THE JOURNAL OF BIOLOGICAL CHEMIBTRY Vol. 249, No. 19, Issue of October 10, PP. 6062-6066, 1974

Printed in U.S.A.

The Oxidation State of Copper in Resting Tyrosinase*

(Received for publication, April 15, 1974)

NOBUO MAKINO, PAUL MCMAHILL, AND HOWARD S. MASON

From the Department of Biochemistry, liniversity of Oregon Medical School, Portland, Oregon 97%‘01

THOhlAS H. Moss

From the IBM T. J. Watson Research Center, Yorktown Heights, New York 10598

SUMMARY

Resting tryosinase was diamagnetic between 1.4 K and 200 K. Redox titration showed that it, but not apotyrosinase, contained a titrable group, E’, = $0.36 volt (pH 7.0), R = 2. Upon denaturation with acid under strictly anaerobic conditions, the EPR-detectable copper increased from less than 5 % of the copper present to about 100%. It is con- cluded that the active site of the enzyme contains a pair of antiferromagnetically coupled Cu2+ ions.

Tyrosinase (EC 1 10 3 .I ; o-&phenol : oxygen oxidoreductast)

is an osygcil- and 4-electroll-traiisferrilig phenol osidase which

catalyzes phenol o-hydroxylation and &hydrogenation in plants

and animals (1)

Monophenol + 02 = o-quinone + Hz0

2 o-Diphenol + 0, = 2 o-quinone + 2HsO

The enzyme (from ilgaricus bisporus) contains 4 g atoms of

copper per 1.2 x 10; g atoms of protein (2). The oxidation state of the copper in the resting enzyme is not known. It has been

suggested that in the preparations we have studied, it is pre- dominantly in the osidized, antiferromagnetically c,oupled bi-

cupric state (3) on the grounds that the enzyme has no intrinsic

EPR absorption (a), that the apoenzyme can be wholly recoil- stituted with Cu2+ to an active product without an El’R signal

(4-6), and that upon reaction with Hz02 tyrosinase forms an 02- and NO-binding compound which has chemical and spec-

troscopic characteristics similar to those of the bicuproprotcin,

hemocyanin (3, 7, 8). ln this study we have examined mag-

netic susceptibility, oxidatioli-reduction potentials, and denatu- ration under anaerobic conditions of resting tyrosinase’ in order

to characterize further the oxidation state of its copper.

EXPERIMENTAL PROCEl)URES

Preparation of ?‘yrosinase-Tyrosinase isozymes were prepared from the common commercial mushroom, A. bisporus, by the method of Bouchilloux et al. (2) as modified by Nelson and Mason

* This research was supported by Grant AM 0718 from the United States Public Health Service and by Grant BC-IK from the American Cancer Society.

1 Resting tyrosinase, the enzyme as prep&red.

(9) and Balasingam and Ferdinand (10). A brief description of the procedure was reported by Jolley el al. (3). The principal enzyme preparations which were employed in this study are char- acterized in Table I; other preparations are described in legends to figures. Methods of assay are described by Nelson and Mason (‘3).

Apotyrosinase was prepared by dialyzing the enzyme against 0.01 M KCN in 0.026 M borate buffer, pH 8.6. The preparation used in this study retained 7.0% of its original copper, and showed 0.17y0 of its original activity. The enzyme could be reconstituted with Cu2+ ions by the method of Kertesz et al. (G); 85y0 of the original activity was recovered. Intrinsic oxytyrosinase in the preparations was determined from the difference spectrum, (aerobic enzyme) - (anaerobic enzyme), using ~345 = 1 X 10’ M-l cm-’ (3).

Iieagents and Methods-Potassium molybdicyanide (KaMo. (CN)a) was synthesized by the methods of Olsson (11) and Kolt- hoff and Tomisicek (12). NAI>H was obtained from the Sigma Chemical Co.; its concentration was determined from its ab- sorbance at 340 nm, using ea4,, = 6.22 x lo3 M-’ cm-’ (13). All other reagents were the best grade commercially available. Copper was estimated with a Techtron type AA atomic absorption spectrophotometer, using a Fisher Scientific Co. reference stan- dard. Il:PIt spectra were recorded with a Varian V-4502 15PIt spectrometer at -175”, modulation amplitude of 9.7 G, and modu- lation frequency of 100 kc. Low temperatures at the samples were produced and regulated by a Varian variable temperature accessory and Dewar, using Nz cooled by liquid Nn. Ii;Pli signal intensities were quantitated with the use of standard Cu(II)- EDTA solutions. Anaerobic Ii:PIt experiments were carried out in a closed ISPR tube with two sidearms, similar to the design described by Palmer (14). The reagents were added separately to the sidearms and cooled in ice water. The system was then deoxygenated by 10 cycles of vacuum (10 to 15 mm Hg) followed by flushing with argon (Matheson, 99.999~0). The solutions were finally mixed and transferred to the bottom of the EPIt tube, then frozen at -190” after a 3.min incubation, and spectra deter- mined.

