Anions binding to bilirubin oxidase from Trachyderma tsunodae K-2593

ELSEVIER

lnorganica Chimica Acta 273 I 1998 ) 204-212

Anions binding to bilirubin oxidase from Trachyderma tsunodae K-2593 Junzo Hirose *, Kaori Inoue, Hirokuni Sakuragi, Mituru Kikkawa, Masayoshi Minakami, Tsutomu Morikawa, Hiroyuki Iwamoto, Keitaro Hiromi Department o/'Food Science and Technology. Faculty ol'Engineerin~, Fukuvama ~hziversity. Fukuyama. Hiroshima 729-0251. Japan Received 28 April 1997: revised 29 May 1997: accepted 15 October 1997

Abstract Bilirubin oxidase obtained from Trachyderma tsunodae K-2593 is a multi-copper enzyme which has type l :type 2:type 3 copper atoms in the ratio 1:1:2. Anion binding properties of bilirubin oxidase were investigated to determine the structural and functional properties ofbilimbin oxidase. The anions N:~ , S C N , F , CI , and Br were non-competitive inhibitors against the substrate, bilirubin ditaurate, and the inhibition constants of the anions were in the following order: N~ < SCN << F
1. Introduction

In a multi-copper e n z y m e , type I copper has an intense absorption band ( e - - - 3 0 0 0 - 5 0 0 0 M - ~ cm ~) around 600 nm, ascribed to a ligand S ( C y s ) --+ Cu 2. charge transfer transition and a distinctive E P R signal, characterized by a particularly low' All value ( < 10 roT) [ 1 ]. The function of type I copper is to accept electrons from the substrate and to relay them to the o x y g e n reduction site. This latter site is a trinuclear cluster, constituted by a type 2 copper ion and a type 3 c o p p e r pair, formed by two magnetically coupled cupric ions, silent in the E P R spectrum and responsible for an absorption shoulder at 330 nm [ I , 2 ] . Type 2 copper is always detectable by EPR [ 11. *C~rresponding author. Fax: +81 849 36 2023: e-mail: hirose@ fubac, fukuyama-u.ac.jp 0020 1603/gg/$10.(X) 4"~ 1998 Elsevier Science S.A. All rights reserved. P1180020-1693(97)06183 5

Bilirubin oxidase ( B O D ) (bilirubin o x y g e n oxidoreductase [EC 1.3.3.5]) catalyzes the oxidation o f bilirubin to biliverdine. T w o kinds of B O D obtained from Trachyderma tsunodae K-2593 and M v r o t h e c i u m t:errucaria have been used for m e a s u r e m e n t of human serum bilirubin in clinical chemistry [ 3,4 ]. The molecular weight of B O D isolated from Trachyderma tsunodae K-2593 (T.t. B O D ) was determined to be 64 kDa [5]. It was reported by Matui et al. that T.t. B O D contained four c o p p e r atoms [ 4 ]. In our previous paper [ 5 ], T.t. B O D was shown to be a multi-copper e n z y m e which had type 1:type 2:type 3 copper atoms in the ratio 1:1:2. The spectrophotometric properties of T.t. B O D (e~m ..... = 3 8 7 0 M ' c m ~ and A , ( t y p e 1 c o p p e r ) = 8 . 4 m T ) were very similar to those o f fungal multi-copper laccases. These facts indicate that the coordination structure of copper atoms of T.t. B O D is similar to that of laccase, but the substrate specificities of the e n z y m e are quite different [ 5 ]. Recently,

J. Hirose et al. / Im,rganica Chimica AcI[1273 (1998) 204 212

lwamoto et al. isolated the clone of T.t. BOD and deduced the amino acid sequence [6,7] ~. The coordination residues of type 1, type 2, and type 3 copper were predicted by the homology of the amino acid sequence between T.t. BOD and ascorbate oxidase obtained from zucchini [ 6,7 ]. Every l igand of type 2 and type 3 copper ions in T.t. BOD was shown to be His and was completely consistent with those of zucchini ascorbate oxidase [6,7]. The ligands of type I copper in T.t. BOD were Cys, His, His, and Phe. Met which coordinates with type 1 copper in zucchini ascorbate oxidase [2] was replaced by Phe in T.t. BOD [ 6,7 ]. Koikeda et al. [8] isolated the clone of BOD obtained from Mvrothecium verrucaria (M.:'. BOD) and deduced the amino acid sequence. According to the homology-based alignment, it was predicted that the enzyme was multi-copper enzyme and the ligands of type I copper were Cys, His, His, and Met [8]. Xu et al. [9,101] characterized M.~,. BOD in the redox potential of type 1 copper, the substrate specificity and the anions inhibition. The redox potential of the type 1 copper was 0.49 V and slightly larger than that in zucchini ascorbate oxidase [ 101. BOD obtained from Trachyderma tsunodae K-2593 ( T.t. BOD) was the multi-copper enzyme but type I, type 2 and type 3 copper ions in T.t. BOD have not been sufficiently characterized. In this paper, the anion binding properties in T.t. BOD were investigated to determine the structural and functional properties of T.t. BOD by the inhibition kinetics of the enzyme activity, spectrophotometric, EPR, and stopped-flow methods.

