Generation of superoxide during the enzymatic action of tyrosinase

ARCHIVES

OF BIOCHEMISTRY

Vol. 292, No. 2, February

AND

BIOPHYSICS

1, pp. 570-575,1992

Generation of Superoxide during the Enzymatic Action of Tyrosinase Satoshi Koga,*,t Minoru

Nakano,*Tl and Shozo Tero-Kubotaz

*Photon Medical Research Center, School of Medicine, Hamamatsu University, Hamamatsu, Handa-cho 3600, Shizuoka 431-31, Japan; TTaiho Pharmaceutical Co., Ltd., Kandanishiki-cho, Chiyoda-ku, Tokyo 101, Japan; and *Institute for Chemical Reaction Science, Tohoku University, Sendai 980, Japan

Received July 8, 1991, and in revised form October 4, 1991

Evidence for the generation of superoxide anion in an enzymatic action of tyrosinase is reported. In the dopatyrosinase reaction, 1 mol of Oz is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and a mol of 0,. Superoxide dismutase and a-methyl6-phenyl-3,7-dihydroimidazo[l,2-alpyrazin-3-one (a chemiluminescence probe and Oz trap) do not inhibit the rate of dopachrome formation from dopa in the presence of tyrosinase, indicating that free 0, is not utilized for metabolizing dopa. ESR studies for the accumulation of semiquinone radicals generated from tyrosine and Nacetyltyrosine in the presence of tyrosinase imply that 0; is not generated by the semiquinone + O2 reaction. Since the addition of HzOz and dopa to tyrosinase promotes the release of 0, and formation of dopachrome, the Cu(II)O;Cu(I) complex could be formed as a intermediate (an active form of tyrosinase); [CU(II)]~ + H202 = CU(I)O~CU(II) f 2H’. 0 1992 Academic Press, Inc.

Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme widely distributed in nature and mainly involved in the biosynthesis of melanin and of other polyphenols (1). The copper-containing active site is binuclear in mushroom (2-4) and in human tyrosinase (5). Most of the enzyme is in the oxidized cupric form, which is activated by reduction by dihydric phenols (metabolites of monohydric phenols). The Cu(1) form then binds oxygen to form an oxytyrosinase (6). Three active oxygen-coordinated complexes have been proposed as active intermediates in the tyrosinase reaction (3). If active oxygen species (lo2 and 0;) are released from the active oxygen-coordinated complexes, they should be detectable by a specific and sensitive chemiluminescence method using a cypridina ’ To whom correspondence

should be addressed.

luciferin analog. The present work was undertaken to explore the generation of 0; in the tyrosinase reaction using 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2-u]pyrazin-3one (CLA)’ as a chemiluminescence probe and to confirm the generation of semiquinone radicals in these reactions, using a Zn2+-stabilizing method. MATERIALS

AND

METHODS

Chemicals. 3,4-Dihydroxy-L-phenylalanine (dopa), L-tyrosine (tyrosine), and N-acetyltyrosine were purchased from Sigma Chemical Co. 4-Hydroxyanisole and 2-methyl-6-phenyl-3,7-dihydroimidazo[l,2alpyrazin-3-one (CLA) were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan) and Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), respectively. Partial purification of tymsinuse. A commercial mushroom tyrosinase (2200 units/mg, Sigma Chemicals) dissolved in 20 mM potassium phosphate buffer at pH 7.0 (250 ml containing 300 mg of protein) was applied to a column (2.5 X 30 cm) of DEAE-cellulose equilibrated with 20 mM potassium phosphate buffer at pH 7.0 (buffer A). The activity was eluted by application of a linear gradient of NaCl (mixing flask, 300 ml of buffer A; inlet flask, 300 ml of buffer A containing 0.5 M NaCl). Sevenmilliliter fractions were collected. The peak of tyrosinase activity (20 fractions) was pooled. The specific activity in the pooled solution was fourfold greater than that in the starting enzyme solution. Enzyme activity was measured in a system containing 1 mM dopa, 35 mM sodium phosphate buffer at pH 6.8, and enzyme, in a total volume of 3 ml at 35°C. One unit of enzyme activity was defined as that amount which produced 1 pmol of dopachrome/ml/min (7). Protein concentration was estimated by the method of Lowry et al. (8). Other enzymes. Catalase from bovine liver and superoxide dismutase from bovine erythrocytes were purchased from Sigma Chemicals. The former, dissolved in 50 mM potassium phosphate buffer at pH 7.0 (1 ml), was dialyzed against 3 liters of the same buffer overnight before use. The incubation systems and assays. The systems for investigating a relationship between CLA-dependent luminescence and cytochrome c reduction (0; generation) contained 43 PM hypoxanthine, 40 PM fer-

