hand, this chromophore might require that both metal ions be oxidized. The present results can not discriminate between these possibilities. Another epr signal from type 3 Cu(II) has recently been produced as an intermediate during reoxidation of the native enzyme by peroxide or by the reduction of the newly reoxidized native or type 2 copper-depleted laccase [23]. This signal exhibits similar g values but a lower A IIvalue than the signal in Figure 1D. Whether these two signals each represent one type 3 Cu(II) or the same copper ion, despite the unequality in epr parameters, is not yet known. This work was supported by grants from the Swedish Natural Research Council. The author is indebted to Miss A.-C. Carlsson for the preparation o f the tree laccase.
REFERENCES 1. B. Reinhammar and B. G. Malmstr'6m, in Copper Proteins, Vol. 3 of Metal Ions in Biology, T. G. Spiro, Ed., John Wiley & Sons, New York, 1981, pp. 107-149. 2. L.-E. Andr6asson, R. Bfiind4n, B. G. Malmstr'6n, and T. V~/nngSrd,FEBS Lett. 32, 187 (1973). 3. R. Brh'nd~n and J. Deinum,FEBSLett. 73,144 (1977). 4. R. Bfiind~n, J. Deinum, and M. Coleman, FEBSLett. 89, 180 (1978). 5. R. Bfiind6n and J. Deinum, Biochim. Biophys. Acta 524,297 (1978). 6. O. Farver, M. Goldberg, and I. Pecht, Eur. J. Biochem. 104, 71 (1980). 7. B. Reinhammar and Y. Oda, J. Inorg. Biochem. 11,115 (1979). 8. R. Malkin, B. G. Malmstr6m, and T. V~inng~rd,Eur. J. Biochem. 7,253 (1969). 9. M.T. Graziani, L. Morpurgo, G. Rotilio, and B. Mondov(,FEBSLett. 70, 87 (1976). 10. L. Morpurgo, M. T. Graziani, A. Finazzi-Agr8, G. Rotflio, and B. Mondov(,Biochem. J. 187, 361 (1980). 11. L. Morpurgo, M. T. Graziani, A. Desideri, and G. Rotilio, Biochern. J. 187,367 (1980). 12. L. Morpurgo, A. Desideri, G. Rotilio, and B. MondivLFEBSLett. 113,153 (1980). 13. L. Morpurgo, M. T. Graziani, L. Avigliano, A. Desideri, and B. Mondovi, lsraelJ. Biochem. 21, 26 (1981).
121
S p e c t r o s c o p i c P r o p e r t i e s o f M e t a l - D e p l e t e d T r e e Laccase
14. C.D. Lu Bien, M. E. Winkler, T. J. Thamann, R. A. Scott, M. S. Co., K. O. Hodgson, and E. I. Solomon, J. Amer. Chem. Soc. 103, 7014 (1981). 15. B.M. Kanne, personal communication, April 1979. 16. E.M. Fielden, P. B. Roberts, R. C. Bray, D. J. Lowe, G. N. Mautner, G. Rotilio, and L. Calabrese, Biochem. J. 139, 49 (1974). 17. B. Reinhammar, Biochim. Biophys. Acta 205, 35 (1970). 18. P.E. Brumby and V. Massey, inMethods ofEnzymology, vol. 10, R. W. Estabrook and M. R. Pullman, Eds., Academic Press, New York, 1967, p. 463. 19. B.G. MalmstriSm, B. Reinhammar, and T. Vanngard, Btochtm. Biophys. Acta 205, 48 (1970). 20. B. Reinhammar, R. Malkin, P. Jensen, B. Karlsson, L.-E. Andre'asson, R. Aasa, T. Vanngard, and B. G. MalmstRim, J. Biol. Chem. 225, 5000 (1980). 21. J.A. Larrabee, G. Woolery, B. Reinhammar, and T. Spiro, unpublished results. 22. F.E. Dwulet and F. W. Putnam, Proc. Natl. Acad. Sci. USA 78, 2805 (1981). 23. B. Reinhammar, J. lnorg. Biochem. 15, 27 (1981). 24. A. L. M. Schott Uiterkamp, H. van der Deen, H. C. J. Berendsen, and J. F. Boas, Biochim. Biophys. Acta 372,407 (1974). ..