Protein was estimated by the method of Lowry (1951) using a bovine serum standard (Labtrol). Spectrophotometry was carried out with a Cary model 14 spectrophotometer; the tem- perature of the optical cuvette was maintained at 25” + 0.2”.

Oxidation-reduction potentials were measured with a Radiom- eter P 101 platinum electrode, a K 401 calomel reference electrode, and an Orion Research potentiometer, model 801. The anaerobic titration v&se1 (1 ml) was essentially the same as that described by Dutton (15) and the system was maintained oxygen-free with argon gas. The midpoint potential of the 4-lert-butylcatechol I- terl-butyl-o-benzoquinone system was measured by titrating 4-tert-butylcatechol with 02 in the presence of a catalytic amount of tyrosinase. Its E, value (pH 7.0, 25”) was +0.346 volt (initial tert-butylcatechol concentration, 0.1 mM).

Magnetic susceptometry was carried out with a vibrating sample magnetometer (16) by a procedure already described (17), except that samples were not deoxygenated; instead, correction

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TABLE I Characteristics of tyrosinase preparations employed in

this study

Preparation No.

HA 11/3-a HA 12/24-a. HA S/18-cu. HA l/9$3.. . HA 11/27-01.

-

Cate- choke

1068 1315 1168

447 _-

l-Butyl- cate- Cresolase

cholase

units/mg

190 122 303 141 343 288

1374 98 266 115

-

-

Total copper

EPR-de- tectable copper

% 0.16 0.01 0.200 0.00 0.23 0.01 0.19 0.01 0.20 0.01

THEORETICAL

I I I 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0

I/T (OK)-’ 18

FIG. 1. The temperature dependence of the susceptibility of tyrosinase and of buffer blanks. The buffer for the protein solu- tions and blanks was 0.026 M sodium borate, pH 8.6: X--X, protein buffer blank: 5% bovine serum albumin in borate buffer equilibrated with air; protein buffer blank: 5qb bovine serum albumin in borate buffer, deoxygenated with dithionite; +, tyro- sinase, preparation HA 12/24, [Cu] = 2.56 mM; 0, tyrosinase, preparation HA/8/18, [Cu] = 1.9 mM; 0, buffer blank, borate buffer only, equilibrated with air.

for the susceptibility of dissolved O2 was estimated by measuring the susceptibilities of air-equilibrated and deoxygenated buffers in the presence and absence of a protein standard (bovine serum albumin). The buffer, 0.026 M sodium borate, pH 8.6, contained 0.02 PM copper. A 5y0 solution of the bovine serum albumin in H20 contained [Cu] = 15 PM. Therefore, the contribution of cupric copper to the experimental system from either buffer or protein was negligible, since the concentrations of enzyme copper were about 2 orders of magnitude greater.

RESULTS

Magnetic Susceptibility of Tyrosinase-Magnetic susceptibility measurements are shown in Fig. 1; values for buffer blanks are also shown. The susceptibilities were very low, i.e. the samples were essentially diamagnetic. Data for two independently pre- pared samples are given to illustrate the reproducibility of even the low susceptibilities measured. Data for buffer with and without a large molecular weight solute (5% bovine serum al- bumin) are included to show the problems involved in estimating 02 contribution to diamagnetic or nearly diagmagnetic samples (see “Discussion”). In our procedure magnetic susceptibilities (x) at various temperatures from 201 K to 1.4 K were plotted versus l/T so that the Curie constant (x = C/T) could be deter- mined directly from the slope. The equation holds when the condition PH < < < kT is valid, which was true in this study

045 r-

I

04 \

1

2 I

s \

G \ I

0.35 1

03

I

” 0.5

ELECTRON EQUIVALENTSKu

1

6063

I

FIG. 2. Oxidation-reduction titrations of holo- and apotyro- sinase: 1.0 ml holotyrosinase, 92.6 pM total copper, 6.5 PM EPR- detectable copper (0) or apotyrosinase (82.6 pM original total copper) (A) were titrated anaerobically with 0.58 mM or 0.7 mM NADPH, respectively, in the presence of 3 PM phenaeine metho- sulfate in phosphate buffer, pH 7.0, at 25”. Mediators were used: 4-tert-butylcatechol (3 PM) and dichloroindophenol (3 /IM). -, calculated titration curve for holoenzyme based on the assump- tions described in the text.