’ Abbreviations used: SOD, superoxide dismutase; CLA, 2-methyl-6phenyl-3,7-dihydroimidazo[l,2-a]pyrazin-3-one; dopa, 3,4-dihydroxy-Lphenylalanine.

570 All

0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.

GENER,$TION

OF SUPEROXIDE

DURING

THE

ricytochrome c (for the cytochrome c method) or 6 pM CLA (for the chemiluminescence method), a range of xanthine oxidase concentrations (162-486 units/ml), which were determined by the Roussos method (9), and 0.1 M potassium phosphate buffer at pH 6.5 in a total volume of 1 ml. The reaction was initiated by addition of the enzyme at 37°C. The initial rate (maximal rate) of cytochrome c reduction or the maximal luminescence intensity was determined by measuring the increase of absorption at 550 nm (t,,,. = 2.11 X lo4 Mm’ cm-‘) (10) or the maximal count/min in a luminescence re.ader (1 l), respectively. Calibration curves were then made by plotting maximal light intensity/min versus [enzyme] or maximal intensity/min versus initial rate of cytochrome c reduction (Fig. 1). All values were corrected for controls lacking xanthine oxidase. The standard reaction mixture contained 0.3 mM substrate (tyrosine, N-acetyltyrosine, dopa, 4-hydroxyanisole), 4 or 40 munits of tyrosinase/ ml, k6 FM CLA (for luminescence measurement), and 0.1 M potassium phosphate buffer at pH 6.5, in a total volume of 1 ml (for luminescence measurement), 0.6 ml (for o-dopaquinone measurement), 3.8 ml (for oxygen consumption), or 3.0 ml (for other assays). The reaction was initiated by the addition of tyrosinase and maintained at 37°C with a vigorous agitation. Production of o-dopaquinone was monitored in terms of ascorbate oxidation followed at 265 nm (t,., = 15.3 X lo3 Mm’ cm-‘) (12,13). Dopachrome formation was monitored by measuring A171m(~max = 3.7 X lo3 Mm’ cm-i) (14,15). Gxygen consumption was measured with a Clark-type electrode in an Instech oxygenometer (Model 102) assuming 217 nmol/ml for [O,] in the initial incubation mixture at 37°C. CLAdependent luminescence was measured in a luminescence reader (Aloka, BLR-102) as described previously (11). Integrated chemiluminescence intensity was obtained by tracing chemiluminescence intensity change (as a function of time) on homogeneous paper and weighing; it was expressed in terms of relative value. The HsOs concentration was calculated from the absorbance at 230 nm, assuming an extinction coefficient of 81 Mm’ cm-i (16). ESR spectra were observed with a Varian E-109 X-band spectrometer at 100 KHz field modulation, at 20°C. A magnetic field of 3360 G, a m:icrowave frequency of about 9.4 GHz, a microwave power of 5 mW, and a modulation width of 0.63 G were adopted. Semiquinone radicals were detected by the ESR-spin stabilization method (17).

RESULTS

(1) Generation of 0; in the Dopa-Tyrosinase

System

When dopa was incubated with tyrosinase in the presence of oxygen, O2 was generated and could be monitored

FIG. 1. Relationship between. CLA-dependent chemiluminescence and cytochrome c reduction (0; formation in hypoxanthine and xanthine oxidase). The reaction mixtures, incubation conditions, and assays were described under Materials and Methods. MAX.CL, maximal chemiluminescence; HX-XOD SYST, hypoxanthine-xanthine oxidase system.