O
.
.
..
Received April 5, 1982; accepted June 4, 1982
O
Technology, Gothenburg, Sweden ABSTRACT Due to conflicting reports on the properties of Rhus laccase depleted in type 2 copper a further investigation of this protein derivative has been undertaken. In contrast to most other reports it is shown that the type 3 copper site retains its absorbance at 330 nm when type 2 copper is removed. The type 3 copper ions are oxidized in the resting protein and part of the type 3 Cu(ll) can be made electron paramagnetic resonance (epr) detectable on reduction by ascorbate. This new epr signal is highly rhombic and the epr parameters are comparable to those found in other metalloproteins containing Cu(ll) in binuclear sites. Certain preparations of type 2 deficient protein exhibit lower extinction coefficients at 330 nm. Since these protein derivatives have lost some type 3 copper, it is inferred that the absorbance at 330 nm is dependent on a native type 3 copper site. Also in contrast to other reports, it is found that the extinction coefficient at 614 nm of the type 1 Cu(lI) decreases from 5700 to 4700 M - l c m -I when type 2 copper is removed. The oxidized-reduced difference spectrum also shows a substantial decrease in the absorbance between 700 and 800 nm. The changes in absorbance above 600 nm are probably due to a modification of the type I Cu(ll) site on removal of type 2 copper. The present results also suggest some explanations to the apparent discrepancies among the earlier reports,
INTRODUCTION Laccase is a blue copper-containing oxidase that catalyzes the reduction of dioxygen to water. It contains four copper ions of three different types, all of which take part in the catalytic transfer of electrons from one-electron donating reductants to dioxygen. Two of the metal ions, types 1 and 2 copper, are electron paramagnetic resonance (epr) detectable in the oxidized enzyme and thus Cu(II). The two epr nondetectable ions, type 3 copper, are thought to constitute an antiferromagnetically coupled Cu(H)-Cu(II) pair, Address reprint requests to Dr. Bengt Reinhammar, Department of Biochemistry and Biophysics, Chalmers Institute of Technology, S-412 96 Gothenburg, Sweden.
JournaloflnorganicBiochemistry18,
! 13-121 (1983) (~ Elsevier Science Publishing Co., Inc. 1983 52 Vanderbilt Ave., New York, NY 10017
113 0162-O134/83/020113-0953.00
1 14
B. Reinhammar
which is associated with a 330 nm absorption band with an extinction coefficient of 2800 M ~cm ~. (For a recent review see ref. [1].) Extensive mechanistic studies have been performed in order to elucidate the role of the different metal sites in the catalytic mechanism and several models have been presented for the participation of type 2 copper in the reduction and reoxidation processes. These models are conflicting in many respects. For example, this metal was thought to be involved in the stabilization of a proposed peroxide intermediate [2] or the epr detectable oxygen intermediate [3-5] until the intermediates were further reduced to water. In another study it was inferred tha,t this metal takes part in the breaking of the O-O bond of a peroxide intermediate, which would be bound between the oxidized type 3 copper site and reduced type 2 copper [6]. However, the type 2 copper-depleted protein is reoxidized in a manner similar to that of the native enzyme, which would seem to preclude type 2 copper being involved in 02 reduction [7]. It was previously shown that it is possible to reversibly remove only the type 2 copper in both Polyporus [8] and Rhus [9] laccases. It is therefore now possible to investigate the role of this metal ion in the catalytic mechanism. Several laboratories have published the properties of their type 2-depleted tree laccase [7, 9-14]. These preparations have rather different characteristics. For example, most laboratories report that the 330 nm absorption band disappears following removal of type 2 copper [9-15]. In one case, this is suggested to depend on a reduction of the type 3 copper pair during preparation [ 14]. Other reports claim that the type 3 copper pair is still oxidized, although most of the 330 nm absorption has disappeared [10-13]. Another study shows that the 330 nm chromophore is unaffected in the type 2-depleted protein [7]. The present study is a further investigation of the properties of type 2 copper-depleted Rhus laccase. The results are in accord with an earlier report [7]. It is confirmed that the type 3 copper ions are oxidized in the resting type 2-deficient protein and that they are associated with an absorption band at 330 nm with the same extinction coefficient as the native enzyme. Samples with lower absorbancy at this wavelength contain less that two type 3 copper ions per protein molecule. The disappearance of this chromophore is theretore not due to the loss of type 2 copper per se, although