EPR studies on the anaerobic reduction of fungal laccase

Biochimica et Biophysica Acta, 405 (1975) 236-242

© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37160 EPR STUDIES ON T H E A N A E R O B I C R E D U C T I O N OF F U N G A L LACCASE E V I D E N C E F O R P A R T I C I P A T I O N OF TYPE 2 C O P P E R IN T H E R E D U C T I O N MECHANISM

ROLF BR.~NDI~N and BENGT REINHAMMAR Institutionen f6r Biokemi, Gi~teborgs Universitet och Chalmers Tekniska H6gskola, Fack, S-402 20 Gi~teborg 5 (Sweden)

(Received March 6th, 1975)

SUMMARY 1. In anaerobic reduction studies on fungal laccase B (p-diphenol:O2 oxidoreductase, EC 1.14.18.1) with the EPR and stopped-flow techniques it was found that the type 2 copper of the enzyme is rapidly undergoing a reduction-oxidation cycle which is followed by a slower reduction in a couple of seconds. An intermediate EPR signal of unknown origin is formed in the same time-range as the initial reduction of type 2 copper and disappears again when this copper ion is reoxidized. 2. The rate of the anaerobic reoxidation of type 2 copper is similar to the reduction rate of the two-electron acceptor, suggesting that they are interacting in the electron transfer of the enzyme. 3. The changes in the reaction rates of both type 2 and type 3 copper apl~ear to be affected in a similar way by changes in pH. 4. The EPR signal of the type 2 Cu 2+ suggests that this ion is liganded to one or more nitrogens.

INTRODUCTION The blue copper-containing oxidases contain three types of Cu z+ [1-3]. Two of these, type 1 Cu 2÷ and type 2 Cu 2+, are paramagnetic. The non-paramagnetic copper ions are supposed to exist as a pair of divalent Cu '+ and to be associated with a two-electron acceptor which can be observed by its absorbance at 330 nm [2-4]. This pair of Cu 2+ (called type 3 in this report) as well as type 1 are thought to change valence during the catalytic transfer of electrons from substrate to molecular oxygen [2, 5]. Whether type 2 copper participates in the electron transfer between substrate and oxygen or if it has some other function in the enzyme is not well understood. This lack of knowledge mostly depends on the experimental difficulties involved in the study of this copper. However, it is essential for the activity of the enzyme which has been demonstrated by its reversible removal from fungal laccase [6], and its specific interaction with the inhibitor F - [7]. The presence of a two-electron acceptor, as well as kinetic evidence, has led

237 to the suggestion that the reduction of 02 to water, catalyzed by the enzyme, would involve two consecutive two-electron transfer steps [8]. H202 or one of its ions would then be formed as an intermediate which is bound to the enzyme. Type 2 Cu 2+ which is known to bind anions such as F - and N3- strongly [9], and also interact with H202 in the native enzyme [7], has been supposed to have the role of stabilizing this peroxide intermediate until it is further reduced to water [8, 10]. In this communication we present a new experimental approach to the study of copper-containing oxidases. By anaerobic rapid-freeze technique it is found that the type 2 copper of fungal laccase is rapidly reduced and seems to be reoxidized in a mechanism where its electron is transferred to type 3 copper. Therefore, type 2 copper probably takes part in the electron transfer mechanism, at least under anaerobic conditions. MATERIALS AND METHODS Fungal laccase B was prepared according to the method of F~thraeus and Reinhammar [11]. The preparations used were freed from contaminating F - as described earlier [7], and the protein concentrations were determined spectrophotometrically at 610 nm on the basis of an absorbance coefficient of 4.9 m M - l . c m -1 [11]. Analytical grade ascorbic acid was purchased from Merck and Co. (Darmstadt). Acetic acid buffers (0.05 M) were prepared from deionized water and were used in all experiments. Anaerobic rapid-freeze experiments were made with isopentane which had been bubbled with N2 (99.999 ~ pure) for a couple of hours at 170 °K before use. To avoid contact with air, the mixing chamber and the connecting tubes were flushed with N2 until about 10 s before use. When the enzyme solution was to be squirted into the cold isopentane, the jet nozzle was dipped into it 1 s or less before the mixing. This procedure gave very nice crystals for packing into EPR tubes. The reproducibility between different samples, including all operations, was within 5 ~. The rapidfreeze equipment used in these experiments has been described earlier [12]. The oxygen concentration in the solutions within the mixing chamber and the connecting tubes was tested by the following procedure: Anaerobic N,N,N',N'-tetramethyl-p-phenylenediamine (100 #M) and laccase (5/~M) were mixed and the intensely colored product formed was taken as a measure of the amount of oxygen consumed. This test revealed that initially very little 02 was present in the system, less than 5 #M. However, oxygen was continually leaking into the system and after about 30 s the product formation indicated that about 10/~M O2 had been consumed. Another test was made to control the amount of oxygen in the isopentane. Fungal laccase B (400 #M), which had been anaerobically titrated with four electron equivalents of ascorbate, was squirted into the anaerobic isopentane solution. EPR spectral measurements revealed that less than 10/zM of the enzyme was reoxidized. Aerobic rapid-freeze experiments have shown that the reoxidation of fully reduced fungal laccase is very rapid (Br/and6n, R., unpublished). The tests described above indicate that the anaerobicity of the system is satisfactory and the small amount of oxygen present does not disturb the results obtained in this study. Anaerobic, stopped-flow experiments were performed at 25 °C. The equip-