ENZYMATIC

ACTION

INCUBATION

TIME,

571

OF TYROSINASE

min

TYROSINASE,

m unit/ml

FIG. 2. (A) O;-induced chemiluminescence in the dopa-tyrosinaseCLA system and the effect of possible inhibition on the chemiluminescence. The standard reaction mixture containing 0.3 mM dopa, 6 pM CLA, 4 munits of tyrosinase/ml, and 0.1 M potassium phosphate buffer at pH 6.5 was used. Additive were catalase (20 pg/ml), 20 mM histidine (HIS), 10 mM dimethylfuran (DMF), and 0.5 pM SOD. CL, chemiluminescence. (B) Double-reciprocal plot of the maximal light intensity against the concentration of dopa. The standard reaction mixtures containing 1, 2, and 6 munits of tyrosinase/ml, 6 pM CLA, and 0.1 M potassium phosphate buffer at pH 6.5 and various concentrations of dopa were used. (C) Relationship between 0, formation and tyrosinase activity. The standard reaction mixture was used, save that the enzyme concentration was varied from 0 to 10 munits/ml.

by CLA-dependent chemiluminescence. As shown in Fig. 2A, CLA-dependent luminescence appeared promptly just after the addition of enzyme, reached a maximum, and decreased exponentially thereafter. Since 0; reacts with CLA to emit light (18), CLA-dependent luminescence corresponds to the rate of 0; generation. Under the same experimental conditions, except for CLA, the rate of dopachrome formation reached a maximum within about 20 s and decreased to about 80% at 60 s, which is in good agreement with that for chemiluminescence intensities (data not shown). This luminescence was not influenced by histidine, dimethylfuran, or catalase, but was completely eliminated by a catalytic amount of SOD. The K,,, of tyrosinase for dopa was found to be 0.2 mM, as shown in Fig. 2B. Under the same experimental conditions, save that O2 consumption and dopachrome formation were used for the assays of the enzyme activities, K,,, values for the enzyme were found to be 0.5 mM with O2 consumption and 0.8 mM with dopachrome formation (data not shown). Korytowski et al. (17) have reported that the K,,, for dopa obtained with O2 consumption is 0.9 InM. With a fixed concentration of dopa, 0; generation was a linear function of enzyme concentration up to about 4 munits of the enzyme/ml and was calculated to be 0.55 nmol/min/ml (Fig. 2C). Cytochrome c reduction and chemiluminescence were linearly related, as shown in Fig. 1. The relationship between O2 consumption and product formation is shown in Table I. Under the standard reaction conditions, with 4 munits of tyrosinase/ml, 1 mol of Oz is required for the production of 2 mol of dopaquinone, 1 mol of dopachrome, and i mol of 0,. SOD at the

572

KOGA, TABLE

NAKANO,

AND

I

O2 Consumption and Metabolite Formation in the DopaTyrosinase System” Substance

[nmol/min/ml]b

0; Dopachrome Dopaquinone H,O, O2 consumption

0.55 2.12 4.00 Negligible 2.01

Note. Values were expressed as means of five experiments. ’ The standard reaction mixture with 4 m units of tyrosinase/ml 0.3 pmol of dopa/ml. * Initial rates.

catalytic amount had no effect on the dopachrome mation (data not shown).

and

for-

(2) Generation of 0; in the Tyrosine (or 4Hydroxyanisole)-Tyrosinase System To confirm the generation of 0, from tyrosinasemonohydric phenols, tyrosine and 4-hydroxyanisole, which possess a strong cytotoxicity on malignant melanoma (19), were used. Since CLA-dependent chemiluminescence was too weak to detect when 0.3 mM tyrosine was incubated with tyrosinase at an enzyme concentration of 4 munits/ml, a tyrosinase concentration of 40 munits/ ml was used in the tyrosine system. On the other hand, CLA-dependent chemiluminescence in the 4-hydroxyanisole system was detectable using tyrosinase at the lower concentration. As shown in Fig. 3A, the chemiluminescence from the tyrosine and 4-hydroxyanisole systems had lag periods. The lag was very much greater with tyrosine than with 4-hydroxyanisole. The CLA-dependent luminescence from both systems was completely quenched by a catalytic amount of SOD. Under the same experimental conditions, integrated chemiluminescence in the tyrosine system was not parallel to the dopachrome formation from tyrosine (Fig. 3B). This indicates that 0, generation is not directly related to the conventional enzyme activity. (3) ESR Spectrometry and O;-Induced Chemiluminescence It has been known that, in the tyrosinase-catalyzed reaction, tyrosine is oxidized to dopaquinone which rapidly cyclizes to generate dopa, while N-acetyltyrosine is oxidized by a noncyclizing pathway (14). Since both tyrosine and dopa are substrates for tyrosinase, tyrosine, when used as a substrate, would provide more electrons than would N-acetyltyrosine. Consequently 0; generation would be greater with tyrosine than with N-acetyltyrosine. To investigate this, two parameters such as semiquinone

