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Two forms of copper (II) in fungal laccase
BIOCHIMICA ET BIOPHYSICA ACTA
67
BBA 25911 TWO FORMS OF C O P P E R (II) IN F U N G A L LACCASE BOG. MALMSTROM, BENGT REINHAMMAR AND TORE VANNG~RD Depar/ment of Biochemistry, University of GSteborg and Chalmers Institute of Technology, Gdteborg (Sweden)
(Received September I9th, 1967)
SUMMARY I. The nature of different forms of copper in fungal laccase (p-diphenol:O 2 oxidoreductase, EC 1.10.3.2 ) and their possible role in the catalytic activity has been studied b y electron paramagnetic resonance (EPR) and proton relaxation measurements. 2. By comparisons between experimental and simulated E P R spectra at 9 and 35 GHz it is shown t h a t both laccase A and B contain two forms of Cu 2+, each corresponding to I atom per mole of protein. One (represented b y Signal I) corresponds to the spectrally unique form described earlier and shows a small deviation from axial symmetry, while the other one (Signal 2) represents the form earlier attributed to denatured molecules. D a t a obtained here suggest that both forms are present in the same protein molecule. 3- Proton relaxation measurements show that neither form binds easily exchangeable water. However, the form with Signal 2 gives the largest contribution to the enhancement, indicating that it is more available. This is supported by the finding that only this form is affected b y various solutes. 4. Denaturation of the enzyme with guanidine-HCI destroys Signal I as well as the characteristic absorption at 61o m/z. Treatment with azide, on the other hand, decreases Signal 2 without affecting the blue colour. 5. The rates of reduction b y quinol of Cu 2+ giving Signals I and 2, as well as of the corresponding forms in ceruloplasmin, and of added Cu 2+ have been measured. Signal I is reduced much more rapidly than Signal 2, but this still reacts considerably faster than added Cu 2+. 6. Treatment of the enzyme with cyanide produces, with concomitant loss of oxidase activity, an entirely new E P R spectrum corresponding to 4 ° % of the original intensity. The treatment is entirely reversible which indicates that Cu 2+ is still at its original binding site. The new spectrum suggests coordination of Cu 2+ to three to four N atoms.
INTRODUCTION Earlier studies (see ref. I for a summary) have established that fungal laccase (p-diphenol:O 2 oxidoreductase, EC I.IO.3.2), as well as the related protein, ceruloAbbreviation: EPR, electron paramagnetic resonance. Biochim. Biophys. Acta, 156 (1968) 67-76
68
BO. G. MALMSTROM, B. REINHAMMAR, T. V~.NNGARD
plasmin, contains at least two forms of copper. One gives a characteristic electron paramagnetic resonance (EPR) signal ~ and is consequently Cu 2+, while the other one is not detectable by the E P R technique 3 and appears 4 to be Cu +. The E P R signals of all purified samples of both proteins show, in addition to the usual 4 hyperfine lines of the Cu~+ spectrum, an extra line at low magnetic field, as first described for ceruloplasmin 3. One way to explain this is to assume that there is a superposition of spectra from Cu 2+ in two different environments, as recently discussed in the case of ceruloplasnlin 5. In the present communication we intend to demonstrate that the laccase E P R spectrum is also due to two forms of Cu 2+. Data concerning the relation between these two forms and their role in the catalytic activity will be presented. These data involve the nature of the E P R signal at 9 and 35 GHz, the saturation behaviour of the X-band signal, the enhancement of proton relaxation and the rate of reduction of Cu 2+ by substrate. In addition, certain effects of treatment with guanidine, azide or cyanide have been utilized. The latter has also given some information concerning ligand groups involved in the binding of the metal. MATERIALS AND METHODS
Proteins and chemicals Fungal laccase A and B were prepared by the method of F2XHRAEUSAND REINHAMMAR6. It was stored frozen in o.I M sodium phosphate buffer, pH 6.0, and most experiments have been performed with this buffer. When other buffers were used, the buffer change was accomplished by dialysis in the cold of a few ml of laccase solution against I 1 of the new buffer with stirring for several hours. Ceruloplasmin was obtained from AB Kabi, Stockholm (we are indebted to Mr. H. BJ6RLING for generous gifts of this material; cf. refs. 3 and 7). Deionized water was used for making solutions. Buffer solutions were purified by dithizone extraction (o.oi % dithizone in CC14). Guanidine- HC1 was prepared from the carbonate and purified according to ANSONs. In all other cases, reagent grade chemicals were used without purification.
E P R and other spectral measurements E P R measurements were made at 77 °K in a Varian E- 3 spectrometer at X-band and at about IiO °K in a Varian V-45o3 spectrometer at about 35 GHz. Visible and ultraviolet spectra were recorded at 25 ° in a Zeiss RPQ 2oA recording spectrophotometer with a I-cm light path.
Measurements of proton relaxation times The longitudinal relaxation time T 1 of the water protons was measured by the fast adiabatic passage method using a Varian DP-60 spectrometer. The measurements have been described in detail elsewhere 9.
Analytical methods Copper analyses were performed with tile biquinoline method 10, as described earlier 3 except that ascorbie acid rather than quinol was used as the reducing agent. The enzyme activity was determined at 25 ° as described earlier 3 except that a Zeiss PMQ I I spectrophotometer, coupled to a potentiometric recorder, was employed. Biochim. Biophys. Acta, 156 (1968) 67-76
COPPER (II)
69
IN FUNGAL LACCASE
RESULTS
Spectral properties of the untreated enzyme The E P R spectra at 9 and 35 GHz of a typical preparation of laccase A are given in Figs. Ia and 2a. At X band, in addition to the spectrum with the narrow hyperfine splitting reported earlier (Signal 1) 3, a relatively strong line is seen at about 2700 gauss (part of Signal 2), which represents the form earlier attributed to denatured molecules n. This indicates that the recorded spectrum represents the superposition of signals from two forms of Cu 2+. A reasonable description of the spectrum can be made on the basis of this assumption, as shown by the simulated spectra ~2 in Figs. Ib and 2b. These have been calculated using the resonance parameters given in the caption to Fig. I. The total intensities of Signals I and 2 have been assumed to be the same. In the experimental 35 GHz spectrum, Fig. 2a, the "perpendicular" peak shows a splitting that cannot be accounted for in case of axial symmetry. Thus, Signal I has Igx--gv[ ~ 0.02. Signal 2 has such low amplitude that only an upper limit can be given for its deviation from axial symmetry, rgx--gvl < 0.o4. All our purified preparations of laccase A examined so far have shown the same relative amounts of the two signals, within the accuracy of this estimation. For example, in two cases analyzed with particular care, the total E P R absorption corresponded to 44 and 45 %, respectively, of the total copper content, while Signal I, /,
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Fig. I. E x p e r i m e n t a l (a) and s i m u l a t e d (b) E P R X - b a n d spectra of laccase. S p e c t r u m (a) w a s recorded at 77 ° K w i t h 0.3 mM laccase in o , i o M s o d i u m p h o s p h a t e buffer, p H 6.o. In S p e c t r u m (b) Signal I (dotted) h a s g , = 2.19o, g± = 2.o42, IA,,I = 88 gauss, [1.1 = ~o gauss and t h e line w i d t h 37-5 gauss; Signal 2 ldashed) has g, = 2.262, g± = 2.036, ]A Ill = ~ I 6 9 gauss, IA±l = 3 ° gauss and t h e line w i d t h 7 ° gauss. B o t h spectra s i m u l a t e d using Gaussian shape. T h e y correspond to t h e s a m e n u m b e r of u n p a i r e d spins a n d t h e full line in (b) is their sum. M i c r o w a v e frequency, 92o6 M H z . P a r t of spectra also s h o w n w i t h i o t i m e s higher gain (a' and b'). Fig. 2. E P R spectra of laccase recorded at 34.I G H z , (a) e x p e r i m e n t a l and (b) p a r t of a s i m u l a t e d s p e c t r u m . S p e c t r u m (a) recorded at a b o u t I i o ° K w i t h t h e s a m e s a m p l e as in Fig. IS. S p e c t r u m b is t h e s u m of Signals I and 2 w i t h t h e s a m e p a r a m e t e r s as in Fig. 1, except t h a t the line w i d t h s are 60 and 15o gauss for Signals I and 2, respectively.