the procedure to remove t h i s m e t a l ion might cause a loss of or a modification of the type 3 copper site. It is also shown that one of the type 3 copper ions can be selectively reduced and that the other Cu(II) then becomes epr detectable in certain preparations of type 2 copper-deficient enzyme. This metal shows epr parameters similar to those in other bimetallic sites where the Cu(ll) is coordinated in a rhombically distorted site. The present study also confirms an earlier result [7] that the 614 nm absorption band of type 1 Cu(ll) decreases by about 1000 M ~cm ~when type 2 copper is taken away. There is also a considerable change in the absorbance at 700 to 800 nm. These changes are probably due to a perturbation of the type 1 Cu(ll) site as an effect of a conformational change when type 2 copper is removed.
MATERIALS AND METHODS
Rhus vernicifera laccase was prepared according to previously published methods [7, 17]. Removal of type 2 copper was performed by a recently published method [7] or with such modifications as are mentioned in the results. Optical absorption spectra were recorded on a Beckman ACTA M IV spectrophotometer. The anaerobic reduction experiments were made in an optical quartz cell with a Thunberg top containing the
Spectroscopic Properties of Metal-Depleted Tree Laccase
1 15
reductant. The cell was made anaerobic by several cycles of evacuating and flushing with O2-free nitrogen gas. Electron paramagnetic resonance (epr) spectra were recorded on Varian E-3 or E-9 instruments at about liquid nitrogen temperature. The concentration of paramagnetic copper was determined by double integration of epr spectra. Total copper in the protein samples was determined by the biquinoline method [18]. Protein concentration was determined from the absorption at 280 nm, using the molar extinction coefficient value previously published [ 19].
RESULTS
epr Properties of Type 2 Copper-depleted Laccase Several prepartions of type 2-depleted laccase were examined, epr spectral measurements were performed with the same protein samples that were subjected to optical absorption measurements. The epr samples were frozen in liquid nitrogen at the same time as the optical absorption spectra were recorded. Figure I shows the epr spectra of native (A) and type 2 copper-depleted samples (B and C). Sample B represents a normal type 2-depleted protein with properties as reported earlier [7]. Sample C originates from a preparation of type 2-depleted protein that showed the presence of a low-field line at about 0.265 T similar to the line at this field in C, but smaller. Several laboratories have reported the presence of this line in their preparations of type 2-depleted laccase and it has been suggested to have come from residual type 2 copper [ 12, 15]. Since there are no other copper hyperfine lines characteristic for this Cu(ll), it cannot come from this metal. In an order to remove this impurity, the sample was dialyzed for 24 h against anaerobic potassium phosphate buffer, pH 7.4, ionic strength 0.25 M, which also contained I mM sodium ascorbate and l mM ethylenediaminetetraacetate (EDTA). The protein sample was thereafter dialyzed against several changes of the same buffer in the presence of air. Sample C contains 1.0 type l Cu(II) but only 1.6 type 3 copper. The low-field line in C consists of at least two signals. One of the signals belongs to the type 3 Cu(II) shown in sample D. It is estimated that sample C contains about 0. I mol of signal D per mol of protein. The other contribution is probably from Fe(CN)63, since metal analyses show that there is 0.4 mol of iron per mol of protein in this sample. Since iron is still present after the extensive dialysis, it must be very strongly bound to some site in the protein. There is also a weak band at about 420 nm in the absorption spectrum (not shown) that indicates the presence of Fe(CN)63. Preparations of type 2 copper-depleted tree laccase from other laboratories show similar spectral properties [10, 12, 13, 15] and the low-field line was inferred to originate from type 2 Cu(Il). The reported values of this copper in several preparations of type 2-depleted laccase [10, 12] are therefore presumably too high. Sample D is the same as sample C but after reduction for about l min with 5mM ascorbate at 20°C. The integrated epr intensity corresponds to 0.4 Cu(II) per protein molecule. This signal must originate from the oxidized type 3 copper pair, since it can be made epr detectable on reduction. It is very similar to the new Cu(ll) epr signal that is obtained on reduction of type 2-depleted Polyporus laccase [20] and shows a Cu(II) that is coordinated in a rhombically distorted site. The epr spectrum can be simulated with the parameters given in Table 1; the parameters from Cu(II) in bimetallic centers found in some other proteins are also presented.