TERO-KUBOTA

accumulation and quinone production were measured and compared with chemiluminescence. As shown in Fig. 4A, chemiluminescence with tyrosine and with N-acetyltyrosine had lag periods and similar kinetics, but was of significantly different intensity; i.e., the maximal light intensity in the tyrosine system was approximately 10 times that in the N-acetyltyrosine system. With the same systems, save that CLA was excluded, quinone accumulation detected in the presence of ascorbate in the tyrosine systems was approximately 1 that in the N-acetyltyrosine system (Fig. 4A, inset). Using higher enzyme concentrations at 2O”C, ESR signal heights (Fig. 4B) of Zn*+-stabilized radicals in N-acetyltyrosine and tyrosine systems were increased after lag periods, reached maximum at about 30 min, and then decreased, identical to the time course of chemiluminescence intensities in Fig. 4A. Such symmetric intensity curves for ESR study may indicate little or no production of their corresponding second radicals. Furthermore, the results showed that chemiluminescence and the semiquinone signal with tyrosine and N-acetyltyrosine were inversely related; i.e., high 0; generation correlated with low semiquinone accumulation. The ESR spectrum obtained during the incubation of tyrosine with tyrosinase in the presence of Zn2+ is shown in Fig. 5A. The hyperfine structure could be interpreted with four kinds of protons (a: = 4.0 G, a! = 0.63 G, a; = 2.25 and 4.25 G) as confirmed by computer simulation. These values are in good agreement with those of the Zn2+-stabilized dopaquinone anion radical, obtained by horseradish peroxidase-catalyzed dopa oxidation at pH 6.4 (20). Assignment of hyperfine splitting could reason-

A

z

-5% INCUBATION

TIME,min

4-HO-ANISOLE

TYR I

IO

I

20 30 INCUBATION TIME, mm

40

‘0 1

FIG. 3. (A) Time courses of CLA-dependent chemiluminescence in the tyrosine (or 4-hydroxyanisole)-tyrosinase system. The standard reaction mixture containing 0.3 mM tyrosine (or 4-hydroxyanisole), 6 pM CLA, 4 (. . . ) or 40 (-1 munits of tyrosinase/ml, and 0.1 M potassium phosphate buffer at pH 6.5 was used. (B) Relationship between relative integrated chemiluminescence (REL. ICLI) and dopachrome formation. The standard reaction mixtures containing 0.3 mM tyrosine, 40 munits of tyrosinase/ml, 6 pM CLA (for luminescence measurement) or none, and 0.1 M potassium phosphate buffer at pH 6.5 were used. The values at 40 min were taken as unity. TYR, tyrosine; 4-HO-ANISOLE, 4-hydroxyanisole; CL, chemiluminescence; muE, munits of enzyme.

GENERATION

OF SUPEROXIDE

DURING

ably be given by spin density calculation for the 4-alkylo-quinone anion radical (21). The hyperfine splitting due to the proton at the 3-plosition of the quinonoid ring is unresolved because of the smallness of its value (<0.2 G). Since the ESR spectrum. clearly shows line-width alternation owing to the hindered rotation of the methylene group, the unequivalent protons could be distinguished from the ring protons. A similar ESR spectrum was obtained during the incubation of N-acetyltyrosine instead of tyrosine under the salme conditions (Fig. 5B). From the simulated spectrum, the hyperfine splitting constants were determined to be af;’ = 3.94 G, a: = 0.63 G, and a; = 2.3 and 3.8 G, respectively. The difference of uH values of this radical from those of the Zn2+-stabilized dopaquinone anion radical suggests that the conformation of the methylene group is affected by the N-acetyl group. Thus, the radical derived from N-acetyltyrosine under our experimental conditions is considered to be the Zn2+-stabilized N-acetyldopaquinone anion radical.