2. Experimental 2.1. Materials

BOD (T.t. BOD) from Trachyderma tsunodae K-2593 was purchased from Takara Shuzo. The enzyme was used after purification with a Phenyl-Sepharose column [5]. The copper content of the enzyme was measured by an atomic absorption, and the content was 3.5-4.0 atoms per enzyme molecule. Sodium azide, sodium fluoride and potassium thiocyanate (reagent grade) were purchased from Nakarai, and used without further purification. Bilirubin ditaurate disodium used as a substrate for BOD was purchased from Funakoshi. The concentration of BOD was calculated from the molar absorption coefficient of BOD (3870 M ~cm ~) at 610 n m [ 5 ]. Li= [ Co(11) ( 2,6-pyridinedicarboxylate ) =] used for measuring the redox potential of type 1 copper in T.t. BOD was synthesized by the method of Williams and Yandell

llll. ' The nucleic acid sequences of the biliruhine oxidase obtained from Trachydelvml tsum~dae K-2593 have been reported to the DNA Data Bank of Japan I the accession number AB006824).

2(t5

2.2. Methods 2.2.1. Inhibition o f enzyme activi O' by various anions In studying the inhibition of the reaction by various anions, the reaction mixture contained the substrate (concentration s; bilirubin ditaurate disodium), the enzyme (E), and the inhibitor (concentration i; various anions (NAN,, KSCN, NaF, NaC1, and KBr) ), in the following final concentrations: [E1~,=5.6× 10 -~ M, s = 5 - 4 0 t-tM, [NaN3] = 0 - 2 0 t-tM, [KSCN]=0-60mM, [NaF]=0-60mM, [NaCII=0200 mM, and I KBr] = 0-400 raM. The reaction in which the enzyme oxidized bilirubin ditaurate to biliverdine ditaurate in 0.05 M phosphate buffer (pH 6.8) at 25°C was followed at 450 nm on a Waken-Otsuka Electronics micro-stopped flow apparatus interfaced with a microcomputer system for data acquisition and manipulation. At least tire kinetic traces were averaged for each measurement at a given reagent concentration. The determination of the reducing rate of the substrate (bilirubin) was obtained from the initial velocity ( r l of the reaction ( A e ( 4 5 0 n m ) = 3 4 1 0 0 M ~cm ') traced by a micro-stopped flow. Prior to the inhibition experiments, the Michaelis constant. Kin, was determined at 25~'C, pH 6.8 and was 15 txM which was calculated from the relationship between the initial velocities (~) and the concentrations of the substrates (s) by the non-linear least-squares method. On the basis of this value, the substrate concentrations in the inhibition experiments of the anions were selected. 2.2.2. Measurements o f absm7~tion and EPR spectral changes by addition c~["various anions The absorption spectral changes of BOD caused by various anions were measured with a double beam spectrophotometer, Shimadzu UV-3100, in 0.05 M phosphate buffer ( pH 6.8, 25°C). The X-band EPR spectra were respectively measured at room temperature and at 77 K on an EPR spectrometer (JES-FE2XG, Jeol Tokyo). A capillary cell for aqueous solution was used for the measurements at room temperature. The amount of the EPR detectable Cu 2 ~ was determflled using Cu-EDTA as a standard. The copper concentrations of Cu-EDTA and T.t. BOD were measured with an atomic absorption spectrophotometer. Spectral g values were calibrated with Mn 2 + as a standard. 2.2.3. Measurement ~?]'stopped-flow The anion binding to T.t. BOD was studied kinetically by monitoring the increase and decrease in absorbancc ( 610 or 410 nm) of the characteristic type 1 of the multi-copper enzyme, T.t. BOD, under pseudo-first-order conditions. Kinetic experiments were perfiwmed with the Waken-Otsuka Electronics micro-stopped flow apparatus interfaced with a microcomputer (NEC PC-9801 RX) li)r data accumulation and manipulation I 12]. At least five kinetic traces were averaged for each measurement at a given reagent concentration. The overall process was treated as a single exponential reaction. All kinetic studies were done in 0.05 M phosphate buffer, pH 6.8, at 25°C.