1 16
B. Reinhammar
g-VALUE 26
24
I
I
2.2
20
I
D
I
l
0.25
I
I
I
I
1
I
1
030
1
0.35
MAGNETIC FLUX DENSITY (T) FIGURE 1. epr spectra of native (A) and type 2 copper-depleted (B and C) Rhus laccase, (D) The same as C but after reduction with ascorbate. All samples in potassium phosphate buffer, pit 7.4, ionic strength 0.25 M. Protein concentration about 0.5 raM. Spectra were recorded at 77°K and 9.1 GHz with a microwave power of 20 mW and a modulation amplitude of 2 roT.
Optical Absorption Properties of Type 2 Copper-depleted Laccase The optical absorption difference spectra from 300 to 800 nm of the oxidized-reduced protein samples are shown in Figure 2. The type 3 copper site is generally very slowly reduced by ascorbate in the type 2-deficient protein, but it can be reduced within a few hours w i t h excess sodium dithionite. However, about 40% of the 330 nm band is sample C can be reduced within 1 rain with ascorbate, as shown in D. Further reduction with this reductant was extremely slow and no change in the 330 nm absorption was observed even after 30 rain of reaction. Full reduction was, however, possible within 15 rain with dithionite. In agreement with the native enzyme (A), the type 2-depleted samples (B and C) show the presence of an absorption band at 330 nm. The extinction coefficient of this band in sample B is almost comparable to the native protein but is only about 805 of the native in
Spectroscopic Properties of Metal-Depleted Tree Laccase
1 17
T A B L E 1. epr Parameters for Cu(II) Signals from Bimetallic Centers in
Various Proteins gx
gy
gz
2.03
2.15
2.277
13.2
This study
2.025 2.04
2.148 2.09
2.268 2.30
13.2 13.2
20 24
2.025
2.103
2.257
13.9
16
2.052
2.109
2.278
10.8
20
2.05
2.15
2.305
8.4
23
Protein
Az × ( 103 c m - l )
References
Rhus laccase, type 2-depleted
Polyporus laccase, type 2-depleted Half-met-hemocyanin Bovine superoxide dismutase Cytochrome c oxidase Native Rhus laccase
sample C. Since this latter sample contains only 1.6 type 3 copper per protein molecule, it seems likely that about 20% of the molecules have lost the type 3 copper pair and thus the absorption at 330 nm. All samples display similar extinction coefficients at 450 nm where the blue proteins have a weak absorption band [l]. The extinction coefficient at 614 nm, calculated from the concentration of epr detectable type 1 Cu(II) and the optical absorbance, is 5500 M-~cm t in the native enzyme, in accordance with earlier results [19]. The type 2-depleted samples show lower values, 4400 M ~cm -~, and also a significantly lower absorbance at higher wavelengths, as first reported by Morpurgo et al. [10]. These
FIGURE 2. Oxidized-reduced difference spectra of native (A) and type 2-depLeted samples (B-D). The same samples as in Figure 1A-D but diluted to about 0.1 mM protein. Samples A-C were reduced at 20°C for 15 min to 5 hr with a few crystals of sodium dithionite under anaerobic conditions. Curve D shows sample C reduced for 30 min with 1.5 mM sodium ascorbate.