238 ment used as well as the techniques for the p r e p a r a t i o n o f a n a e r o b i c enzyme and sut;strate solutions have been described earlier [12, 13]. T o t a l E P R intensities were o b t a i n e d by integrations o f the spectra using u1~treated enzyme as a reference. E P R m e a s u r e m e n t s were m a d e at 77 °K in a V a r i a n E-3 s p e c t r o m e t e r at a b o u t 9.2 G H z . RESULTS A n a e r o b i c rapid-freeze reductions o f fungal laccase B were carried o,~t at pH 5.0, 5.2 and 5.6. The r e d u c t i o n o f type 1 c o p p e r at these p H values is so fast t h a t no contril:t~ticn from this Cu z+ is okserved in the E P R spectra even at the shortest observation time used (10 ms}. In F ik~. 1 the E P R spectra at three different times after I

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Fig. 1. EPR spectra of anaerobically reduced fungal laccase B with ascorbic acid at pH 5.2. The concentrations of enzyme and ascorbic acid after mixing were 400/~M and 6 mM, respectively. The figure shows the spectral changes after 10 ms (B), 30 ms (C) and 150 ms (D). (A) shows the spectrum of the preparation used. Microwave frequency was 9.15 GHz and modulation amplitude was 10 G. Gain settings for the right part of the figure were 2.105, (A x 10); 3.2.105, (B x 10); 5. l0 s, (C × 10); and 3.2-10 s, (D × 10). mixing the enzyme and a s c o r b a t e at p H 5.2 are shown. The low-field p a r t of the spectra d e m o n s t r a t e t h a t the type 2 E P R signal r a p i d l y d i s a p p e a r s and t h a t an intermediate E P R signal is formed, which at m o s t c o r r e s p o n d s to a b o u t 0.3 Cu 2+ per enzyme molecule. A f t e r a b o u t 150 ms m o s t o f the t y p e 2 E P R signal is again recovered and the intermediate signal has decreased to less t h a n h a l f o f its intensity at 30 ms. Then, type 2 c o p p e r reduces once a g a i n within a few seconds. Similar results were o b t a i n e d at p H 5.0 and 5.6. In Fig. 2, the results at all three p H values are s u m m a r i z e d . A p p a r e n t l y , the rate o f b o t h the first and second r e d u c t i o n o f the type 2 c o p p e r signal increases with the H + concentration. Also, the rate o f r e a p p e a r a n c e o f the type 2 c o p p e r signal seems to be p H dependent. A n estim-

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(E3--E3); 5.2, ( 0 - - 0 ) ; and 5.6, (A--A). Black symbols represent the amount of copper/enzyme molecule of the intermediately formed EPR signal at the same corresponding pH values.

ated half time for the reoxidation of about 100 ms is obtained at pH 5.0 and 5.2. At pH 5.6 the half time for reoxidation appears to be about 0.5-1 s. Superhyperfine lines are observed in the high-field part of the spectrum when the enzyme is reduced under anaerobic conditions. Fig. 3 shows the spectrum 100 ms after mixing with ascorbate at pH 5.6. At this pH very little of the intermediate EPR signal is formed. Therefore, the spectrum represents almost only type 2 copper. Nine lines with a separation of about 15 G are observed in the high-field spectral region. These lines probably arise from nitrogens liganded to the type 2 copper. Earlier resuits from cyanide-treated enzyme also indicate that this Cu z+ is bound to 3-4 nitrogens supplied by the protein [1, 9]. Similar spectra were also obtained at pH 5.2 (cf. spectrum D in Fig. 1) and at pH 5.0 about 100-150 ms after mixing.

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Fig. 3. E P R spectrum of anaerobically reduced fungal laccase at pH 5.6, 100 ms after mixing. Ascorbic acid was used as substrate. Concentrations of enzyme and substrate were similar to those in Fig. 1. Microwave frequence was 9.155 G H z and the modulation amplitude was 2.5 G.