Bioahim. Biophys. Acta, 156 (1968) 6 7 - 7 6
70
BO. G. MALMSTROM, B. REINHAMMAR, T. VANNGARD
estimated as in ref. 5, accounted for 55 and 53 %, respectively, of the total signal. It has been found that laccase B also shows the same distribution among the various forms of copper. Thus, one sample gave a Cu ~+ content which represented 47 % of the total copper, and 52 % of the total Cu2+ corresponded to Signal I. It has not been possible to remove either Signal I or Signal 2 by passage of the protein solution through a column of chelating resin (Chelex ioo) equilibrated with 0.5 M sodium acetate at pH 6.0. The E P R spectrum obtained is dependent on the buffer used, as shown in Fig. 3. It appears that only Signal 2 is affected by the buffer. The saturation behaviour at X-band of the two signals, as well as of the signal from Cu2+ added to the enzyme, was studied in o.i M triethylamineacetic acid buffer, pH 5-5, the same buffer as has been used in all kinetic experiments. Added Cu2+ is probably present mostly as the acetate complex under these conditions. No absolute measurements of the saturation properties have been made as these are difficult with the present design of the spectrometer bridge. However, it is clear that the signal from added Cu 2+ saturates much more strongly than either Signal I or 2. These two signals show the same saturation behaviour. Earlier results 3 with the related protein, ceruloplasmin, would suggest that the strong blue colour of laccase (see Fig. 6) is related only to Cu2+ giving Signal I. Therefore, the extinction coefficient was calculated on the basis of this fornl of copper. A total of twelve different preparations of laccase A were examined but, as already mentioned, particularly careful integrations and analyses were performed in two cases. For these two samples, the estimated extinction coefficients at 61o mff for Cu2+ giving Signal I are 5200 and 5300 l'mole -~'cm -~, respectively. For one sample of laccase B a value of 5000 was found. [
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2800 3000 3200 Magnetic field ( g a u s s )
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Fig. 3. E P R spectra at 77 ° K of laccase in three different buffers, sodium phosphate (a), sodium acetate (b), and sodium citrate (c), all o . i M, p H 5.5. Microwave frequency, 915o MHz. T h e lowfield part also s h o w n with higher gain (a', b ' a n d c'). Fig. 4. E P R spectra recorded at 77 ° K of untreated laccase (a) and laccase treated with guanidine. HC1, 2 M (b), a n d 3 M (c). Laccase concentration, 0.2 m M in o.i M sodium acetate buffer, p H 5.5. Microwave frequency, 915o M H z . Modulation amplitude in (a), (b) a n d (c), io gauss, in (a'), (b') a n d (c'), 3 ° gauss. Amplifier gain setting in (a), (b) a n d (c), 8- lO 4, in (a'), 2. 5. IO~ and in (b') a n d (c'), IO~.
Biochim. Biophys. Acta, 156 (1968) 67-76
COPPER
(II)
IN FUNGAL LACCASE
71
The results of proton relaxation measurements on the untreated enzyme are given in Table I, which also includes effects of various treatments described in later sections. The effect of E D T A is probably not due to removal of extraneous copper, as it has been found that EDTA can cause reduction of laccase copper, accompanied by a bleaching. TABLE i PROTON RELAXATION OF LACCASE (2.95 mM IN TOTAL COPPER) AT 60 MHz, AND 25 ° The e n h a n c e m e n t v a l u e s are referred to a 2.95 mM a q u e o u s s o l u t i o n of CuCI~.
Sample
Enhancement factor
Untreated enzyme W i t h 5 mM E D T A W i t h 25 m M quinol, I rain W i t h 25 mM quinol, 9 mill W i t h 56 mM ascorbic acid, 1.25 m i n W i t h 56 mM ascorbic acid, 7.25 rain W i t h 20 mM CN-, I rain W i t h 20 mM CN-, 11. 5 rain
o.45 o.37 o.28 0.20 0.33 O.ll 0.30 0.28
Denaturation with guanidine. HCl Treatment of the enzyme with guanidine. HC1 destroys Signal I, as shown in Fig. 4. At 2 M guanidine concentration (Fig. 4b) almost all Cu 2+ appears to be converted to a form giving a spectrum similar to Signal 2. Also some Cu + seems to be oxidized, as the total intensity increases b y 30 %. At the same time as the E P R spectrum is changed, the strong blue colour is lost. There is little evidence of denaturation b y other criteria, such as ultraviolet difference spectra or sedimentation velocities 13 at this low guanidine concentration (2 M). At 3 M guanidine concentration (Fig. 4 c) the change is more rapid, but there is also other evidence of denaturation.
The effect of azide and cyanide Treatment of the enzyme with azide causes a large reduction of Signal 2 (Fig. 5) without any decrease in colour (Fig. 6). Thus, the extinction coefficient at 61o mff calculated on the basis of the total concentration of Cu ~+ seen in E P R increases b y a factor of 1.26. The treatment is accompanied b y an increase in absorption at shorter wavelengths (Fig. 6). When higher concentrations of azide and longer times are used, both Signals I and 2, as well as the absorption at 61o mff, are decreased while the short wavelength band continues to increase; there is an isosbestic point close to 530 mff. These spectral changes seem to be irreversible. Treatment of the enzyme with cyanide causes marked changes in the E P R spectrum. At low concentrations (3 raM) Signal I is reduced, as seen in Fig. 7b (note the higher gain in Fig. 7 b as compared to Fig. 7a). The low-field line of Signal 2 changes its shape, but it is seemingly also reduced in intensity. On further treatment (longer times and higher concentrations), however, there is not a complete reduction. Instead the remaining Cu 2+ is converted into a new form, which gives an entirely different E P R spectrum (Fig. 7c), with g,---- 2.16, g± = 2.04, IA,l ~- 22o gauss and Biochim. Biophys. Acta, 156 (1968) 6 7 - 7 6
72
BO. G. MALMSTROM, B. REINHAMMAR, T. V,'~_NNGARD
w i t h N hyperfine structure. The E P R i n t e n s i t y corresponds to a b o u t 4o % of t h e original Cu ~+ content. On r e m o v a l of t h e c y a n i d e b y dialysis, or just s h a k i n g at
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Fig. 5. E P R s p e c t r a of u n t r e a t e d laccase (a) a n d laccase t r e a t e d w i t h azide (b) recorded a t 77 ° K a n d a m i c r o w a v e f r e q u e n c y of 915oM Hz. P r o t e i n c o n c e n t r a t i o n , o.I raM. S a m p l e for S p e c t r u m (b) t r e a t e d w i t h 0.89 nlM s o d i u m azide for 2 rain, all in o.i M s o d i u m p h o s p h a t e buffer, p H 6.0. Fig. 6. Optical a b s o r p t i o n of u n t r e a t e d laccase (a), a n d laccase t r e a t e d w i t h azide (b), a n d c y a n i d e (c a n d d). All s p e c t r a n o r m a l i z e d to a p r o t e i n c o n c e n t r a t i o n of o.19 m M w h i c h was t h e a c t u a l c o n c e n t r a t i o n for t h e c y a n i d e e x p e r i m e n t ; half this c o n c e n t r a t i o n w a s u s e d for t h e azide experim e n t s . F o r S p e c t r u m b, laccase w a s t r e a t e d w i t h 0.89 m M s o d i u m azide for 2 rain, for Spectra c a n d d, laccase was t r e a t e d w i t h p o t a s s i u m cyanide, 3 m M for 7 ° m i n a n d 4 ° m M for 175 min, respectively. All s o l u t i o n s c o n t a i n e d o.i M p h o s p h a t e buffer, p H 6.0.
