Peroxide binding to the type 3 site in Rhus vernicifera laccase depleted of type 2 copper

Vol. 108, No. 1, 1982 September

8lOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

16, 1982

Pages

273-278

PEROXIDE BINDING TO THE TYPE 3 SITE IN RHUS VERNICIFERA LACCASE DEPLETED OF TYPE 2 COPPER 0.

Farver,

P. Frank

and I.

Pecht*

Department of Chemical Immunology The Weizmann Institute of Science Rehovot, Israel Received

July

19,

1982

between hydrogen peroxide and oxidized SUMMARY: The interaction Rhus vernicifera lactase from which the type 2 copper has been For that end, the circular dichroic removed, was investigated. spectrum of the modiliied enzyme has been-measured in the presence of increasing concentrations of hydrogen peroxide. The characteristic band observed upon binding peroxide to native lactase is also observed for the type 2 copper depleted enzyme. However, there are several quantitative differences in the latter one. the intensity is lower and band width is larger. Secondly, First, from the titrations, it becomes apparent that the affinity for While the lower than that of the native native enzyme is higher than 10e3YgY; it decreases for the type 2 depleted enzyme.

INTRODUCTION The reversible vernicifera of

lactase

significant

nature However,

the

its

conflicting features

of the

the

type

for

investigating copper

this (2-5).

2 copper

depleted

reverse;

namely,

(2), that

the

no change

been

while

report takes

which

about

prepared the

(2-5). spectro-

by essentially

330 nm band reported

(T2D)

and the

claimed

is

and structural

interactions

published

place

Rhus

functional

has been

lactase

from

derivative

and their

derivative

3 site

another

2 copper

an enzyme

have

Thus,

type

type

sites

enzyme

oxidized

unaffected

the

produces

reports of

remained

*Currently Chemistry Technology,

of

(l-3)

different

same procedure

istic in

value

of

scopic

removal

character-

to be absent 615 nm band

essentially

at the

the

330 nm band,

a Sherman Fairchild Distinguished Scholar, Division and Chemical Engineering, California Institute of Pasadena, California 91125.

of

0006-291X/82/170273-06$01.00/0 273

Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 108, No. 1, 1982

yet

a marked

recently,

decrease

evidence

and at

the

that

the

spectral

with

hydrogen

peroxide

indicated

that

the

3 site

at 615 nm (4).

type

3 site

type

at

this in

More

controversy

T2D is

reduced,

330 nm can be recovered

(5).

In

contrast,

of T2D lactase

other

is

in

by

studies

an oxidized

(2,6). The type

coupled

3 copper

binuclear site

site

shown

has been

affinity

binding

one,

and reduction

complex

(7-11).

interesting detail

in

to bind

reported

to be lost the

interaction spectrum

The near

ultra

provide

removal

of the

latter

of

peroxide, that

a characteristic

evidence

copper

site

in

hemocyanins

and tyrosinase

distinction

between

of peroxide. T2D lactase the native

complex

the

We have for

enzyme,

as evidenced

with

rather

high

The role

of that

(7-13). oxidation

of

that

still

by the

is

characteristic

its

proteins

enables

the

of

titration

shown

as other

site

to to the like

a clear

and its

affinity

binding of

compared forming

in

circular

binding

it

to

enzyme

of

been

decreased capab!-

the

course

copper

although

In order

peroxide

Hence,

some

has been

on the

monitored

as well

in

binding

the

an

of T2D lactase

2 copper.

the

is markedly it

(5)

peroxide type

is

examined

study

for

peroxy-

mechanism

has earlier

lactase

found

peroxide

peroxide

during

CD spectrum

binding this

we have

violet

binuclear

dioxygen

modifications

system

a

form,

and has been

of

is

oxidized

affinity

upon

with

dichroic

native,

mentioned

high

which

as the

reduction

issue,

the

effect

its

hydrogen

above

lactase,

proposed

dioxygen

In the

by H202,

of

(7,8,10).

the

oxidation

examine

In

and important (9-11).

site

has been

and specificity

lactase

of

occurs

features

oxidation

its

RESEARCH COMMUNICATIONS

has been made to resolve

by providing

state

BIOPHYSICAL

in extinction

an attempt

least

AND

the

to that

a peroxy-

CD band.