THE

ENZYMATIC

ACTION

of H201pfor Tyrosinase Activity

It has been reported that the oxytyrosinase produced by the addition of Hz02 to resting mushroom tyrosinase is probably a functional species in tyrosinase catalysis (3). If 0; is generated during the enzymatic process, the addition of H20z to the dopa-tyrosinase system should enhance 0, generation. As shown in Fig. 6A, the rate of 0; generation, monitored by measuring maximal chemiluminescence intensity, increased with increasing [H,O,] and reached a plateau above 30 FM. A similar increasing curve was obtained by plotting dopachrome formation against [Hn02] (Fig. 6B). However, an excess of CLA did not affect the production of dopachrome, even though CLA trapped 0; (data not shown). To investigate whether or not the enzyme consumes exogeneously added H202, H202 was added to the dopatyrosinase system prior to or during the incubation and its concentrations were analyzed by the addition of catalase. Judging by the production of O2 from HzOz in the presence of catalase (HzOa = Hz0 + 1 O,), half of the HzOz added (50 PM) was consumed in 4 min under the experimental conditions. However, the rate of consumption was not influenced b!y the addition of HzOz, indicating no catalase activity in tyrosinase, as shown in Fig. 6C. DISCUSSION

We have demonstrated the generation of 0, in tyrosinase-substrate systems. The production of ‘02 in the tyrosinase-dopa system was negligible as shown by complete quenching of luminescence by a catalytic amount of SOD and little or no effect on the luminescence of each of several ‘02 traps. However, it was not possible to detect 0; by the conventional cytochrome c method, because of direct reduction of cytochrome c by dopa.

573

-

I (T/R) =

.^^ I 1w

9

;; a

2 _._...” _..i

50

:.’ .:’ I _:’ ._: ti

“.. 3 c i7i

0

20

5 i I

iv 0 40

22 v Zl d

2 (N-ACT(R) . . .. . ..-e ..- . . ...__..,,

..:\

0

E

03I=

B

2 ..- -‘*. ‘.‘.., i” 1..

H Y2-

2

t

..:6

5

,.::’ ;’

F I -

(4) Requirement

OF TYROSINASE

‘:, i,

!5 0

IO

20

30

40

50

INCUBATION TIME, min FIG. 4. (A) Time course of CLA-dependent chemiluminescence (A) or quinone formation (A, inset) and Zn*+-stabilized radical accumulation (B) in the enzymatic oxidation of tyrosine (TYR) (-) or N-acetyltyrosine (N-AcTYR) (. . . ). The reaction system contained 40 munits of tyrosinase/ml in the standard reaction mixture with (A) and without (A, inset) CLA. For the ESR spectrometry, the reaction mixture con250 munits of tyrostained 1 mM tyrosine (or 1 mM N-acetyltyrosine), buffer at pH 6.8. ESR specinase/ml, 6.1 M Zn2+, and 0.1 M Tris-HCl trometries were done at ambient temperature on solutions contained in a flat cell. CL, chemiluminescence; Q, quinone.

Korytowski et al. (17) demonstrated the production of semiquinone radicals during the oxidation of dopa or Nacetyldopamine by tyrosinase, using spin stabilization with Zn2+. It is known that dopaquinone and dopaminequinone undergo rapid intramolecular cyclization, forming dopachrome and dopaminechrome, respectively, while quinones from N-acetyldopamine (13) and 4-methylcatech01 (22) do not cyclize. For similar rates of enzymatic oxidation of substrates, the semiquinones formed from noncyclizable quinones decay much more rapidly (via the second-order dismutation) (17). Thus, the most plausible pathway of tyrosinase-catalyzed oxidation of dopa is shown in Fig. 7. With slow generation systems for dopaquinone and Nacetyldopaquinone, such as tyrosine-tyrosinase and Nacetyltyrosine systems, respectively, the accumulation of Zn2+-stabilized dopasemiquinone in the tyrosine-tyrosinase system was low compared with that in the N-acetyltyrosine-tyrosinase system even though the rate of reaction in the tyrosine-tyrosinase system was twice that in the N-acetyltyrosine-tyrosinase system as monitored