2600
2~300 BOO0 3200 Magnetic field (gauss)
3400
Fig. 7. E P R s p e c t r a a t a b o u t 9o ° K of u n t r e a t e d laccase (a), a n d laccase t r e a t e d with increasing a m o u n t s of c y a n i d e (b a n d c). C o n c e n t r a t i o n s a n d t i m e s as in Figs. 6a, 6c, a n d 6d, respectively. Microwave f r e q u e n c y , 9206 MHz. T h e gain in (b) is 2.1 t i m e s t h a t in (a). M o d u l a t i o n a m p l i t u d e in (a) a n d (b), 25 gauss, in (c), 8 gauss.
Biochim. Biophys. dcta, 156 (1968) 67-76
(II)
COPPER
IN
FUNGAL LACCASE
73
pH 5.5, the effect on both signals is completely reversed and the spectrum in Fig. 7 a is again obtained. The cyanide-treated enzyme is completely inactive but the activity is also fully regained on removal of the cyanide. Table I gives proton-relaxation data for the cyanide-treated enzyme.
Reduction experiments The relative rates of reduction of Cu 2+ giving Signals I and 2 in laccase and ceruloplasmin, as well as of added Cu 2+, by a substrate, quinol, was studied in simple kinetic experiments. The protein solution (300 tA) was first pipetted into an E P R sample tube. 30/zl of 0.55 M quinol were then rapidly added from a piece of thin plastic tubing attached to a syringe. The solutions were mixed and rapidly frozen. The time from the addition of substrate to complete freezing was about 0.5 min. The E P R spectrum was recorded. The solution was rapidly thawed by immersion of the sample tube in lukewarm water. It was then allowed to stand for 2 min, refrozen, and the E P R spectrum was again recorded. TABLE
II
THE REDUCTION OF VARIOUS FORMS OF C u 2+ IN LACCASE AND CERULOPLASMIN BY QUINOL IN O.I M TRIF,TH•LAMINE--ACETATE BUFFER, pI-I 5.5, AND 223 The protein
concentrations
Treatment
w e r e a p p r o x . 0.20 r a M . A b o u t 0.24 m M Cu ~+ w a s a d d e d in o n e c a s e .
Relative signal Laccase
Untreated 5 ° m M q u i n o l , o. 5 m i n 5 ° m M q u i n o l , 2. 5 m i n
Ceruloplasmin
Signal I
Signal 2
Signal I
Signal 2
lOO <3 <3
IOO 64 45
ioo 3° 5
ioo 65 5°
Added
IOO 90 --
The change with time after addition of substrate of the E P R signals, estimated for Signal I from the amplitudes of the "perpendicular" and hyperfine lines and for Signal 2 from the area of the low-field line 5, is shown in Table II. It is seen that in laccase Signal I is completely reduced before the first measurement, while an estimate of the rate of reduction of all other signals can be made. The colour of laccase is completely lost on reduction of Signal I alone. It should be noted that the residual signal after reduction of Signal I in laccase is not identical with Signal 2. Thus, the presence of substrate affects Signal 2. However, with respect to saturation the residual signal behaves as that of the untreated enzyme. Ascorbic acid was found to reduce Signal 2 more slowly than quinol. Proton relaxation data for samples reduced in different ways are given in Table I. It can be seen that under conditons where Signal I is fully reduced the enhancement is always more than half of that for the untreated enzyme. This is true even in the case of quinol when part of Signal 2 has also disappeared at the shortest time. This shows that Cu 2+ giving Signal 2 makes the largest contribution to the enhancement. Biochim. Biophys. Acta, 156 (1968) 6 7 - 7 6
74
s o . G. MALMSTROM, B. REINHAMMAR, T. VANNGARD
DISCUSSION
The two forms of Cu 2+ described here have been found in all preparations of fungal laccase examined so far. A similar heterogeneity has also been described for Cu 2+ in ceruloplasmin ~& For this protein evidence was presented 5 that both forms exist in a single molecule. The experiments described here do not directly show if this is also true in the case of laccase or if there are instead two types of molecules, one giving Signal i and the other Signal 2. However, various experimental findings taken together give strong indirect support for the view that every laccase molecule contains one ion of each form of Cu2+. It has not been possible, despite extensive efforts, to separate the two signals by protein fractionation procedures. A I : I ratio between the signals has ahvays been obtained. Furthermore, laccase exists as two electrophoretically distinct isoenzymes, laccase A and B, and in the B enzyme there is also one Cu ~+ giving Signal I and one giving Signal 2. It has been repeatedly pointed out (see ref. I) that Signal I represents Cu 2+ in a rather unique chemical environment. This unique bonding is also responsible for the strong blue colour of laccase, which is entirely due to the Cu2÷ giving Signal I, as shown by the denaturation (@ ref. 3) and reduction experiments as well as by the treatment with azide. The molar extinction coefficient at 610 m~ (about 52oo), calculated on the basis of this form of Cu 2+, is probably very close to that for ceruloplasmin; the reported value (44oo) '4 should be corrected upwards, as it was based on total Cu 2÷ content without consideration of the presence of Signal 2 (ref. 5)- As the E P R parameters are essentially the same 2, both proteins appear to contain Cu 2+ bonded in the same manner. It should be noted that in both cases it is this form of Cu 2+ which is most rapidly reduced by substrates (Table II). Signal 2 represents a more normal E P R spectrum for a Cu 2+ complex. However, several lines of evidence show that it is due to a form of specifically bound Cu 2+ and not just extraneous copper. This form is always present in stoichiometric amounts (one ion per molecule), and it is not removed by dialysis or treatment with a chelating resin. Signal 2 shows the same saturation behaviour as Signal I, whereas the signal due to added Cu 2+ is much more easily saturated. While Signal 2 is more slowly reduced by substrate than Signal I, the reduction is faster than that of added Cu 2÷ (Table II). As the enhancement value for laccase under all conditions (Table I) is quite low for protein-bound Cu 2+, neither form of Cu 2+ can bind an easily exchangeable water molecule. On the other hand, several experimental findings indicate that the ion giving Signal 2 is more available than that giving Signal I. First, Cu 2+ giving Signal 2 is responsible for the greater part of the enhancement. Second, only Signal 2 is affected by buffer anions (Fig. 2). Quinol also changes Signal 2 but its possible effect on Signal I cannot be tested as this is too rapidly reduced. Despite the apparent greater availability of Signal-2 Cu 2÷, the other Cu 2+ accepts electrons from a good substrate, such as quinol, more readily (Table II). Azide, on the other hand, reduces Signal 2 befo, e Signal I (Fig. 5). The electron transfer to Signal-I Cu 2+ with a good substrate undoubtedly takes place in a specific enzymesubstrate complex, as indicated by the fact that the oxidation follows MichaelisMenten kinetics (A. LINDBERGAND ]~. G. MALMSTROM,unpublished). Azide, and other reducing agents which are not good substrates, are probably incapable of interacting Biochim. Biophys. Acta, 156 (1968) 67-76