MATERIALS AND METHODS 7.0,

Lactase and the

solutions protein

were made up in 0.1 M HEPES buffer, concentrations were determined from 274

pH the

Vol. 108, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

350

Fimre hydrogen

1.

CD-spectra

peroxide.

400

RESEARCH COMMUNICATIONS

450

X krnl of TZD lactase (23 uM) titrated with a) no peroxide; b) 23 uM; c) 115 PM; and added to solution a. All other conditions

absorbance at 615 nm (E = 5700 M-1 cm-l), Type 2 copper depleted lactase was prepared according to the method of Graziani et al (l-3). Its copper content was determined spectrophotometrically with biquinoline. The two, independently prepared samples of T2D lactase studied in this work contained 2.8 and 2.9 Cu ions per mole protein, respectively. In view of changes caused in the molar extinction at 280 nm of T2D lactase, protein concentration was determined by the Lowry method. This showed that while essentially no change occurs in the intensit of the 615 nm band, that at 280 nm increases to Q99700 M- 4: cm-l (6). T2D lactase preparations were oxidized by excess ferricyanide which was removed by extensive dialysis or gel filtration. Protein concentrations examined ranged from 23 to 70 PM. CD spectra were measured in 1 cm cuvettes on a Roussel Jouan Dichrograph III. Data were collected by means of a Tektronix minicomputer averaging 200 measurements per nm.

RESULTS AND DISCUSSION The circular the

near

Namely, are

UV region both

present

The shorter do look

the with

dichroic is

wavelength

similar.

similar

negative close

spectrum

of

to that

oxidized

T2D

of the native

(447 nm) and positive to

the

same intensity

features,

Upon addition

though of 215

H202,

in

enzyme.

(375 nm) bands (cf.

not

lactase

also

too well

changes

in

ref.

2).

resolved, the

region

Vol. 108, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Scatchard plot derived from titrations of T2D hydrogen peroxide. L is the concentration of free ligand (H202), and N is-the fraction of complex divided by total enzyme concentration. N is determined at (AE - Ae,)/ AE - AEN) of the 330 nm band'for T2D lacease where AE is the intensity without geroxide, As is the intensity of the same band after addition of a 100 fo r d excess of H202, and AE~ denotes the intensities of the various intermediate peroxide concentration.

300-400 these

nm emerge. changes.

Figure

1 provides

A prominent

band

at 442 nm is unaffected. increases of

From

several

T2D lactase shown

in

such

Figure

near

of

of

330 nm, while

that

the

330 nm band a maximum value

of a hundred

fold

excess

done on two independently plot

of H202 prepared

was constructed

and is

2. of

shown by the

as the

presence

a Scatchard

The formation been

at

illustration

concentration,reaching

titrations,

samples,

appears

The intensity

with peroxide M -1 cm-1 in the

0.75

typical

the

peroxy

regeneration

of both

UV and visible

oxidized

TZD lactase

(6)

followed

by extensive

complex

electronic upon

the

original

This

against

by excess

has

CD as well

and EPR spectrum

reduction

dialysis

is reversible.

of

ascorbate,

ferricyanide

and and

buffer. The negative

CD-band

hydrogen

peroxide

to the

one produced

oxidized

native

formed

to oxidized

T2D lactase

when 1 mole

lactase,

at ~330 nm upon

equivalent

and shown

276

bears

addition close

resemblance

of H202 is added

to be a peroxy-lactase

of

to

Vol. 108, No. 1, 1982

BIOCHEMICAL

complex

Conspicuous

(7,8,11).

increased

width,

of peroxide the

and the

in

order

appearance

dependent

of

is

the

exhibited

by the

binuclear

copper

tyrosinase

sites

that

of a partly

several

reasons:

lactase

was dialyzed

with

H220 experiments

the

Prior

was removed

that

via

that

This

Still,

like

a T2D

spectral

as well

proteins

(6)

equivalents

to the

peroxide a large

feature

as by the

hemocyanins

and

or ascorbate

(14)

required

for

the

maximum

is

T2D

titration

shown

fully

in other

reoxidized

as can be seen in

are

is not the

that

that

complete

affected

bands

at

by

at higher

least

the

type

Finally,

anaerobic

with

benzohydro-

T2D lactase

has shown the

the

We have

beginning. of

for

the

before

demonstrating

sample

of

of ferricyanide

450 nm band

and neither

of a similar

are

excess

T2D lactase

the

as a result

titration,

just

the

at

formed

can be excluded

Secondly,

of

oxidized

is

CD spectra.