at the applied fields, H = 0 - 500 G for low temperature points used for data analysis. Inferences about the spin of the metal ions involved were made from the relationship C = S(S + l)p,&/ 3K where X is the ionic spin, pb the Bohr magneton, and g* the average of the square of the ionic g value taken over all spatral orientations. There was no ambiguity arising from the diamag- netic susceptibilities of the protein, the solvent, or the sample holder, as the paramagnetic contribution was inferred only from change in susceptibility with temperature.

Oxidation-Reduction Titration-Fig. 2 shows the potentio- metric titration of tyrosinase with NADH-phenazine methosul- fate as a reducing system, in the presence of 4-tert-butylcatechol and dichloroindophenol as mediators. 4-lert-Uutylcatechol could not be replaced by potassium ferricyanide as a mediator, and dichloroindophenol was necessary to make the end point of the titration firm. It was found that a theoretical curve calcu- lated to fit the titration curve in Fig. 2 on the basis of the Nernst equation, gave a best fit (Fig. 2, p) when the following assump- tions were made: 1, E, = 0.36 volt; 2, n = 2; 3, 90% of the total copper in the sample is reductively titrable; and 4, 3.8% of the total copper is in the cuprous state in the native enzyme, and therefore not reductively titrable. Assumption 4 was based upon a determination of intrinsic ‘I” (oxytryrosinase) in the preparation; upon deoxygenation, T” or oxytyrosinase is con- sidered to give rise to the active site, (Cu’+)z (3). Apotyrosinase, titrated under the same conditions as the resting holoenzyme, showed almost no electron accepting capacity (Fig. 2, A- - -A) in the range of potentials observed. The difference between the titration curves of holo- and apoenzyme can reasonably be

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6064

0

0

.

-\ Xx* ‘x, . ‘X‘$ ‘CA*

x-(r -,x 04 ;: ,&-Butylcotechol

\ . X’, l .

\ . \ .

NADH-Phenozme ‘, .

methosulfo%x It l .

0 0.5 1.0 1.5

Reducing Equivalents Ku

FIG. 3. Oxidation-reduction titration of tyrosinase with tert- butylcatechol: 1.0 ml of tyrosinase (60.6 PM total copper, 1.8 pM

EPR-detectable copper) was titrated anaerobically with tert- butylcatechol (260 PM), without other mediators, in 0.1 M phos- phate buffer, pH 7.0, 25”. - , calculated curve already shown in Fig. 2. X, phenazine methosulfate titration depicted previ- ously in Fig. 2.

attributed to the presence and absence of copper (see “Discus- sion”).

Fig. 3 depicts the result of a potentiometric titration of tyro- sinase with 4-terl-butylcatechol as reductant in the absence of mediator. This titration curve was in good agreement with that obtained with NADH-phenazine methosulfate as the re- ducing system (Fig. 2; also indicated by - - - in Fig. 3). How- ever, as expected from the measured oxidation-reduction poten- tial of 4-tert-butylcatechol, E’ 0 = 0.346 volt (pH 7.0, 25”), which is close to the oxidation-reduction potential here obtained for tyrosinase (E’o = 0.36 volt, pH 7.0, 25”), no sharp decrease in the measured potential was observed during the last half of the titration.

The reversibility of the reductive titration of tyrosinase was checked with oxidants. The enzyme, which had been reduced by NkDH-phenazine methosulfate, was titrated with K3Mo. (CN)s (E, = +0.778 volt) (11) and with O2 (E’. = 0.81 volt for the reaction 02:HzO at pH 7) (18). The potential changes in both cases were slow and loose, and no quantitative relation- ship was observed. Apparently the reactions of reduced tyro- sinase with these oxidants were very slow. It is already known that reduced tyrosinase, T”‘, binds 02 to form the metastable oxytyrosinase, T” (3).