COPPER (II) iN FUNGAL LACCASE
75
with the specific substrate-binding site and then naturally would react most readily with the more available Cu ~+. The finding that ascorbic acid reduces Signal 2 more quickly than quinol despite the fact t h a t it is a much poorer specific substrate (the Michaelis constant is about IO times larger than that for quinol) is also consistent with this view. As only Signal I is rapidly reduced by good substrates, it appears difficult to visualize the function, if any, of the other form of Cu 2+. Signal 2 is similar to the signal obtained from Signal I when laccase is mildly denatured (see Fig. 4; cf. ref. 3), and one m a y raise the question of whether it is in fact formed by partial denaturation of the enzyme (@ ref. II). The results with laccase b y themselves give little justification for such a suggestion, as no preparations giving less (or more) than one Cu 2+ of Signal-2 type have ever been obtained. However, recent findings with ceruloplasmin 5 indicate that this possibility must be considered. The amount of Signal 2 in ceruloplasmin preparations used in the work of ref. 5 corresponded to one Cu ~ . However, in one sample of serum this signal was found to be absent. Thus, it appears that one site is more labile than the other ones. In view of other close similarities between the two proteins, such a situation m a y then exist also in laccase. In such a case the laccase formed by the fungus should initially give only Signal i. However, it has been impossible to test this directly on the culture medium, as this must contain relatively high concentrations of CuSQ. Even if Signal I is relatively stable, the denaturation experiments show that it is destroyed under milder conditions than those required to denature the enzyme grossly. This lability of the binding site m a y be related to the fact1, TM that it is distorted from the usual square planar geometry found in complexes of Cu 2+. This is evident both from optical rotatory dispersion experiments of ceruloplasmin TM and the 35 GHz results of laccase reported here. As this distortion is believed to be the basis of the unique spectral properties, which, in turn, can be related to the oxidase function of the proteins (see ref. I), this lability m a y be a general property of the blue coppercontaining oxidases. Denaturation to different extents m a y then be the cause of apparent discrepancies in spectral properties reported for the same enzyme prepared in different laboratories (cf., for example, refs. 17 and 18). It m a y be noted that published E P R spectra TMof laccase from lacquer trees also show the presence of two forms of Cu 2+. In the cyanide experiments the new signal obtained on treatment at high concentrations of cyanide for long periods of time clearly shows the presence of nitrogen hyperfine coupling. Furthermore, the general appearance of the N hyperfine structure and the resonance parameters are very similar to those of the Cu 2+ complex of triglycylglycine 9 at p H > 9 (g~ = 2.172, g± = 2.o41, [A~I = 206 gauss with the isotope 63Cu). In this complex the Cu ~+ is coordinated to four nitrogens (three from the peptide bonds and one from the ~-amino group). In view of this similarity, it seems likely that Cu 2+ in cyanide-treated laccase is coordinated to three to four nitrogens. As cyanide molecules, unless they form bridges between metal ions TM, are thought to coordinate to metal ions through the carbon end ~9, these nitrogens are probably supplied b y the protein. The cyanide treatment is completely reversible, suggesting that the Cu 2+ in the cyanide-treated protein is still at the original binding site. As the particular cyanide spectrum is obtained at a comparatively low pH, it is somewhat unlikely that all nitrogens would come from peptide bonds (cf. ref. 9), although Biochim. Biophys. Acta, 156 (1968) 67-76
76
BO. G. MALMSTROM, B. REINHAMMAR, T. V.'~NNGARD
t h e specific a r r a n g e m e n t in a p r o t e i n c o u l d m a k e t h i s k i n d of c o m p l e x to f o r m at a m u c h l o w e r p H t h a n w i t h s m a l l p e p t i d e s . T h u s , it s e e m s m o r e r e a s o n a b l e to h a v e n i t r o g e n s f r o m one or m o r e i m i d a z o l e g r o u p s also i n v o l v e d in t h e b i n d i n g . U n f o r t u n a t e l y , it is n o t possible on t h e basis of p r e s e n t d a t a to d e c i d e if it is t h e Cu 2+ s h o w i n g S i g n a l I or 2, or b o t h , in t h e u n t r e a t e d p r o t e i n t h a t g i v e s rise to t h e n e w signal on c y a n i d e t r e a t m e n t . More e x p e r i m e n t s are n e e d e d to c l a r i f y t h i s i m p o r t a n t p o i n t as are e x p e r i m e n t s w i t h Cl5N, w h i c h w o u l d g i v e a d e f i n i t e a n s w e r to t h e q u e s t i o n w h e t h e r t h e n i t r o g e n of t h e c y a n i d e c o o r d i n a t e s or not. ACKNOWLEDGEMENTS W e w i s h to t h a n k Mr. S.-O. FALKBRING for v a l u a b l e t e c h n i c a l assistance. T h e s t u d y has b e e n s u p p o r t e d b y g r a n t s f r o m t h e K n u t a n d Alice W a l l e n b e r g F o u n d a t i o n , t h e S w e d i s h N a t u r a l S c i e n c e R e s e a r c h Council, t h e I n s t i t u t e of G e n e r a l M e d i c a l S c i e n c e s of t h e U.S. P u b l i c H e a l t h S e r v i c e (GM I2 280-03) a n d t h e A g r i c u l t u r a l R e s e a r c h S e r v i c e of t h e U.S. D e p a r t m e n t of A g r i c u l t u r e ( F G - S w - I o 7 ) . O n e of us (T.V.) a c k n o w l e d g e s a special r e s e a r c h p o s i t i o n of t h e S w e d i s h N a t u r a l Science R e s e a r c h Council.
REFERENCES 1 B. G. MALMSTROM, in T. E. KING, 1~. S. MASON AND ),]7. •ORRISON, Oxidases and Related Redox Systems, John Wiley, New York, 1965, p. 207. 2 B. G. MALMSTROM AND T. VANNG.~RD, dr. Mol. Biol., 2 (196o) 118. 3 L. BROMAN, B. G. MALMSTR~M, R. AASA AND T. VANNG.~-RD, J. Mol. Biol., 5 (1962) 3Ol. 4 A. EHRENBERG, B. G. MALMSTR6M, L. BROMAN AND R. MOSBACH,J. iV[ol. Biol., 5 (1962) 45 o. 5 T. V~NNG;XRD, in A. EHRENBERG, B. G. MALMSTROM AND T. VANNG.~RD, i~/Iagnetic Resonance in Biol. Systems, Pergamon Press, Oxford, 1967, p. 213. 6 G. F.~_HRAEUSAND B. REINHAMMAR,Acla Chem. Scan&, in the press. 7 L. BROMAN, B. G. MALMSTROM, R. AASA AND T. VANNG.~RD, Biochim. Biophys. Acta, 75 (1963) 365 • 8 M. L. ANSON, J. Gen. Physiol., 24 (1941) 3999 K.-E. FALK, H. C. FREEMAN, B. G. MALMSTR6M AND T. V~NNG.&RD, J. A m . Chem. Soc., 89
(1967) 6o71. IO G. FELSENFELD, Arch. Biochem. Biophys., 87 (196o) 247. i i B. G. MALMSTRDM,R. AASA AND T. V~NNGARD, Biochim. Biophys. Acta, IiO (1965) 431. 12 T. V£NNG3,RO AND R. AASA, in W. Low, Paramagnetic Resonance, Academic Press, New York, 1963, p. 509. 13 J. BuTzow, to be published. 14 \V. E. BLUMBERG, J. EISINGER, P. AISEN, A. G. MORELL AND 1. H. SCHEINBERG, J. Biol.