(6).

shown),

fully

titrations

enzyme

even reduced

(not

is

new band

reduced

the

of peroxide,

wave length

quinon

indicates

lactase

filtration

intensity

addition

copper

by gel

conditions

1, the

the

against

monitored

these

Figure

other

T2D lactase.

a concentration

produced. of

excess

(12,13).

reoxidation

under

in

in

intensity,

large

band with

clearly

form

a lower

a rather

nm CD band

is indeed

native

RESEARCH COMMUNICATIONS

are

of

that

manner,

The possibility

which

requirement

(-)330

complex

BIOPHYSICAL

differences

to produce

and specific

lactase-peroxy

AND

three

1

electron

reduction

of

the

615 nm

band. Taking full

formation

Scatchard constant (Figure

2).

lactase-peroxide

M-l

cm-l

of a T2D lactase-peroxide

plot of

A& = -0.75

could

10200

M-l

be constructed. a 1:l

complex

In comparison,

the

stability

complex

complex, This

for

was estimated 277

as a measure a linear

yielded

between

a stability the

constant as > 10'

for

reactants for

M-l

the native

(7.8).

Hence,

vol.

108, No. 1, 1982

the

affinity

when the It than

BIOCHEMICAL

of type

is

lactase

that

W region

the

positive

for

T2D lactase

for

native

The same is -0.75

the

M-l

of

cm-'

RESEARCH COMMUNICATIONS

decreases

while

drastically

the

and -0.2 the peroxy native

enzyme,

375 nm is intact

in

the near

significantly

smaller CAE = -0.7 M-1 cm -1

protein M-l

longer

is essentially

oxidized

with

the

at wavelengths

T2D lactase

at

for for

of

CD band

(8,9,11), case

that

the native

compared

lactase

peroxide

to note

CD spectrum

with

BIOPHYSICAL

is missing.

interesting

identical

vs.

towards

2 copper

400 nm, the

AND

cm-l

complexes

for

T2D laccase.1 (AE: = -2.6 M -1 cm -1

and T2D lactase-peroxide

complex,

respectively).

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

Graziani, M. T.; Morpurgo, L.; Rotilio, G. and Mondovi, B. (1976) FEBS Lett., 70, 82-90. Morpurgo, L.; Grazizi, M. T.; Finazi-Agro, A.; Rotilio, G.; and Mondovi, B. (1980) Biochem, J., 187, 361-366. Morpurgo, L.; Graziani, M. T.; Desideri, A. and Rotilio, G. (1980) Biochem. J., 187, 367-370. Reinhammar, B. and OK Y. (1979) J. Inorg. Biochem., 11, 115-122. LuBien, C. D.; Winkler, M. E.; Thamann, T. J.; Scott, R. A.; Co, M..S.; Hodgson, K. 0. and Solomon, E. I. (1981) J. Am. Chem. Sot., 103, 7014-7016. Frank, P. anaecht, I. Submitted for publication. Farver, 0.; Goldberg, M.; Lancet, D. and Pecht, I. (1976) Biochem. Biophys. Res. Commun., 73, 494-500. Pecht, I.; Farver, 0. and Goldberg, M. (1977) Bioinorg. Chem. II. Adv. Chem. Ser. 162 (Raymond K. N., Ed), pp. 179-206, ACS, Washington, D.C. Farver, 0.; Goldberg, M. and Pecht, I. (1980) Europ. J. Biochem., 104, 71-77. Goldberg, M.; Farver, 0. and Pecht, I. (1980) J. Biol. Chem., 255, 7353-7361. Farver, 0.; Goldberg, M. and Pecht, I. (1978) FEBS Lett., 94,

383-386.

Eckerson, K. W. and van Holde, I<. E. (1971) Comp. Biochem. Physiol., 39B, 855-872. Lerch, K. m81) in "Metal Ions in Biological Systems", 13, 143-171, Sigel, H., Ed., Marcel Dekker, Inc., New York. Morpurgo, L.; Desideri, A.; Rotilio, G. and Mondovi, B. (1980) FEBS Lett., 113, 153-186.

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