206

J. Hirose el td. /hlor,v, uni~ o Chimica ,4cta 273 (199,~) 204-212

where i is the inhibitor concentration. The relevant equation for non-competitive inhibition in Dixon plots is as follows:

3. Results 3.1. T y p e o f i n h i b i t i o n a n d i n h i b i t o r c o n s t a n t s b y v a r i o u s {IIliOIIS

1 -=

Prior to the inhibition experiments, the Michaelis constant, K .... and the molecular activity, k. ( = V ....... / I E ] o ) . where V...... is the m a x i m u m velocity and [ E ]o the molar concentration of the enzyme, were determined at 25°C in 0.05 M phosphate buffer ( p H 6 . 8 ) . The values were as follows: K..=lS±3gMandko=163±10s ~.T.t. B O D h a s a h i g h affinity for bilirubin ditaurate. The type of inhibition and inhibitor constants. K,. were determined for the live anions at 25°C. pH 6.8. The type of inhibition was j u d g e d from the C o r n i s h - B o w d e n plots [13,141. in which s / u is plotted against the inhibitor concentration, i. at different suhstrate concentrations, s. and by the Dixon plots 113.14], in which 1/t' is plolted against the inhibitor concentration, i. at different substrate concentration, s. The relewmt equation |or non-competitive inhibition in Cornish-Bowden plots is as follows: s K,,,+s i K,,,+.~ - = - + - ~' V,,.,, Ki V,..,

(1)

(a)

(o
3 x

o

2

"7,

2

-

"

0

-3

-2

-1

0

1

2

i= [N 3- ] X 1 0 s (M) 60

(b)

50

40 o to

K,,,+s

30

2O

i

- -

I'

K,,,+s + - -

V ...... ,Y K i

(2)

V,,,avY

Cornish-Bowden plots and Dixon plots can be derived as follows from the familiar rate equation for non-competitive inhibition [ 13 ]: r-

( V,,,~,x)/( l +i/K,)

(3)

.~"+ K.,

The examples of C o m i s h - B o w d e n and Dixon plots for N~ are shown in Fig. 1 and these plots show good linearity. In Fig. 1, the C o m i s h - B o w d e n plots ( s h ' versus i plots) and Dixon plots ( I / u versus i plots) at different s values show that the intersection points o n the i axis and their inhibition constants were consistent with each other. This behavior strongly indicates that the type of inhibition by N~ is purely non-competitive. For the tive anions ( N:, . S C N , F ,CI a n d B r ) s t u d ied. C o m i s h - B o w d e n plots (x/v versus i plots) and Dixon plots ( l / r versus i plots) at different s values show that the intersection points on the i axis and the inhibition constants obtained from Dixon plots were consistent with those obtained from Cornish-Bowden plots. These results show that the five anions are non-cornpetitive inhibitors for the substrate ( bilirubin ditaurate). The inhibition constants, K,, for various anions obtained from Dixon or C o r n i s h - B o w d e n plots are shown in Table l and were in the following order: N~ < S C N <
10

0

.

-3

-2

-1

,

.

1

,

.

Inhibition constants (K,) for the anions at 25
2

i = [N 3 ] X l 0 s (M)

Fig. I. Inhibition of the enzyme activity by N~ on 25~+Cm 0.05 M phosphatL" bufl~'r at pH 6.8. The concentrations of the suhstratc (bilirubin ditauratc): ,G. 40 FM: A. 2(I p.M: O, I0 heM:O, 5 heM. (a) I)ixon plots in which 1/~ is plotted against the inhibilor concentration (i} al dillErcnt suhstratc con cenlrations. I h) (7ornish-Bowden plols in which ~/t is ploned agains!lhc inhibitor concentration ( i ) at different substralc concentralions.