Oxidation State of Copper Released by Acid from Tyrosinase- Tyrosinase was treated under anaerobic conditions with a variety of denaturants such as sodium dodecyl sulfate, urea, and guani- dine sulfate, but only a small part of the total copper in the en- zyme then appeared as cupric copper, according to the EPR criterion. It had been shown by Uouchilloux et al. (2) that the enzyme, which contains a high proportion of hydrophobic resi- dues, is relatively stable to sodium dodecyl sulfate, possibly behaving as an oily droplet in which the active site complex is stabilized. However, when the enzyme was treated under strict anaerobic conditions (see “Experimental Procedures”) with phosphoric acid to pH 1, the copper was transformed completely to Cu2+ by the EPR criterion. In experiments with three differ- ent enzyme preparations, the following results were obtained:

HA 3/29-a, 115%; HA 6/25-p, 96% and 84%, and HA 11/27-a 88% of the EPR-nondetectable copper was converted into EPR- detectable copper. We can conclude that 95% f 11 y0 of the EPR-nondetectable copper, other than intrinsic T” (cuprous under anaerobic conditions) is in an oxidation state equivalent to Cu(I1). The variability is probably attributable to the method of assay. The presence of neocuproine in the system made no difference to the result. On the other hand, hemocya- nin, prepared from Cancer magister, and denatured under the same conditions revealed less than 3% of its copper in the EPR- detectable form in this protein. This copper is considered to be in the cuprous form (17). Again, the presence of neocuproine made no difference to the result.

DISCUSSION

It has been inferred from the lack of an EPR signal arising from tyrosinase that the copper ions in the enzyme are diamag- netic (2). We have now found (a) that the magnetic suscepti- bility of air-equilibrated solutions of tyrosinase is the same as that of typical diamagnetic buffers containing protein, in equi- librium with air, (b) that the resting enzyme undergoes a reduc- tive titration, with BY0 = 0.36 volt, n = 2, which apotyrosinase does not undergo, and finally, (c) that the EPR-nondetectable copper in resting tyrosinase becomes EPR-detectable upon anaerobic denaturation with phosphoric acid. The diamag- netism and the reducibility of enzyme copper are thus unam- biguously established. The release of cupric ion upon anaerobic denaturation of the resting enzyme is consistent with the hy- pothesis of an active site containing two cupric ions, although it is possible that the protein contains an oxidizing group (e.g. disulfide) which could oxidize Cur+. However, it was not de- tected in the reductive titration of apoenzyme. In addition, resting tyrosinase cannot bind 02, whereas the reduced enzyme can do so (3).

If, as now seems probable, our resting tyrosinase contains pairs of spin-coupled cupric ions or an equivalent state (3, 7, 8), what can be said about the structure of this active center? We have already commented on the spectral and chemical analogies between methemocyanin, oxyhemocyanin, and hemocyanin, and resting tyrosinase, Tr, oxytryrosinase, T”, and cuprotyrosinase, T”’ (338), respectively. Hemocyanin also contains a pair of cuprous ions in close proximity at its active site (7, 8, 17). Oxy- hemocyanin has been examined by resonance Raman spectros- copy (19), and spectral lines have been observed which support the structure, Cu(II)202=. In addition, a peak was observed at 282 cm-l, which was tentatively assigned to a vibrational mode arising from Cu-0 stretching, or Cu-0-Cu symmetrical stretching in a linear or nearly linear Metal-O-Metal system, where the 0 atom could arise from Hz0 or protein hydroxyl. This kind of-O-bridged structure has also been implicated at the 2-iron OS-binding center in hemerythrin (20), and could ac- count for antiferromagnetic coupling in both proteins through the superexchange mechanism (21, 22).

We have interpreted the susceptometric, spectrophotometric, and chemical properties of resting tyrosinase in terms of anti- ferromagnetically coupled Cu(I1) pairs, but, unlike other sys- tems containing such pairs (23-25), resting tyrosinase shows no, or extremely weak, absorption in the 340~nm charge transfer region, and none in the 690-nm region. Intense absorption in the latter region is sometimes attributed to distortion of the normally axial cupric complex ligand field (26, 27). Polyporus lactase contains a pair of Cu(I1) ions which undergo reduction simultaneously; the pair is associated with a moderately intense

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Rhus &case

Polyporus lactase

Ceruloplas- min

Bovine super- oxide dis- mutase

Rhus blue protein

Spinach plas- tocyanin

Pseudomonas azurin

Agaricus bi- sporus tyro- sinase

Redox poterltials of copper proteins

0.415 (1) 0.460 0.434 (1) 0.483 (2 0.767 (1) 0.785 (1) 0.782 (2 0.490 (1) Site re- 0.580 (1) ported

0.184 (1)

0.370 (1)

0.328 (1)

0.42 (1:

0.36 (2:

1

5

1

PIf Reference

7.0 28, 29 7.5 30 6.25 31 5.5 30 5.5 35

6.65 34

7.1

.4-9.’