Chem., 238 (1963) 1675. 15 J. PEISACH AND W. E. BLUMBERG, Federation Pro&, 26 (1967) 834. 16 W. E. BLUMBERG, in J. PEISACH, P. AISEN AND W. E. BLUMBERG, The Biochemistry of Copper, Academic Press, New York, 1966, p. 49. 17 W. E. BLOMBERG, W. G. LEVlNE, S. MARGOLIS AND J. PEISAEH, Biochem. Biophys. Res. Commun., 15 (1964) 277. 18 Z. I~AKAMURAAND Y. OGURA, in J. PEISACH, P. AISEN AND W. E. ]~LUMBERG, The Biochemistry of Copper, Academic Press, New York, 1966, p. 389. 19 D. F. SHRIVER, in C. K. JORGENSEN, J. B. NEILANDS, ~R. S. NYHOLM, D. REINEN AND R. J. p. WILLIAMS, Structure and Bonding, Vol. i, Springer, Berlin, 1966, p. 32. Biochim. Biophys. Acta, 156 (1968) 67-76
67
BBA 25911 TWO FORMS OF C O P P E R (II) IN F U N G A L LACCASE BOG. MALMSTROM, BENGT REINHAMMAR AND TORE VANNG~RD Depar/ment of Biochemistry, University of GSteborg and Chalmers Institute of Technology, Gdteborg (Sweden)
(Received September I9th, 1967)
SUMMARY I. The nature of different forms of copper in fungal laccase (p-diphenol:O 2 oxidoreductase, EC 1.10.3.2 ) and their possible role in the catalytic activity has been studied b y electron paramagnetic resonance (EPR) and proton relaxation measurements. 2. By comparisons between experimental and simulated E P R spectra at 9 and 35 GHz it is shown t h a t both laccase A and B contain two forms of Cu 2+, each corresponding to I atom per mole of protein. One (represented b y Signal I) corresponds to the spectrally unique form described earlier and shows a small deviation from axial symmetry, while the other one (Signal 2) represents the form earlier attributed to denatured molecules. D a t a obtained here suggest that both forms are present in the same protein molecule. 3- Proton relaxation measurements show that neither form binds easily exchangeable water. However, the form with Signal 2 gives the largest contribution to the enhancement, indicating that it is more available. This is supported by the finding that only this form is affected b y various solutes. 4. Denaturation of the enzyme with guanidine-HCI destroys Signal I as well as the characteristic absorption at 61o m/z. Treatment with azide, on the other hand, decreases Signal 2 without affecting the blue colour. 5. The rates of reduction b y quinol of Cu 2+ giving Signals I and 2, as well as of the corresponding forms in ceruloplasmin, and of added Cu 2+ have been measured. Signal I is reduced much more rapidly than Signal 2, but this still reacts considerably faster than added Cu 2+. 6. Treatment of the enzyme with cyanide produces, with concomitant loss of oxidase activity, an entirely new E P R spectrum corresponding to 4 ° % of the original intensity. The treatment is entirely reversible which indicates that Cu 2+ is still at its original binding site. The new spectrum suggests coordination of Cu 2+ to three to four N atoms.
INTRODUCTION Earlier studies (see ref. I for a summary) have established that fungal laccase (p-diphenol:O 2 oxidoreductase, EC I.IO.3.2), as well as the related protein, ceruloAbbreviation: EPR, electron paramagnetic resonance. Biochim. Biophys. Acta, 156 (1968) 67-76
68
BO. G. MALMSTROM, B. REINHAMMAR, T. V~.NNGARD
plasmin, contains at least two forms of copper. One gives a characteristic electron paramagnetic resonance (EPR) signal ~ and is consequently Cu 2+, while the other one is not detectable by the E P R technique 3 and appears 4 to be Cu +. The E P R signals of all purified samples of both proteins show, in addition to the usual 4 hyperfine lines of the Cu~+ spectrum, an extra line at low magnetic field, as first described for ceruloplasmin 3. One way to explain this is to assume that there is a superposition of spectra from Cu 2+ in two different environments, as recently discussed in the case of ceruloplasnlin 5. In the present communication we intend to demonstrate that the laccase E P R spectrum is also due to two forms of Cu 2+. Data concerning the relation between these two forms and their role in the catalytic activity will be presented. These data involve the nature of the E P R signal at 9 and 35 GHz, the saturation behaviour of the X-band signal, the enhancement of proton relaxation and the rate of reduction of Cu 2+ by substrate. In addition, certain effects of treatment with guanidine, azide or cyanide have been utilized. The latter has also given some information concerning ligand groups involved in the binding of the metal. MATERIALS AND METHODS
Proteins and chemicals Fungal laccase A and B were prepared by the method of F2XHRAEUSAND REINHAMMAR6. It was stored frozen in o.I M sodium phosphate buffer, pH 6.0, and most experiments have been performed with this buffer. When other buffers were used, the buffer change was accomplished by dialysis in the cold of a few ml of laccase solution against I 1 of the new buffer with stirring for several hours. Ceruloplasmin was obtained from AB Kabi, Stockholm (we are indebted to Mr. H. BJ6RLING for generous gifts of this material; cf. refs. 3 and 7). Deionized water was used for making solutions. Buffer solutions were purified by dithizone extraction (o.oi % dithizone in CC14). Guanidine- HC1 was prepared from the carbonate and purified according to ANSONs. In all other cases, reagent grade chemicals were used without purification.
E P R and other spectral measurements E P R measurements were made at 77 °K in a Varian E- 3 spectrometer at X-band and at about IiO °K in a Varian V-45o3 spectrometer at about 35 GHz. Visible and ultraviolet spectra were recorded at 25 ° in a Zeiss RPQ 2oA recording spectrophotometer with a I-cm light path.
Measurements of proton relaxation times The longitudinal relaxation time T 1 of the water protons was measured by the fast adiabatic passage method using a Varian DP-60 spectrometer. The measurements have been described in detail elsewhere 9.
Analytical methods Copper analyses were performed with tile biquinoline method 10, as described earlier 3 except that ascorbie acid rather than quinol was used as the reducing agent. The enzyme activity was determined at 25 ° as described earlier 3 except that a Zeiss PMQ I I spectrophotometer, coupled to a potentiometric recorder, was employed. Biochim. Biophys. Acta, 156 (1968) 67-76
COPPER (II)
69
IN FUNGAL LACCASE
RESULTS
Spectral properties of the untreated enzyme The E P R spectra at 9 and 35 GHz of a typical preparation of laccase A are given in Figs. Ia and 2a. At X band, in addition to the spectrum with the narrow hyperfine splitting reported earlier (Signal 1) 3, a relatively strong line is seen at about 2700 gauss (part of Signal 2), which represents the form earlier attributed to denatured molecules n. This indicates that the recorded spectrum represents the superposition of signals from two forms of Cu 2+. A reasonable description of the spectrum can be made on the basis of this assumption, as shown by the simulated spectra ~2 in Figs. Ib and 2b. These have been calculated using the resonance parameters given in the caption to Fig. I. The total intensities of Signals I and 2 have been assumed to be the same. In the experimental 35 GHz spectrum, Fig. 2a, the "perpendicular" peak shows a splitting that cannot be accounted for in case of axial symmetry. Thus, Signal I has Igx--gv[ ~ 0.02. Signal 2 has such low amplitude that only an upper limit can be given for its deviation from axial symmetry, rgx--gvl < 0.o4. All our purified preparations of laccase A examined so far have shown the same relative amounts of the two signals, within the accuracy of this estimation. For example, in two cases analyzed with particular care, the total E P R absorption corresponded to 44 and 45 %, respectively, of the total copper content, while Signal I, /,
/" / \j'
~l
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/ II
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/., X 10
I
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/
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b /'
/
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I
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~ 10000
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11000 Magnetic field (gauss]
J
I
12000
L
Fig. I. E x p e r i m e n t a l (a) and s i m u l a t e d (b) E P R X - b a n d spectra of laccase. S p e c t r u m (a) w a s recorded at 77 ° K w i t h 0.3 mM laccase in o , i o M s o d i u m p h o s p h a t e buffer, p H 6.o. In S p e c t r u m (b) Signal I (dotted) h a s g , = 2.19o, g± = 2.o42, IA,,I = 88 gauss, [1.1 = ~o gauss and t h e line w i d t h 37-5 gauss; Signal 2 ldashed) has g, = 2.262, g± = 2.036, ]A Ill = ~ I 6 9 gauss, IA±l = 3 ° gauss and t h e line w i d t h 7 ° gauss. B o t h spectra s i m u l a t e d using Gaussian shape. T h e y correspond to t h e s a m e n u m b e r of u n p a i r e d spins a n d t h e full line in (b) is their sum. M i c r o w a v e frequency, 92o6 M H z . P a r t of spectra also s h o w n w i t h i o t i m e s higher gain (a' and b'). Fig. 2. E P R spectra of laccase recorded at 34.I G H z , (a) e x p e r i m e n t a l and (b) p a r t of a s i m u l a t e d s p e c t r u m . S p e c t r u m (a) recorded at a b o u t I i o ° K w i t h t h e s a m e s a m p l e as in Fig. IS. S p e c t r u m b is t h e s u m of Signals I and 2 w i t h t h e s a m e p a r a m e t e r s as in Fig. 1, except t h a t the line w i d t h s are 60 and 15o gauss for Signals I and 2, respectively.