Anion

Kb ( M )

N~ SCN F C1 Br

1,9 × I0 0.4 × IIt 2.6XI0 1.1×10 4.3 × 10

Type o f " • ~ ~ ~

inhibition

non-competitive mm
d. Hirose et al / hmr.~,,anica Chimica At'to 273 (1998) 204-212

0.30

m o ill .o

0.30

(a)

0.20

0,20

0.10

0.10

0.00

I

I

I

400

600

800

Wavelength

207

(b)

0.00

I

t

t

400

600

800

(nrn)

Wavelength

(nm)

0.30

}

(c)

0.20

E 0 0.10

0.00

400

600 Wavelength

800

(nm)

Fig. 2. Spectra of T.I, B e D vdth the addition of anions al 2 5 ( ' ill [).05 M phosphate huffei" ( pit 6.8 ). The concentration ',if lhe e n z y m e was 4.13 x 10 ~ M. ( a i S p c c t r a o f T . I . B O D i n i h e p r e s c n c c o f N ~ (OM I ( I m M ) : ( b l s p c c t r a l ) f / . t . BOD in the presence of SCN q ' ( l M - 5 3 m M ) : ( c ) s p e c t r a o l 7 " . t . B O D i n the presence of F ([) M - 1 2 6 mM ).

itive inhibitors against bilirubin ditaurate. The types of inhibition by the anions in T.t. B e D were purely non-competitive. This suggests thai the anions bind to the type 2/type 3 cluster site.

3.2. The c[fe('t q/atHotl,v (m the absoq~liou spectra ~1" resting B O D

absorption bands around 610 aud 330 nm. In Fig. 3, the absorbance changes at 610 nm in the binding of the resting T.t B e D with F , SCN , and N~ are plotted as a function of the concentration of the wtrious anions. The relationship between the concentration of anions and the absorbance E

eQ

0.05 0.04

The addition of anions to the resting T.t. B e D affected its absorption spectrum. The spectra obtained when various concentrations of the anions (N~ . SCN , and F ) were added to the resting T.t. B e D are shown in Fig. "~ With the addition of N~ to the resting 7".1. B e D (Fig. 2 ( a ) ) , the absorption around 610 nm, which is characteristic of the type 1 cupric ion, increases, whereas the absorption shoulder around 330 nm, which is characteristic of the type 3 cupric ion, slightly decreases. In Fig. 2 ( b ) . the same behavior is also observed on addition of SCN Io the resting T.t. BeD. In Fig. 2( c I, the addition of I: , m contrast, resulted respectively in the decreasing arid increasing of the

¢D CO c ~1 (.9 ¢=

o

o

<

• "

oO

0.03

/

0.02

t ,' !

0,01

i1© O'

." ~o.o.(~'

0.00

A

- -%~.

-0.01 -7

-6

-5

.4

log [ C o n c e n t r a t i o n Fi~. 3. Anhm~, binding to T./. B O I ) at , . ( (pH6.81:O,N~

:(3. SCN

:M,.F

.

-3

-2

-1

of anion] in 0.05 M ph(~:,phate bultm"

,L Hirose et al. / hun~¢anica (Tdmica Acla 273 (1998) 2(M-212

208

Table 2 The dissociation constanls of Ihe anions frmn T.I. B O D and kinetic parameters of the anion binding to T.t. BOD. All experiments were done in 0.05 M phosphate buffer {pH 6.8) at 25~'C Anion

K,(M)

K~,,(M

k3is 0.356 0.207

N~

1.8×10

4

1.0XI() ~

SCN F

3.2:<10 1.7X10

' ~

3,0×102 k.K,,. : I . 5 M

~s

-

Eli

ilK d + 1

~:,

k eis

(4)

-

where Kd, ~,, ~,,_, A A , Eo and i are the dissociation constant of the anion from T.t. BOD, the molar absorbances of Ei and E, the absorbance change, the total concentration of T.t. BOD, and the concentration of anions, respectively. In Fig. 3, the theoretical curves which are calculated from Eq. (4) are consistent with the data plots obtained from the spectral changes with the addition of the anions. N, , S e N . and F are implied to bind to the resting T.t. BOD in the molar ratio of I:1. By titration of Rhus I'ernic~f~'ra laccase and C u c m M t a p~7m ascorbate oxidase with N~ , the binding of two azide molecules with different affinities ( K , d R h u s t'ernic(lt'ralaccase): 1 . 3 × 1 0 5 M a n d 1 . 4 × 1 0 2M 115]" Kd( Cucurt?ita pepo ascorbate oxidase) : < 2.0 X l0 4 M and ca. 1.0x 10 2 M [ 16] ) was observed. Therefore, the dissociation constant ( l . 8 X l 0 a M ) of N~ of T.I. BOD in "Fable 2 may correspond to the dissociation constant at a high aflinity site. The absorption spectra were also determined in lhe presence of 0.2 M KBr and NaCI, but these anions did not inlluence the absorption spectra of "l'.t. BOD. The dissociation constants of the anions are as follows: N, < SCN <
The EPR spectra of type I and type 2 copper in T.t. BOD were measured in the presence of the various anions ( N 3 ,