6000
A
4000 A
2000
300
400
I
l
I
I
500
600
700
800
WAVELENGTH ( n m )
l 18
B. Reinharnmar
6000 A
/..000
2000
....... 300
I 400
t 500
t 600
I 700
t 800
WAVELENGTH ( n m ) FIGURE 3. Optical absorption spectrum (A) of type 2-depleted laccase in 0.1 M sodium acetate buffer, pH 5.0. (B) Oxidized dithionite-reduced difference spectrum recorded after 5 hr of reduction. (C) Difference spectrum (reoxidized-reduced) after reoxidation with air for 5 min. authors report a decrease in absorbance at 750 nm of 300 M - tcm- t. The results in Figure 2 show that it is even higher, or 500 M-~cm -~ . Figure 3 shows the optical absorption spectra of a sample that was dialyzed for 50 h to remove the type 2 copper. In addition to the loss of this metal, type 3 copper was also partially removed. The sample contains 1.0 type 1 Cu(II) and 1.3 type 3 copper per protein molecule. The oxidized-reduced difference spectrum (B) shows that the 330 nm band retains only about 45% of the absorbance of the native protein. Similar results were obtained from the same sample in 0.1 M sodium acetate buffer, pH 5.0, or in 0.1 M potassium phosphate buffer, pH 7.4. On reoxidation by air for 5 min, this band reappears but the absorbance at 614 nm returns by only 45%. This result indicates that the loss of absorbance at 330 nm is due to the removal of some type 3 copper and that the type 1 Cu(I) is only reoxidized in molecules having a functioning type 3 copper pair. Since only 45% of the molecules appear to contain a native copper pair, the remaining type 3 copper is either in a modified oxidized or reduced, metal pair that is unreactive or as a single metal site where the copper would be reduced, since it is not detected by epr. DISCUSSION
The results in this study demonstrate that the type 3 copper ions are oxidized and associated with a 330 nm absorption band with the same molar extinction coefficient as in the native protein and also in the present preparation of type 2 copper-depleted tree laccase. In samples containing less than two type 3 copper ions per protein molecule (Figs. 2C and 3), this absorption band shows a diminished value that is not unexpected. The reported disappearance of this absorption band in other preparations of type 2-depleted laccase is therefore not likely due to the removal of the type 2 copper as suggested [10, 13], but to some other reason. It is notable that several investigators
Spectroscopic Properties of Metal-Depleted Tree Laccase
1 19
report that their type 2-depleted laccase has lost some type 3 copper (and also part of the type 1 copper). Thus, Morpurgo et al. [10, 12] report between 1.25 and 1.79 type 3 copper in nine samples of their type 2-depleted protein. If the 330 nm chromophore depends on the presence of both type 3 Cu(II) ions, which seems reaonable, the 330 nm absorbance would be only 25-90% compared to the native protein. Since they report that the samples have only 20-45% residual absorbance at this wavelength, it seems likely that the loss of type 3 copper is responsible for the disappearance of the 330 nm absorption band, although this idea was rejected [10]. The results in this investigation show, however, that the extinction coefficient of the 330 nm absorption band is apparently unaffected following removal of type 2 copper. Therefore, this metal does not seem to contribute significantly to the absorbance of this chromophore. The type 2-depleted laccase prepared by Lu Bien et al. [14] lacks the 330 nm absorption band, and is shown that the type 3 copper ions are in the Cu(I) oxidation state even in the presence of dioxygen. This result indicates that the copper pair contains modified protein, since other preparations can be rapidly reoxidized by dioxygen [7, 12]. Since the 330 nm band reappears on reoxidation of the type 3 copper ions with hydrogen peroxide, it again shows that this chromophore depends on the type 3 Cu(II) pair and not on the type 2 Cu(II). The present study also confirms the earlier results which show that the molar extinction coefficient of the 614 nm chromophore decreases from 5500 to 4400 (oxidized-reduced protein) when type 2 copper is removed. It should be noted that this extinction coefficient is calculated from the optical absorbance and the integrated epr intensity of type 1 Cu(II) in the same samples. Since the optical spectra were recorded at the same time as the epr samples were frozen in liquid nitrogen, the