Bioahim. Biophys. Acta, 156 (1968) 6 7 - 7 6
70
BO. G. MALMSTROM, B. REINHAMMAR, T. VANNGARD
estimated as in ref. 5, accounted for 55 and 53 %, respectively, of the total signal. It has been found that laccase B also shows the same distribution among the various forms of copper. Thus, one sample gave a Cu ~+ content which represented 47 % of the total copper, and 52 % of the total Cu2+ corresponded to Signal I. It has not been possible to remove either Signal I or Signal 2 by passage of the protein solution through a column of chelating resin (Chelex ioo) equilibrated with 0.5 M sodium acetate at pH 6.0. The E P R spectrum obtained is dependent on the buffer used, as shown in Fig. 3. It appears that only Signal 2 is affected by the buffer. The saturation behaviour at X-band of the two signals, as well as of the signal from Cu2+ added to the enzyme, was studied in o.i M triethylamineacetic acid buffer, pH 5-5, the same buffer as has been used in all kinetic experiments. Added Cu2+ is probably present mostly as the acetate complex under these conditions. No absolute measurements of the saturation properties have been made as these are difficult with the present design of the spectrometer bridge. However, it is clear that the signal from added Cu 2+ saturates much more strongly than either Signal I or 2. These two signals show the same saturation behaviour. Earlier results 3 with the related protein, ceruloplasmin, would suggest that the strong blue colour of laccase (see Fig. 6) is related only to Cu2+ giving Signal I. Therefore, the extinction coefficient was calculated on the basis of this fornl of copper. A total of twelve different preparations of laccase A were examined but, as already mentioned, particularly careful integrations and analyses were performed in two cases. For these two samples, the estimated extinction coefficients at 61o mff for Cu2+ giving Signal I are 5200 and 5300 l'mole -~'cm -~, respectively. For one sample of laccase B a value of 5000 was found. [
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-
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2800 3000 3200 Magnetic field ( g a u s s )
:3400
L~oo
2~oo ~o'oo ~2oo Magnetic
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field (gauss)
Fig. 3. E P R spectra at 77 ° K of laccase in three different buffers, sodium phosphate (a), sodium acetate (b), and sodium citrate (c), all o . i M, p H 5.5. Microwave frequency, 915o MHz. T h e lowfield part also s h o w n with higher gain (a', b ' a n d c'). Fig. 4. E P R spectra recorded at 77 ° K of untreated laccase (a) and laccase treated with guanidine. HC1, 2 M (b), a n d 3 M (c). Laccase concentration, 0.2 m M in o.i M sodium acetate buffer, p H 5.5. Microwave frequency, 915o M H z . Modulation amplitude in (a), (b) a n d (c), io gauss, in (a'), (b') a n d (c'), 3 ° gauss. Amplifier gain setting in (a), (b) a n d (c), 8- lO 4, in (a'), 2. 5. IO~ and in (b') a n d (c'), IO~.
Biochim. Biophys. Acta, 156 (1968) 67-76
COPPER
(II)
IN FUNGAL LACCASE
71
The results of proton relaxation measurements on the untreated enzyme are given in Table I, which also includes effects of various treatments described in later sections. The effect of E D T A is probably not due to removal of extraneous copper, as it has been found that EDTA can cause reduction of laccase copper, accompanied by a bleaching. TABLE i PROTON RELAXATION OF LACCASE (2.95 mM IN TOTAL COPPER) AT 60 MHz, AND 25 ° The e n h a n c e m e n t v a l u e s are referred to a 2.95 mM a q u e o u s s o l u t i o n of CuCI~.
Sample
Enhancement factor
Untreated enzyme W i t h 5 mM E D T A W i t h 25 m M quinol, I rain W i t h 25 mM quinol, 9 mill W i t h 56 mM ascorbic acid, 1.25 m i n W i t h 56 mM ascorbic acid, 7.25 rain W i t h 20 mM CN-, I rain W i t h 20 mM CN-, 11. 5 rain
o.45 o.37 o.28 0.20 0.33 O.ll 0.30 0.28
Denaturation with guanidine. HCl Treatment of the enzyme with guanidine. HC1 destroys Signal I, as shown in Fig. 4. At 2 M guanidine concentration (Fig. 4b) almost all Cu 2+ appears to be converted to a form giving a spectrum similar to Signal 2. Also some Cu + seems to be oxidized, as the total intensity increases b y 30 %. At the same time as the E P R spectrum is changed, the strong blue colour is lost. There is little evidence of denaturation b y other criteria, such as ultraviolet difference spectra or sedimentation velocities 13 at this low guanidine concentration (2 M). At 3 M guanidine concentration (Fig. 4 c) the change is more rapid, but there is also other evidence of denaturation.
The effect of azide and cyanide Treatment of the enzyme with azide causes a large reduction of Signal 2 (Fig. 5) without any decrease in colour (Fig. 6). Thus, the extinction coefficient at 61o mff calculated on the basis of the total concentration of Cu ~+ seen in E P R increases b y a factor of 1.26. The treatment is accompanied b y an increase in absorption at shorter wavelengths (Fig. 6). When higher concentrations of azide and longer times are used, both Signals I and 2, as well as the absorption at 61o mff, are decreased while the short wavelength band continues to increase; there is an isosbestic point close to 530 mff. These spectral changes seem to be irreversible. Treatment of the enzyme with cyanide causes marked changes in the E P R spectrum. At low concentrations (3 raM) Signal I is reduced, as seen in Fig. 7b (note the higher gain in Fig. 7 b as compared to Fig. 7a). The low-field line of Signal 2 changes its shape, but it is seemingly also reduced in intensity. On further treatment (longer times and higher concentrations), however, there is not a complete reduction. Instead the remaining Cu 2+ is converted into a new form, which gives an entirely different E P R spectrum (Fig. 7c), with g,---- 2.16, g± = 2.04, IA,l ~- 22o gauss and Biochim. Biophys. Acta, 156 (1968) 6 7 - 7 6
72
BO. G. MALMSTROM, B. REINHAMMAR, T. V,'~_NNGARD
w i t h N hyperfine structure. The E P R i n t e n s i t y corresponds to a b o u t 4o % of t h e original Cu ~+ content. On r e m o v a l of t h e c y a n i d e b y dialysis, or just s h a k i n g at
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400
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Wavelength ( m y }
Fig. 5. E P R s p e c t r a of u n t r e a t e d laccase (a) a n d laccase t r e a t e d w i t h azide (b) recorded a t 77 ° K a n d a m i c r o w a v e f r e q u e n c y of 915oM Hz. P r o t e i n c o n c e n t r a t i o n , o.I raM. S a m p l e for S p e c t r u m (b) t r e a t e d w i t h 0.89 nlM s o d i u m azide for 2 rain, all in o.i M s o d i u m p h o s p h a t e buffer, p H 6.0. Fig. 6. Optical a b s o r p t i o n of u n t r e a t e d laccase (a), a n d laccase t r e a t e d w i t h azide (b), a n d c y a n i d e (c a n d d). All s p e c t r a n o r m a l i z e d to a p r o t e i n c o n c e n t r a t i o n of o.19 m M w h i c h was t h e a c t u a l c o n c e n t r a t i o n for t h e c y a n i d e e x p e r i m e n t ; half this c o n c e n t r a t i o n w a s u s e d for t h e azide experim e n t s . F o r S p e c t r u m b, laccase w a s t r e a t e d w i t h 0.89 m M s o d i u m azide for 2 rain, for Spectra c a n d d, laccase was t r e a t e d w i t h p o t a s s i u m cyanide, 3 m M for 7 ° m i n a n d 4 ° m M for 175 min, respectively. All s o l u t i o n s c o n t a i n e d o.i M p h o s p h a t e buffer, p H 6.0.