k ,/(k~K,,,) ( M ) 2.9X10 2.3×10 3.3XI0

4

~ -~

S e N , and F ). These spectra are shown in Fig. 4. In Fig. 4, the addition of N 3- and F to the resting T.t. BOD causes dramatic changes in the EPR spectra of type 2 copper, and the EPR spectra of type 2 slightly changes with the addition of S e N . The EPR spectra of' type I copper does not change in the presence of various anions. The EPR spectra clearly showed that the anions bound to the type 2 / t y p e 3 cluster site of the resting T,t. BOD and not to its type I copper. The EPR spectra and the enzyme kinetic data showed that the anions did not bind to type 1 copper. However, the anions affected the absorption spectrum originating from the type 1 cupric ion. Why do the anions affect the characteristic spectrum of the type 1 cupric ion? To determine the reason, the redox potential of type 1 copper in the resting T.t. BOD was measured, and the oxidation of type 1 copper was attempted by H202 to determine whether type I copper was further oxidized [ 17 ].

f

3.3. The eff;:ct q / a n i o n s on the electron .v)in resommce (EPRJ .spectra o f the resting T.t. B O D

')

0.105 0.140 0.05

i

change at 610 nm was shown by clear sigmoidal curves. On the basis of Eq. (4) derived from the assumption that anions bind to the resting T.t. BOD in a molar ratio of I:1, the dissociation constants (Kd) of the anions to resting BOD are calculated by the non-linear least-squares program l12] and are shown in Table 2. ,_kA ,:~i/K~ + ~:~

i)

0.25 Magnetic

I

!

0.30

0.35

Field

(Tesla)

l-Zig. 4. EPR spectra of l'.t. B O D in lhe presence of anions ( pH 7.4, 0.05 M phosphate buffer, T.t. BOD; 5 × 10 " M ): t a) T.t. BOD: (b) T.t. B O D in the presence of 6 × 10 4 M N~ ; ( c ! 7".t. B O D in the presence of 0.02 M S e N : (d) T.;. B O D m the presence of 0.05 M F .

J. Hirose et al. / Inorganica Chimica Acta 273 (1998) 204-212

3.4. The redox potential of ~. pe 1 copper and oxidized type 1 copper content in resting T.t. BOD The redox potential of type 1 copper was measured under anaerobic conditions using Li2[Co(II)(2,6-pyridinedicarboxylate)_,] (Li2Co(ll)[2,6-PA]2) as a mediator [18-20]. The spectra of T.t. BOD were changed by the titration with Li2Co(II) [2,6-PA2]. At each titration, the approach to the equilibrium between the oxidized and reduced forms of T.t. BOD was observed by monitoring the absorbance at 700 nm where [ C o ( I I ) ( 2 , 6 - P A ) 2 ] - ' - and [Co(III)(2,6-PA)2] had no absorbance but type I cupric ion in T.t. BOD did. The redox reaction between Co( IIl) (2,6-PA) _,and type I copper of the enzyme is shown as follows. Co( II ) (2,6-PA) 2 + ECu ( II ) ~ Co(III) (2,6-PA)2 + ECu(I)

[Co(lIl) (2,6-PA) 2] [ C ° ( I I ) (2'6-PA)212

0.059 n

(6)

[ECu(II) ] log

= E~C.ull)iE(.u(ll) + - -

[ECu(I) ]

where k,~L<>(.)lCo¢lll) is the redox potential (747 mV) of [Co(II)(2,6-PA)2I/[Co(III)(2,6-PA)2I [11], E4~o~,,,/E,-<,,~,, is the redox potential of type 1 copper in the enzyme, and n is the number of electrons involved in the reaction. The Nemst plots on the basis of Eq. (6) are shown in Fig. 5. In Fig. 5. the redox potential of type 1 copper is 615 mV at pH 6.8 and the number n of electrons involved in Eq. (6) is 1.5. The potential was also measured at pH 5.0 in 0.2 M acetate buffer (25°C) and was 642 inV. Type 1 copper in T.t. BOD had a high redox potential, so that the type 1 copper of

0.66 ©

d 0.64

o

8 o

0.62

o o~ ltl o o + p,.