suggestion [12, 13] that this lower value is due to denaturation or slow autoreduction of the type 1 Cu(II) above pH 7 is not sound. Moreover, the same preparations have been analyzed at pH 5.0 and 7.4. No difference in the molar extinction of the type 1 chromophore was detected at the two pH values. In an earlier report [7] no significant changes of the optical absorbance were observed above 700 nm following removal of type 2 copper. Two other laboratories have obtained similar results. In the article by Graziani et al. [9], the e750nm decreases by only about 10% and the type 2-depleted laccase prepared by Lu Bien et al. [14] exhibits almost the same extinction as the native enzyme at this wavelength. However, a later report by the Italian group [10] shows that the e decreases by 300 at 750 nm. The oxidized-reduced difference spectra in Figures 2 and 3 confirm that there are substantial absorbance decreases at 700-800 nm in the type.2-deficient protein. It was inferred that the decrease in the extinction coefficient at 750 nm is due to type 2 Cu(II) contribution [10]. It could also depend on a change in the type 1 Cu(II) coordination when type 2 is removed. This is likely to occur, since the extinction of the 614 nm absorption band is altered and the epr parameters are somewhat changed following removal of type 2 copper [7]. A resonance Raman band at about 390 cm- ~also loses about half of its intensity, which indicates that a small angular perturbation of the type 1 Cu(II) site occurs when type 2 copper is removed [21]. The new epr signal that appears on reduction of the modified type 2-deficient laccase (sample D in Fig. 1) originates from one of the type 3 Cu(II). The signal shows strong rhombic character and appears to be almost identical to the type 3 Cu(II) signal appearing on reduction of type 2-depleted Polyporus laccase [20]. This indicates that the metal is possibly bound in similar sites in both laccases. Very similar rhombic Cu(II) epr signals
120
B. Reinhammar
have also been observed in other proteins containing bimetallic centers, such as superoxide dismutase, half-met-hemocyanin, and cytochrome c oxidase (see Table 1). There is also a pronounced amino acid sequence homology between a blue oxidase (ceruloplasmin) and cytochrome c oxidase with the copper-binding site in superoxide dismutase [22]. This similarity of Cu(II) epr signals and amino acid sequences indicate that the metals might be co-ordinated in a similar way in the different proteins. Since it is possible to obtain this new Cu(II) signal on reduction of type 2-depleted laccase the type 3 copper ions must be oxidized in the resting protein. This result is in accord with earlier suggestions that these metal ions consist of a pair of contiguous Cu(II) ions that are strongly antiferromagnetically coupled [ 1]. The physical demonstration of such a pair has, however, not been reported for any laccase. The treatment leading to the modified type 2-depleted sample in Figure 1C apparently changes the reactivity of the type 3 copper pair in some molecules, since one of the Cu(II) ions can be rapidly reduced in about 40% of the molecules with ascorbate. This is not possible with the usual preparation of laccase [7]. The modified type 3 site seems to retain its absorbance at 330 nm, since the absorption decrease occurs simultaneously with the development of the new epr signal in Figure 1D. Since the decrease in 330 nm absorbance matches the reduction of the type 3 Cu(II) ions, the chromophore might depend on this metal ion, with the other Cu(II) then making only a negligible contribution to the 330 nm absorbance. On the other hand, this chromophore might require that both metal ions be oxidized. The present results can not discriminate between these possibilities. Another epr signal from type 3 Cu(II) has recently been produced as an intermediate during reoxidation of the native enzyme by peroxide or by the reduction of the newly reoxidized native or type 2 copper-depleted laccase [23]. This signal exhibits similar g values but a lower A IIvalue than the signal in Figure 1D. Whether these two signals each represent one type 3 Cu(II) or the same copper ion, despite the unequality in epr parameters, is not yet known. This work was supported by grants from the Swedish Natural Research Council. The author is indebted to Miss A.-C. Carlsson for the preparation o f the tree laccase.