240 Another type of spectrum appears when the type 2 copper signal disappears and the intermediate EPR signal reaches its maximum intensity (cf. spectrum C in Fig. 1). This EPR spectrum is more like that of a complex without nitrogens involved. Earlier anaerobic reductions of fungal laccase B with about 5/~M protein concentration and 50-200 # M ascorbate showed that the rate of reduction of the type 3 copper was independent of the substrate concentration. A rate constant of 1.0 s-I was obtained at pH 5.5 [13]. The protein and ascorbate concentrations used in the rapid-freeze experiments reported in this communication were, however, much higher. We therefore performed anaerobic stopped-flow experiments with about 150/zM protein and 25 m M ascorbate at pH 5.5 and 5.0. The rate constant for the reduction of type 3 copper under these conditions was 0.8 s -1 at pH 5.5 in comparison to the data with lower concentrations [13]. However, at pH 5.0 a rate constant of 2.5 s -1 was obtained which demonstrates that the reduction rate of this electron acceptor also appears to be pH dependent. DISCUSSION In the anaerobic reductions of fungal laccase B carried out in this study the type 1 Cu 2÷ is reduced so fast that no E P R signals from this copper are involved in the observed changes of the EPR spectra discussed in this report. The most plausible explanation for the rapid decrease of the remaining type 2 signal is that type 2 Cu 2+ is reduced. Another possibility would be that type 2 Cu 2+ interacts with another paramagnetic component in the enzyme during the reduction process with the result of a broadening of its EPR signal, making it non-detectable. This seems to be a much less probable alternative, however. If it is assumed that the decrease of the EPR signal is caused by the reduction of type 2 Cu 2÷, electrons must rapidly enter this copper in the anaerobic reduction of the enzyme. Whether type 2 copper receives electrons directly from substrate or from another electron acceptor in the enzyme is not yet known. However, type 1 copper appears to be a possible electron donor as it is reduced much faster than type 2 copper, at least at pH 5.5 (cf. ref. 13 and Fig. 2). The second, slow, reduction of type 2 copper has also been observed in aerobic experiments when samples were frozen after the exhaustion of oxygen [1]. This second reduction is even slower than the reduction of type 3 copper which in its case is reduced too slowly to account for the turnover of laccase [10]. It was therefore earlier proposed that the type 2 copper in the blue oxidases would not be reduced but would have the role of stabilizing a peroxide intermediate which was thought to t~e formed during the catalytic turnover including two consecutive two-electron transfers from the enzyme to oxygen [8, 10]. Type 3 copper would in this mechanism be the donor of two electrons to 02 and water, respectively. Under anaerobic conditions type 3 is reduced in a slow intramolecular process and probably receives its electrons from two other electron acceptors in the enzyme [13]. Until now only one such acceptor has been found, namely type 1 copper. Results in this study indicate that type 2 copper can be a candidate for an additional electron acceptor-donor. For example, the half times for reduction of type 3 copper are about 0.3 s at pH 5.0 and 0.9 s at pH 5.6, respectively. From the results in Fig. 2 a half time value of about 0.1 s can be estimated for the reoxidation of type

241 2 copper at p H 5.0 while a value at p H 5.6 is more difficult to estimate. However, it seems to be about 0.5-1 s. The rates of reoxidation of type 2 copper thus seem to be rather similar to the rates of reduction of type 3 copper at both p H values. It therefore appears that type 2 copper might be a possible electron acceptor-donor functioning in the reduction of type 3 copper. Whether the rate of the initial reduction of type 2 copper, under the conditions of this work, is fast enough to be a step in the catalytic mechanism is difficult to answer as no data for the turnover number of the enzyme is known at the high protein and substrate concentrations used in this study. With 5/~M laccase and 1 m M quinol a turnover of 30 s -~ was obtained [12]. However, the value seems to beinverselydependent on the protein concentration which indicates that it might be lower with the protein concentration used here. Moreover, the enzyme oxidizes quinol about twice as rapidly as ascorbate at p H 5.5 under saturating substrate concentrations (Str6mberg, C., unpublished). According to Fig. 2 the half time for the initial reduction of type 2 appears to be about 50 ms at pH 5.6. Therefore, if the reduction of type 2 should not be rate limiting, the turnover number must be smaller than 1.4 s-1 under the conditions used here. The existence of a rapid and a slower reduction of type 2 copper indicates that its reactivity is dependent on the oxidation state of other electron acceptors in the enzyme or on conformational changes accompanying the reactions of other sites. Of particular interest is the finding that it seems to be rapidly reduced only when type 3 copper is in its oxidized state. The intermediately formed EPR signal (see Fig. Ic) appears to have a rather small linewidth and probably arises from a complex, without nitrogen ligands involved. It seems to form at about the same rate as type 2 is initially reduced and to disappear when this copper ion is again oxidized. It therefore appears to have some connection to the electron transfer process involving type 2 copper. However, it is not possible at the present to elucidate the origin of this signal. The results in this communication suggest that type 2 copper might have a more complex role in the laccases than earlier results have suggested. Hopefully, similar studies on tree laccase, currently being performed in this laboratory, might further elucidate the role of the different electron acceptors present in the laccases. ACKNOWLEDGEMENTS The authors are indebted to Professors Bo G. Malmstr6m and Tore V/inng~trd and to Dr Lars-Erik Andr6asson for fruitful criticism and valuable discussions. We are also indebted to Miss Ann-Cathrine Carlsson for the preparation of laccase from fungal cultures. This study was supported by grants from Statens naturvetenskapliga forskningsr~.d. REFERENCES I MalmstrSm, B. G., Reinhammar, B. and V/inng~rd, T. (1968) Biochim. Biophys. Acta 156, 67-76 2 Fee, J., Malkin, R., MalmstrSm, B. G. and V/inng~rd, T. (1969) J. Biol. Chem. 244, 4200-4207

242 3 4 5 6 7 8 9 10

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