0.4

o.,

0.3

J~

c

o

0.2

0.1

0

I

I

400

600 Wavelength

800 (nm)

Fig. 6. Optical spectra of T.t. BOD treated with H~O2. The enzyme concentration was 3 . 9 × 10 5 M in 0.05 M phosphate buffer (pH 6.8/ at 25°('. H202 (0 M-8.6 × 10 ~ M) was added to T.t. BOD.

(5)

where ECu (l) and ECu (lI) are respectively the oxidized and the reduced type I copper of the enzyme. On the basis of Eq. (5), the redox potential of type 1 copper is represented by the following Nernst plots [ 18 ] : ~'-,,ill~/c,,, l . ) + 0 . 0 5 9 log

209

0.60

the enzyme should be easily reduced. Therefore, there is a possibility that some part of the copper ions in the enzyme is in the reduced state• This is observed in some of the multicopper enzymes [ 15,17 ]. The full oxidation of the copper ions in the enzyme by H202 which has a high redox potential was tried to determine the reduced content of type 1 copper in the enzyme [ 17 ]. These results are shown in Fig. 6. In Fig. 6, the absorbances at 610, 400 and 330 nm are increased by the addition of H202. This result shows that some part of the copper ions in the enzyme was present in the reduced state; this was calculated to be 13t~ from the increase in the absorbance at 610 nm. To confirm this result, the cupric content of T.t. BOD was measured at room temperature by EPR. The fraction of the cupric state obtained by EPR in overall copper ions of T.t. BOD was about 36%. Therefore, some of the copper ions in BOD were present in the reduced state. If some of the copper ions m T.t. BOD were present in the reduced form, the disproportionation of electrons between type 1 and type 2/type 3 cluster should occur when anions bind to the type 2/type 3 cluster. A similar result was observed in the interaction of ceruloplasmin with chloride ions [17,21]. The absorbance changes in type 1 and type 3 copper with the addition of anions is the result of the disproportionation of electrons among the copper ions in T.t. BOD, because the binding of the anion to the type 2/type 3 cluster should change its redox potential 122 ]. The opposite absorbance change observed in N~ and F - binding to T.t. BOD (Fig. 4) may be attributed to the opposite changes in the redox potentials of the copper ions in the type 2/type 3 cluster site by the binding of the anions to the cluster site.

0.58

-0.6

-0.4 0.059

-0.2

0

0.2

0.4

0.6

0.8

log ([ECu(II)]/[ECu(I)])

Fig. 5. Nernst plots of the redox reaction between L i z I C o ( I I ) ( 2 , 6 - P A ) , ] and type 1 copper in T,t. BOD at 25°C in 0.05 M phosphate buffer ( pH 6.81 ( enzyme concentratkm, 5.0 :X I 0 ~ M ) : ©, experiment 1: D, experiment 2.

3.5. Anion binding kinetics The kinetics of anions binding to BOD was followed by the absorbance changes at 610 and 410 nm with a microstopped flow apparatus at 25°C. The reactions of BOD with

210

J. ttiro,~e et al, I l n o r g a n i c a C h i m i c a A~'ta 2 73 (1998) 2 0 4 - 2 1 2

Time

E ~.

~0

50

(sec)

100

f~

150

(a)

g "d

200

=

g,

N3 -

e,, e..

SCN-

u e,, ,',

g

F-

W .J~ o

,

I,

,

the type 2-N3 complex were also measured and are shown in Fig. 8 ( a ) . Pseudo-first-order rate constants k,,h~ obtained at 410 nm were consistent with those measured at 610 nm due to type 1 copper. This result also indicates the disproportionation of the electrons among the copper ions in the enzyme. Non-linear dependences of koh~ on anion concentration were observed for N~ and SCN . This behavior, also found in laccase anions reaction by Holwerda et al. [ 23 ], is consistent with the mechanism involving rapid formation of an outer-sphere complex ( B O D . a n i o n ) followed by a ratelimiting dissociative interchange to give the inner-sphere complex ( B O D - a n i o n ) product [23,24].