6.4

7.0

30

32

33

Present work

a Values in parentheses are 1~ values of the titration.

absorption band at 330 nm, and a weak band at Cl0 Iun, and exhibits no EL'R signal in the oxidized or reduced forms at 77 K (24) (2.electron accepting site). Theso (‘u(II) pairs have a high, positive redox potential, n = 2 (Table I I).

The Cu(II)-Cu(II) pair in tyrosinasc is not this strw;tural type because it is silent both optically and magnetically. It appears to be a unique structural cantity, which o11 operational grounds could be called type IV cupric pairs. The failure to detect a charge transfer band in the region of 330 II~ irl the rest- ing enzyme suggest,s that there is no l&and, like -0- or -S--, with a sufliciently high clcctron potclntial to servo as a charge transfer donor to the (‘~(11)~ pair. The lacak of a band iI1 the 600 nm region suggests that the complex is symmetricsal, since it shows 110 d-d transition (26). A dirert Cu(II)-Cu(I1) inter- action as in a copper--copper bond would produce thcsc prop- crties, but the copper ions would have to be very close to each other (-1.5 to 2 A) in order to yield tlic singlet ground state. If this is the case, reduction of the site would require a drawing apart of the copper ions to permit 02.binding and superexchange in the oxycomplex. This hypothesis is under test. The dis- tance, approximately 3.9 A, between copper ions in the NON treated reduced tyrosinase (S), inferred from the g values of the dipole coupled cupric EI’R signal, is fully compatible with a center capable of accommodatin g a bridging 0% when exposed to oxygen.

Tyrosinase contains 1 disulfide group per copper ion (a) and it is possible (although there is 110 evidence) that the active site of tyrosinase consists of a disulfide-copper pair complex. Zuber- buhler and Mason (36) havr reported that the hinuclear C”u(II) peptide complex, Cu(lI)2 L-cystinyl-bis-glycinc, has an I<;I’R spectrum which does not account for the copper present in the complex; an atomic model showed that a configuration in nhich the two copper ions are about 5 A apart, each bound to 1 sulfur atom of the cystine disulfide bridge, would permit coupling through superexchange. If this structure existed ill resting tyrosinase, an oxidation-reduction system similar to that discussed by Hemmerich (37) would be possible:

fully oxidized

iai

half reduced

lb)

SCHISMN 1

l’hcre is at, present no direct cvidencc upon which to determine which of the two structures, u or 6, if either, rcprcsents resting tyrosinasc or which of the reduced structures, b or c, if either, represents the (&binding form of tyrosinasc. In the related case of hemocyanin, no significant amount of thiol groups is re- vealcd upon removing copper with cyanide (38, 39).

A final point applies to the susceptomct,ric results with buffer blanks (Fig. l), and illustratc,s the pitfalls involved in estimating the contribution of dissolved 02 in weakly magnetic protein solutions. The air-equilibrated borate buffer alone is completely diamagnetic and dots not show the paramagnetism expected for O2 at roughly 0.25 mM (air saturated at 25”). This para- magnetism has actually been observed on our susceptometer for air-saturated phosphate and ‘I’ris buffers. The buffer plus ty- rosinasc does show a small pararnagnetism because of the small concentrations of El’R-detectable copper, 39 p and 80 pi, pres- ent in the enzymes. However, these concentrations of cupric ion are small cornpared with the concentration of free spins due to oxygen in air-saturated buffers. However, air-equilibrated buffer solutions containing the same concentration of bovine serum albumin as tyrosmase (5%) gave a normal paramagnetic contribution from the OZ. The bovine s(Irum albumill-involved magnetism did not come from trace amounts of copper (15 PM), or high spin iron or manganese, because ditliionite treatment completely removed the paramagnetism. Our view is t>hat the actual paramagnetic contribution of O2 in frozen solutions de- pends strongly on the structure of the frozen mat,rix, c~hiefly on whether OZ is dispersed in the frozen structure or collected in nonmagnetic aggregates. Presumably, a large molecular weight solute in the borate buffer helps maintain the O2 in the dispersed form. In any case, it appears that buffer blanks for frozen protein solutions must duplicate the ice matrix state by contairl- ing a large molecular weight solute in o&r to provide a valid comparison.

Acknowledgments-We wish to thank ‘l’hcresa Wagner and Glenn Gross for their dcvotcd assistance in preparing the erl- zyme, and Dr. Russell Jolley for suggesting phosphoric acid as a denaturing agent.

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Nobuo Makino, Paul McMahill, Howard S. Mason and Thomas H. MossThe Oxidation State of Copper in Resting Tyrosinase

1974, 249:6062-6066.J. Biol. Chem. 

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