KOGA,

574

-

NAKANO,

AND

TERO-KUBOTA

6

5G

INCUBATiON

[Hz021 ~JM

k

5G

1

FIG. 5. ESR spectra of Zn’+-stabilized dopasemiquinone radical (A) and of Zn’+-stabilized N-acetyldopasemiquinone radical (B). The spectrum for (A) was taken 23 min after the initiation of the reaction (scan time, 10 min). The spectrum for (B) was taken 19 min after the initiation of the reaction (scan time, 4 min). The incubation mixture and conditions were essentially the same as those in the legend to Fig. 4B. The signal height for (B) corresponds to twofold that for (A).

by their corresponding quinone formation (Fig. 4A). Under similar conditions, CLA-dependent chemiluminescence, indicating the rate of O;, was inversely related to Zn2+-stabilized semiquinone accumulation (Fig. 4). It seems likely therefore that the rapid cyclization reproduces a substrate catechol (dopa), thereby increasing the chance for the reaction with the enzyme molecule to generate 0; in this process. Pulse radiolysis and ESR studies carried out on epinephrine orthosemiquinone and other catecholamine orthosemiquinones imply that reactions of oxygen with catecho1 semiquinones are very slow (23). These findings suggest that the 0, trapped by CLA in the tyrosinasesubstrate system is released from the active form of tyrosinase. As reported by others (17,24), 1 mol of O2 was required for the production of 1 mol of dopachrome or the production of 2 mol of dopaquinone in the presence of ascorbate, without H,O, generation, in the dopa-tyrosinase system. In addition to these two products, a mol of 0,

TIME,min

FIG. 6. Effect of exogeneously added H,O, on CLA-dependent chemiluminescence (A) and dopachrome formation (B) in the dopa-tyrosinase system, and HzOz consumption in the dopa-tyrosinase system (C). The standard reaction mixtures containing 4 munits of tyrosinase/ ml with CLA (for luminescence measurement) or 40 munits of tyrosinase/ml (for dopachrome measurement) were used, except that H,O, at a variety of the concentrations was added. The reaction mixture for (C) 0.1 M potassium contained 0.3 mM dopa, 200 munits of tyrosinase/ml, phosphate buffer at pH 6.5, and 100 pM H202 (---) or none (-) in a total volume of 3.8 ml. Catalase, 20 fig/ml, was added at the time indicated by the arrow (---). Both 20 pg of catalase/ml and 100 pM H,O, were added during the reaction at the time indicated by the arrow (-). CL, chemiluminescence; DOPAC, dopachrome.

was produced from 1 mol of Oz. In the absence of CLA, 1 mol of 0, should disappear by disproportionation at the second rate constant of 1 X lo6 ~-9’ at pH 6.5, yielding i mol of O2 and i mol of Hz02. Hydrogen peroxide generated in the reaction would be utilized for the pro-

2 ‘WT (DOPASEMIOUINONE)

2H+

HEp

+

COT H2

(DOPACHROME)

FIG. 7. zyme.

tEUKODOPACHROME)

Plausible mechanism of dopa oxidation

by tyrosinase.

E, en-

GENERATION

OF SUPEROXIDE

DURING

duction of oxytyrosinase. The rate constant of oxytyrosinase formation in the presence of H202 has been reported to be 675 ~-9-l (3). Since CLA or SOD did not inhibit dopachrome formation from dopa in the presence of tyrosinase, free 0, was not utilized for oxidizing dopa. Both tyrosinase and hemocyanin are known to be binuclear metal (Cu) complexes. Jolley et al. (3) have suggested that the bicupric sites of resting tyrosinase and hemocyanin have an oxidation-reduction potential in the same general range and the reaction with HzOz is then formulated as Hz02 + [Cu(II)],

= oxytyrosinase

ENZYMATIC

ACTION

OF TYROSINASE

[Cu(I)lZ + OZ + 2H+.

[l]

Since the addition of HZOZ and dopa to tyrosinase promotes the release of 0; and formation of dopachrome (Figs. 6A and 6B), the Cu(II)O;Cu(I) complex could be formed as an intermediate: + 2H+.