I

K~,~

(b)

E r.

k2

BOD + anion ~ BOD. anion ~ BOD-anion

(7)

o

g

The observed pseudo-first-order rate constant (k,,h~) implied by this mechanism is easily shown to be:

o

O) f= (-

g

k,h~=k ~+

¢.) U r-

K,,~-

g

,JO

k2K,,~l anion I 1 + K,,~[ anion [

[ BOD. anion l [ BOD] [ anion ]

(8)

(9)

I,=

0 o~ .0

,<

I

5

,

I

10

,

I

15

20

Time (sec)

Fig. 7. Anion binding reactions to l'.t. B O D at 2 5 ' C in 0.05 M phosphate buflcr ( p H 6 . 8 ) . (a) Anion binding reaction in the presence of N~ (l.0X10 :M},SCN (l.0Xl0 :Mi,andF (5.{)X10 ' M ) . T h e c o n cenlration of the e n z y m e was 2 . 0 × 10 5 M. (b) N~ binding reaction to l.I. BOD: . N, binding reaction curve. The concentrations of N~ and lhe enzyme were 1 . 0 × 10 2 M and 2 . 6 × I(1 • M, respectively: . . . . , liuing c u r \ e o | the pseudo-first order reaction.

N, , SCN , and F are shown in Fig. 7 ( a ) . At 610nm, increases in the absorbance were observed in the reactions of T.t. BOD with N~ and SCN , and a decrease was observed in the reaction with F . These results were consistent with those observed by the spectrophotometric method ( Fig. 2 ). The reactions of T.t. BOD with N3 (Fig. 7 ( b ) ) and SCN gave only the pseudo-first-order reaclions, but the reaction with F gave two phases. The fast phase reaction was the binding of F to the type 2 / t y p e 3 cluster, and the slow phase reaction may be the denaturation o f the enzyme. In the reaction of F with the enzyme, only the fast phase reaction data (pseudo-first-order reaction) were separated from the overall reaction by a microcomputer [ 12 ]. Observed pseudo-lirst-order rate constants (k,,t,~) in the reaction of T.I. BODwithN~ ,SCN-,andF are shown in Fig. 8, Non-linear dependences of k,,b~ on anion concentration were observed in N) and SCN , with k,,b~ approaching the saturation limit at high concentrations. On the other hand, the dependence of/",,b~ on the F concentration was linear with Ihe intercept. In the reaction of the enzyme with N~ , pseudo-first-order rate constants (k,,b~) followed at 410 nm which originate from

If a large excess of anions over T.t. BOD is present, k2, k 2 and K,,~ can be calculated lbr Ns and SCN by the nonlinear least-squares program using Eq. (8). The theoretical curves obtained by Eq. (8) are shown in Fig. 8 ( a ) and (b) and are consistent with k,,b~ values obtained from the experiments. The values of k2, k 2, and K,,~ in the reaction of BOD with N~ and SCN are shown in Table 2 with the dissociation constants obtained by the spectrophotometric method. The value of K,,~ in F may be very small, so that the term of (1 +K,,~[anion]) in Eq. (8) is almost unity in the F concentration between 0,01 and 0.35 M. Therefore, Eq. (8) is reduced to the following equation:

k,,h, =k 2 +k2K,,~[ anion I

(10)

In Eq. (10), the relationship between k,,~,~and [anionl is a straight line with an intercept ( k 2) and a slope (k2K,,~). The values of k ~ and k:K,~ are also shown in Table 2. The dissociation constant. Kj, obtained by the spectrophotometric method is represented by k. 2, k_~,and K,,~ obtained from the kinetic data.

Ka=k 2/(k2K,,~)

(11)

The values of k ,_/(k:K,,~) calculated from kinetic data are also shown in Table 2. In N~ and SCN , the values of K,~ were almost consistent with those of k ,/(k:KoD. This behavior indicates the validity of Eq. (8). In F , K a was not consistent with k .:/(k2K,,~), because the data obtained by the spectrophotometric method shown in Fig. 3 contains the absorbance change which occurred due to the slow denaturation reaction of the enzyme in the presence of F .

.I. Hiro,~' el al. / Im~r,vanica C h i m i c a Acla 273 (1998) 204 212

0.4

0.5

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(b) o

tO

0.4

0.3

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o

21 I

Y

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0.1 0.1

,

0.0

0.0

,

.

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.

.

.

.

0.0050

i

0.0 0.00

.

0.0100

Concentration of N a

,

I

,

0.02

I

0.04

,

I

o.o6

Concentration of SCN- (M)

(M)

0.6

(c)

0.5

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0.4

0

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o

0.2

0.1

0.0 0.0

0.1

0.2

C o n c e n t r a t i o n of F

0.4

0.3

(M)

Fig. 8. The dependence of the pseudo-lirst order rate conslants ( k,,,,,I on the concentration of the anions. All experiments were done in 0.05 M phosphate buffer ( p H 6 . 8 ) a t 2 5 ° C : ( a j N~ . 6 1 0 n m ( O l , 4 1 0 n m ( @ l : ( b ) SCN : ( e l F .