2600
2~300 BOO0 3200 Magnetic field (gauss)
3400
Fig. 7. E P R s p e c t r a a t a b o u t 9o ° K of u n t r e a t e d laccase (a), a n d laccase t r e a t e d with increasing a m o u n t s of c y a n i d e (b a n d c). C o n c e n t r a t i o n s a n d t i m e s as in Figs. 6a, 6c, a n d 6d, respectively. Microwave f r e q u e n c y , 9206 MHz. T h e gain in (b) is 2.1 t i m e s t h a t in (a). M o d u l a t i o n a m p l i t u d e in (a) a n d (b), 25 gauss, in (c), 8 gauss.
Biochim. Biophys. dcta, 156 (1968) 67-76
(II)
COPPER
IN
FUNGAL LACCASE
73
pH 5.5, the effect on both signals is completely reversed and the spectrum in Fig. 7 a is again obtained. The cyanide-treated enzyme is completely inactive but the activity is also fully regained on removal of the cyanide. Table I gives proton-relaxation data for the cyanide-treated enzyme.
Reduction experiments The relative rates of reduction of Cu 2+ giving Signals I and 2 in laccase and ceruloplasmin, as well as of added Cu 2+, by a substrate, quinol, was studied in simple kinetic experiments. The protein solution (300 tA) was first pipetted into an E P R sample tube. 30/zl of 0.55 M quinol were then rapidly added from a piece of thin plastic tubing attached to a syringe. The solutions were mixed and rapidly frozen. The time from the addition of substrate to complete freezing was about 0.5 min. The E P R spectrum was recorded. The solution was rapidly thawed by immersion of the sample tube in lukewarm water. It was then allowed to stand for 2 min, refrozen, and the E P R spectrum was again recorded. TABLE
II
THE REDUCTION OF VARIOUS FORMS OF C u 2+ IN LACCASE AND CERULOPLASMIN BY QUINOL IN O.I M TRIF,TH•LAMINE--ACETATE BUFFER, pI-I 5.5, AND 223 The protein
concentrations
Treatment
w e r e a p p r o x . 0.20 r a M . A b o u t 0.24 m M Cu ~+ w a s a d d e d in o n e c a s e .
Relative signal Laccase
Untreated 5 ° m M q u i n o l , o. 5 m i n 5 ° m M q u i n o l , 2. 5 m i n
Ceruloplasmin
Signal I
Signal 2
Signal I
Signal 2
lOO <3 <3
IOO 64 45
ioo 3° 5
ioo 65 5°
Added
IOO 90 --
The change with time after addition of substrate of the E P R signals, estimated for Signal I from the amplitudes of the "perpendicular" and hyperfine lines and for Signal 2 from the area of the low-field line 5, is shown in Table II. It is seen that in laccase Signal I is completely reduced before the first measurement, while an estimate of the rate of reduction of all other signals can be made. The colour of laccase is completely lost on reduction of Signal I alone. It should be noted that the residual signal after reduction of Signal I in laccase is not identical with Signal 2. Thus, the presence of substrate affects Signal 2. However, with respect to saturation the residual signal behaves as that of the untreated enzyme. Ascorbic acid was found to reduce Signal 2 more slowly than quinol. Proton relaxation data for samples reduced in different ways are given in Table I. It can be seen that under conditons where Signal I is fully reduced the enhancement is always more than half of that for the untreated enzyme. This is true even in the case of quinol when part of Signal 2 has also disappeared at the shortest time. This shows that Cu 2+ giving Signal 2 makes the largest contribution to the enhancement. Biochim. Biophys. Acta, 156 (1968) 6 7 - 7 6
74
s o . G. MALMSTROM, B. REINHAMMAR, T. VANNGARD
DISCUSSION
The two forms of Cu 2+ described here have been found in all preparations of fungal laccase examined so far. A similar heterogeneity has also been described for Cu 2+ in ceruloplasmin ~& For this protein evidence was presented 5 that both forms exist in a single molecule. The experiments described here do not directly show if this is also true in the case of laccase or if there are instead two types of molecules, one giving Signal i and the other Signal 2. However, various experimental findings taken together give strong indirect support for the view that every laccase molecule contains one ion of each form of Cu2+. It has not been possible, despite extensive efforts, to separate the two signals by protein fractionation procedures. A I : I ratio between the signals has ahvays been obtained. Furthermore, laccase exists as two electrophoretically distinct isoenzymes, laccase A and B, and in the B enzyme there is also one Cu ~+ giving Signal I and one giving Signal 2. It has been repeatedly pointed out (see ref. I) that Signal I represents Cu 2+ in a rather unique chemical environment. This unique bonding is also responsible for the strong blue colour of laccase, which is entirely due to the Cu2÷ giving Signal I, as shown by the denaturation (@ ref. 3) and reduction experiments as well as by the treatment with azide. The molar extinction coefficient at 610 m~ (about 52oo), calculated on the basis of this form of Cu 2+, is probably very close to that for ceruloplasmin; the reported value (44oo) '4 should be corrected upwards, as it was based on total Cu 2÷ content without consideration of the presence of Signal 2 (ref. 5)- As the E P R parameters are essentially the same 2, both proteins appear to contain Cu 2+ bonded in the same manner. It should be noted that in both cases it is this form of Cu 2+ which is most rapidly reduced by substrates (Table II). Signal 2 represents a more normal E P R spectrum for a Cu 2+ complex. However, several lines of evidence show that it is due to a form of specifically bound Cu 2+ and not just extraneous copper. This form is always present in stoichiometric amounts (one ion per molecule), and it is not removed by dialysis or treatment with a chelating resin. Signal 2 shows the same saturation behaviour as Signal I, whereas the signal due to added Cu 2+ is much more easily saturated. While Signal 2 is more slowly reduced by substrate than Signal I, the reduction is faster than that of added Cu 2÷ (Table II). As the enhancement value for laccase under all conditions (Table I) is quite low for protein-bound Cu 2+, neither form of Cu 2+ can bind an easily exchangeable water molecule. On the other hand, several experimental findings indicate that the ion giving Signal 2 is more available than that giving Signal I. First, Cu 2+ giving Signal 2 is responsible for the greater part of the enhancement. Second, only Signal 2 is affected by buffer anions (Fig. 2). Quinol also changes Signal 2 but its possible effect on Signal I cannot be tested as this is too rapidly reduced. Despite the apparent greater availability of Signal-2 Cu 2÷, the other Cu 2+ accepts electrons from a good substrate, such as quinol, more readily (Table II). Azide, on the other hand, reduces Signal 2 befo, e Signal I (Fig. 5). The electron transfer to Signal-I Cu 2+ with a good substrate undoubtedly takes place in a specific enzymesubstrate complex, as indicated by the fact that the oxidation follows MichaelisMenten kinetics (A. LINDBERGAND ]~. G. MALMSTROM,unpublished). Azide, and other reducing agents which are not good substrates, are probably incapable of interacting Biochim. Biophys. Acta, 156 (1968) 67-76
COPPER (II) iN FUNGAL LACCASE
75