[a]

Moss et al. (25) have reported that a complex of singlet oxygen or a superoxide complex, Cu(II)O;Cu(I), may contribute to the structure of oxyhemocyanin. Even though 0; once released from the active enzyme is not directly involved in enz:yme activity, its production indicates the possible formation of Cu(I)O&u(II) in the catalytic process. Hamilton and Libby (26) have proposed that in the Dgalactose oxidase (an enzyme containing one atom of copper per molecule) reaction, 0; dissociates from Cu(II)O,, which is out of the catalytic cycle and inactive catalytically. In their reports, 0; is apparently involved in the conversion of Cu(I1) to Cu(II1) (an active form of the enzyme). However, free 0; is not utilized in the conversion of resting tyrosinase to oxytyrosinase.

575

2. Schoot-Uiterkamp, A. J. M., and Mason, H. S. (1973) PFOC. N&l. Acad. Sci. USA 70,993-996. 3. Jolley, R. L., Evans, L. H., Makino, Biol. Chem. 249,335-345.

N., and Mason, H. S. (1974) J.

4. Makino, N., McMahill, P., Mason, H. S., and Moss, T. H. (1974) J. Biol. Chem. 249, 6062-6066. 5. Nishioka,

K. (1978) EUF. J. Biochem. 85, 137-146.

6. Jolley, R. L., Evans, L. H., and Mason, H. S. (1972) Biochem. Biophys. Res. Commun. 46,878-884. 7. Pomerantz, S. H., and Li, J. P. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. W., Eds.), Vol. 17, Part A, pp. 620-626, Academic Press, New York. 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, (1951) J. Biol. Chem. 193, 265-275.

=

[CU(II)]~ + HzOZ := Cu(I)02Cu(II)

THE

R. J.

9. Roussos, G. G. (1967) in Methods in Enzymology (Grossman, L., and Moldave, V., Eds.), Vol. 12, pp. 5-16, Academic Press, New York. 10. McCord, 6055.

J. M., and Fridovich,

I. (1969) J. Biol. Chem. 244, 6049-

11. Nakano, M., Sugioka, K., Ushijima, Biochem. 159, 363-369.

Y., and Goto, T. (1986) Anal.

12. Miller, W. H., Mallette, F., Roth, L. J., and Dawson, C. R. (1944) J. Am. Chem. Sot. 66, 514-519. 13. Pomerantz,

S. H. (1963) J. Biol. Chem. 238, 2351-2357.

14. Graham, D. G., and Jeff, P. W. (1977) J. Biol. Chem. 252, 57295737. 15. Mason, H. S. (1948) J. Biol. Chem. 172, 83-99. 16. Weiss, S. J., Klein, R., Slivka, A., and Wei, M. (1982) J. Clin. Znuest. 70,598-607. W., Sarna, T., Kalyanaraman, B., and Sealy, R. C. 17. Korytowski, (1987) Biochim. Biophys. A& 924, 383-392. 18. Gotoh, N., and Niki, E. (1990) Chem. Lett., 1475-1478. 19. Riley, P. A. (1969) J. Pathol. 97, 193-206. 20. Kalyanaraman, B., and Sealy, R. G. (1982) Biochem. Biophys. Res. Commun. 106, 1119-1125. 21. Pederson, J. A., and Spanget-Larsen, 35,41-45. 22. Tse, D. C. S., McCreery, Chem. 19,37-40.

J. (1975) Chem. Phys. Lett.

R. L., and Adams, R. N. (1976) J. Med.

23. Land, E. J. (1988) Reu. Chem. Zntermed. 10, 219-240.

ACKNOWLEDGMENT We thank Professor I. Fridovich (Department of Biochemistry, Duke University, North Carolina) for his helpful comments on the manuscript.

F., Garcia-Canovas, F., Iborra, J. L., 24. Jimenez, M., Garcia-Carmona, Lozano, J. A., and Martinez, F. (1984) Arch. Biochem. Biophys. 235, 438-448.

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25. Moss, T. H., Gould, D. C., Ehrenberg, A., Loehr, J. S., and Mason, H. S. (1973) Biochemistry 12, 2444-2449.

1. Lerner, A. B., Fitzpatrick, T. B., Calkins, W. H. (1950) Physiol. Reu. 30,91-126.

E., and Summerson,

26. Hamilton, G. A., and Libby, Commun. 55,333-340.

R. D. (1973) Biochem. Biophys. Res.