4. Discussion Solomon and associates [ 25,26 ], using spectroscopic studies of N~ binding to laccase and ascorbate oxidase, classified this metal binding site as a type 2/type 3 cluster site. Messerschmidt et al. [ 27] clearly show in the X-ray crystal structure that N3 ion binds terminally to one of the type 3 copper. The inhibition constants of halogen anions in T.t. BOD were in the following order: F
shows that the anions binding occur at the type 2/type 3 cluster site. The order of the dissociation constants of anions obtained by the spectrophotometric method in T.t. BOD is almost consistent with that obtained from the inhibition method. However the dissociation constants obtained by the inhibition studies are smaller than those determined spectrophotometrically. This difference will be attributed to the change in the affinity of the type 2/type 3 cluster for anions, because the partly reduced type 2/type 3 cluster in the catalytic cycle has higher affinity for anions than that in the resting state [ 161. The redox potential of type 1 copper in T.t. BOD was about 0.62 V at pH 6.8 and was higher than that of ascorbate oxidascs (0.35 V [ 11) and M.v. BOD (0.49 V [ 10] ). This is because the hydrophobicity of Phe around type I copper in T.t. BOD raises the potential [29-31 ]. The apparent redox potential of O~ calculated nnder the condition (02 ( 0.34 mM) in 0.05 M phosphate buffer ( pH 6.8 ) ) was about 0.71 V. There is a possibility that a small percentage of the copper ions in T.I. BOD are n o t oxidized in spite of the

212

J. H i rose et al. I lnor~,,anica Chimica Acta 27.7 (199
presence of enough O2 in the phosphate buffer (pH 6.8), because the difference in the redox potential between O, and type I copper in T.t. B e D is about 0.09 V. It is proposed that some part of the copper in laccase [ 15 ] and in ceruloplasmin [ 17 ] is in a reduced state. The fact that some part of the copper in T.t. B e D was in reduced form can easily explain the data obtained from anion binding with T.t. B e D . Ozaki et al. [ 22] reported that the redox potentials of the copper ion in superoxide dismutase changed because the anions bound to the copper ions. The phenomenon that the disproportionation of electrons among the copper ions in T.t. B e D occurred when the anions bound to the type 2/type 3 cluster and changed the redox potential of the copper ions implies that the redox potential of the type 2/type 3 cluster is close to that of type I. The opposite absorbance changes of N~ and F binding to B e D , around 610 nm and 330 nm, were attributed to the opposite changes in the redox potentials of the type 2/type 3 cluster site due to the binding of these anions to the cluster site. Recently, Musci et al. [17] reported that chloride ions modulate the redox state of the copper site of human ceruloplasmin. This behavior is very similar to the results obtained in T.t. B e D . The X-ray structure of human serum ceruloplasmin [ 32] showed that the residues around one of the type 1 coppers in ceruloplasmin were Cys, His, His, and Leu. Leucine is a hydrophobic residue. In azurin which contains only type 1 copper, Met l 21 provides a sulfur atom as a copper ligand [ 30,31 ]. Producing a series of Met 121 mutants [ 31 ] showed that large hydrophobic residues would raise the redox potential whereas negatively charged residues lower it. The hydrophobic residues around the type I copper ion in ceruloplasmin and T.t. B e D should cause higher redox potentials of the type 1 copper in the enzymes. The high redox potentials of type I copper cause the disproportionation of electrons among the copper ions in the enzymes. The high redox potential of type I copper in T.t. B e D may also give a special character to the enzyme and the specificity for the substrate in the enzyme activity. The anion binding and dissociation rates to T.t. B e D were very low. The N~ binding rate to type 2 copper in Cu,Znsuperoxide dismutase, which has a narrow channel into the copper ion of the active site, was measured by the stoppedflow method, and its rate was too high to follow by the stopped-flow method (the data are not shown ). On the basis of the influence of the anions on the intramolecular electron transfer rate in ascorbate oxidase, Hazzard el al. 128] suggested that the dissociation of the anion from ascorbate oxidase was very slow ( = 2 s ~). This behavior implies that the anions binding or dissociation rates at the type 2/type 3 cluster site will not be interpreted by sirnple anion binding mechanism. The rate of exchange of a water ligand of the type 2 copper appears to vary depending on whether the 'open" or the "closed" conformational form of the enzyme is present [ 28].

Acknowledgements This work was partly supported by a Grant-in-Aid (No. 09672205) from the Ministry of Education, Science, Sports and Culture.

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