with the specific substrate-binding site and then naturally would react most readily with the more available Cu ~+. The finding that ascorbic acid reduces Signal 2 more quickly than quinol despite the fact t h a t it is a much poorer specific substrate (the Michaelis constant is about IO times larger than that for quinol) is also consistent with this view. As only Signal I is rapidly reduced by good substrates, it appears difficult to visualize the function, if any, of the other form of Cu 2+. Signal 2 is similar to the signal obtained from Signal I when laccase is mildly denatured (see Fig. 4; cf. ref. 3), and one m a y raise the question of whether it is in fact formed by partial denaturation of the enzyme (@ ref. II). The results with laccase b y themselves give little justification for such a suggestion, as no preparations giving less (or more) than one Cu 2+ of Signal-2 type have ever been obtained. However, recent findings with ceruloplasmin 5 indicate that this possibility must be considered. The amount of Signal 2 in ceruloplasmin preparations used in the work of ref. 5 corresponded to one Cu ~ . However, in one sample of serum this signal was found to be absent. Thus, it appears that one site is more labile than the other ones. In view of other close similarities between the two proteins, such a situation m a y then exist also in laccase. In such a case the laccase formed by the fungus should initially give only Signal i. However, it has been impossible to test this directly on the culture medium, as this must contain relatively high concentrations of CuSQ. Even if Signal I is relatively stable, the denaturation experiments show that it is destroyed under milder conditions than those required to denature the enzyme grossly. This lability of the binding site m a y be related to the fact1, TM that it is distorted from the usual square planar geometry found in complexes of Cu 2+. This is evident both from optical rotatory dispersion experiments of ceruloplasmin TM and the 35 GHz results of laccase reported here. As this distortion is believed to be the basis of the unique spectral properties, which, in turn, can be related to the oxidase function of the proteins (see ref. I), this lability m a y be a general property of the blue coppercontaining oxidases. Denaturation to different extents m a y then be the cause of apparent discrepancies in spectral properties reported for the same enzyme prepared in different laboratories (cf., for example, refs. 17 and 18). It m a y be noted that published E P R spectra TMof laccase from lacquer trees also show the presence of two forms of Cu 2+. In the cyanide experiments the new signal obtained on treatment at high concentrations of cyanide for long periods of time clearly shows the presence of nitrogen hyperfine coupling. Furthermore, the general appearance of the N hyperfine structure and the resonance parameters are very similar to those of the Cu 2+ complex of triglycylglycine 9 at p H > 9 (g~ = 2.172, g± = 2.o41, [A~I = 206 gauss with the isotope 63Cu). In this complex the Cu ~+ is coordinated to four nitrogens (three from the peptide bonds and one from the ~-amino group). In view of this similarity, it seems likely that Cu 2+ in cyanide-treated laccase is coordinated to three to four nitrogens. As cyanide molecules, unless they form bridges between metal ions TM, are thought to coordinate to metal ions through the carbon end ~9, these nitrogens are probably supplied b y the protein. The cyanide treatment is completely reversible, suggesting that the Cu 2+ in the cyanide-treated protein is still at the original binding site. As the particular cyanide spectrum is obtained at a comparatively low pH, it is somewhat unlikely that all nitrogens would come from peptide bonds (cf. ref. 9), although Biochim. Biophys. Acta, 156 (1968) 67-76
76
BO. G. MALMSTROM, B. REINHAMMAR, T. V.'~NNGARD
t h e specific a r r a n g e m e n t in a p r o t e i n c o u l d m a k e t h i s k i n d of c o m p l e x to f o r m at a m u c h l o w e r p H t h a n w i t h s m a l l p e p t i d e s . T h u s , it s e e m s m o r e r e a s o n a b l e to h a v e n i t r o g e n s f r o m one or m o r e i m i d a z o l e g r o u p s also i n v o l v e d in t h e b i n d i n g . U n f o r t u n a t e l y , it is n o t possible on t h e basis of p r e s e n t d a t a to d e c i d e if it is t h e Cu 2+ s h o w i n g S i g n a l I or 2, or b o t h , in t h e u n t r e a t e d p r o t e i n t h a t g i v e s rise to t h e n e w signal on c y a n i d e t r e a t m e n t . More e x p e r i m e n t s are n e e d e d to c l a r i f y t h i s i m p o r t a n t p o i n t as are e x p e r i m e n t s w i t h Cl5N, w h i c h w o u l d g i v e a d e f i n i t e a n s w e r to t h e q u e s t i o n w h e t h e r t h e n i t r o g e n of t h e c y a n i d e c o o r d i n a t e s or not. ACKNOWLEDGEMENTS W e w i s h to t h a n k Mr. S.-O. FALKBRING for v a l u a b l e t e c h n i c a l assistance. T h e s t u d y has b e e n s u p p o r t e d b y g r a n t s f r o m t h e K n u t a n d Alice W a l l e n b e r g F o u n d a t i o n , t h e S w e d i s h N a t u r a l S c i e n c e R e s e a r c h Council, t h e I n s t i t u t e of G e n e r a l M e d i c a l S c i e n c e s of t h e U.S. P u b l i c H e a l t h S e r v i c e (GM I2 280-03) a n d t h e A g r i c u l t u r a l R e s e a r c h S e r v i c e of t h e U.S. D e p a r t m e n t of A g r i c u l t u r e ( F G - S w - I o 7 ) . O n e of us (T.V.) a c k n o w l e d g e s a special r e s e a r c h p o s i t i o n of t h e S w e d i s h N a t u r a l Science R e s e a r c h Council.
REFERENCES 1 B. G. MALMSTROM, in T. E. KING, 1~. S. MASON AND ),]7. •ORRISON, Oxidases and Related Redox Systems, John Wiley, New York, 1965, p. 207. 2 B. G. MALMSTROM AND T. VANNG.~RD, dr. Mol. Biol., 2 (196o) 118. 3 L. BROMAN, B. G. MALMSTR~M, R. AASA AND T. VANNG.~-RD, J. Mol. Biol., 5 (1962) 3Ol. 4 A. EHRENBERG, B. G. MALMSTR6M, L. BROMAN AND R. MOSBACH,J. iV[ol. Biol., 5 (1962) 45 o. 5 T. V~NNG;XRD, in A. EHRENBERG, B. G. MALMSTROM AND T. VANNG.~RD, i~/Iagnetic Resonance in Biol. Systems, Pergamon Press, Oxford, 1967, p. 213. 6 G. F.~_HRAEUSAND B. REINHAMMAR,Acla Chem. Scan&, in the press. 7 L. BROMAN, B. G. MALMSTROM, R. AASA AND T. VANNG.~RD, Biochim. Biophys. Acta, 75 (1963) 365 • 8 M. L. ANSON, J. Gen. Physiol., 24 (1941) 3999 K.-E. FALK, H. C. FREEMAN, B. G. MALMSTR6M AND T. V~NNG.&RD, J. A m . Chem. Soc., 89
(1967) 6o71. IO G. FELSENFELD, Arch. Biochem. Biophys., 87 (196o) 247. i i B. G. MALMSTRDM,R. AASA AND T. V~NNGARD, Biochim. Biophys. Acta, IiO (1965) 431. 12 T. V£NNG3,RO AND R. AASA, in W. Low, Paramagnetic Resonance, Academic Press, New York, 1963, p. 509. 13 J. BuTzow, to be published. 14 \V. E. BLUMBERG, J. EISINGER, P. AISEN, A. G. MORELL AND 1. H. SCHEINBERG, J. Biol.
Chem., 238 (1963) 1675. 15 J. PEISACH AND W. E. BLUMBERG, Federation Pro&, 26 (1967) 834. 16 W. E. BLUMBERG, in J. PEISACH, P. AISEN AND W. E. BLUMBERG, The Biochemistry of Copper, Academic Press, New York, 1966, p. 49. 17 W. E. BLOMBERG, W. G. LEVlNE, S. MARGOLIS AND J. PEISAEH, Biochem. Biophys. Res. Commun., 15 (1964) 277. 18 Z. I~AKAMURAAND Y. OGURA, in J. PEISACH, P. AISEN AND W. E. ]~LUMBERG, The Biochemistry of Copper, Academic Press, New York, 1966, p. 389. 19 D. F. SHRIVER, in C. K. JORGENSEN, J. B. NEILANDS, ~R. S. NYHOLM, D. REINEN AND R. J. p. WILLIAMS, Structure and Bonding, Vol. i, Springer, Berlin, 1966, p. 32. Biochim. Biophys. Acta, 156 (1968) 67-76