REFERENCES 1. B. Reinhammar and B. G. Malmstr'6m, in Copper Proteins, Vol. 3 of Metal Ions in Biology, T. G. Spiro, Ed., John Wiley & Sons, New York, 1981, pp. 107-149. 2. L.-E. Andr6asson, R. Bfiind4n, B. G. Malmstr'6n, and T. V~/nngSrd,FEBS Lett. 32, 187 (1973). 3. R. Brh'nd~n and J. Deinum,FEBSLett. 73,144 (1977). 4. R. Bfiind~n, J. Deinum, and M. Coleman, FEBSLett. 89, 180 (1978). 5. R. Bfiind6n and J. Deinum, Biochim. Biophys. Acta 524,297 (1978). 6. O. Farver, M. Goldberg, and I. Pecht, Eur. J. Biochem. 104, 71 (1980). 7. B. Reinhammar and Y. Oda, J. Inorg. Biochem. 11,115 (1979). 8. R. Malkin, B. G. Malmstr6m, and T. V~inng~rd,Eur. J. Biochem. 7,253 (1969). 9. M.T. Graziani, L. Morpurgo, G. Rotilio, and B. Mondov(,FEBSLett. 70, 87 (1976). 10. L. Morpurgo, M. T. Graziani, A. Finazzi-Agr8, G. Rotflio, and B. Mondov(,Biochem. J. 187, 361 (1980). 11. L. Morpurgo, M. T. Graziani, A. Desideri, and G. Rotilio, Biochern. J. 187,367 (1980). 12. L. Morpurgo, A. Desideri, G. Rotilio, and B. MondivLFEBSLett. 113,153 (1980). 13. L. Morpurgo, M. T. Graziani, L. Avigliano, A. Desideri, and B. Mondovi, lsraelJ. Biochem. 21, 26 (1981).
121
S p e c t r o s c o p i c P r o p e r t i e s o f M e t a l - D e p l e t e d T r e e Laccase
14. C.D. Lu Bien, M. E. Winkler, T. J. Thamann, R. A. Scott, M. S. Co., K. O. Hodgson, and E. I. Solomon, J. Amer. Chem. Soc. 103, 7014 (1981). 15. B.M. Kanne, personal communication, April 1979. 16. E.M. Fielden, P. B. Roberts, R. C. Bray, D. J. Lowe, G. N. Mautner, G. Rotilio, and L. Calabrese, Biochem. J. 139, 49 (1974). 17. B. Reinhammar, Biochim. Biophys. Acta 205, 35 (1970). 18. P.E. Brumby and V. Massey, inMethods ofEnzymology, vol. 10, R. W. Estabrook and M. R. Pullman, Eds., Academic Press, New York, 1967, p. 463. 19. B.G. MalmstriSm, B. Reinhammar, and T. Vanngard, Btochtm. Biophys. Acta 205, 48 (1970). 20. B. Reinhammar, R. Malkin, P. Jensen, B. Karlsson, L.-E. Andre'asson, R. Aasa, T. Vanngard, and B. G. MalmstRim, J. Biol. Chem. 225, 5000 (1980). 21. J.A. Larrabee, G. Woolery, B. Reinhammar, and T. Spiro, unpublished results. 22. F.E. Dwulet and F. W. Putnam, Proc. Natl. Acad. Sci. USA 78, 2805 (1981). 23. B. Reinhammar, J. lnorg. Biochem. 15, 27 (1981). 24. A. L. M. Schott Uiterkamp, H. van der Deen, H. C. J. Berendsen, and J. F. Boas, Biochim. Biophys. Acta 372,407 (1974). ..
O
.
.
..
Received April 5, 1982; accepted June 4, 1982
O