Reduction of lactoperoxidase-H2O2 compounds by ferrocyanide: Indirect evidence of an apoprotein site for one of the two oxidizing equivalents

BIOCHEMICAL

Vol. 121, No. 2, 1984 June 15, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 463-470

REDUCTION OF LACTOPEROXIDASE-H202 COMPOUNDS BY FERROCYANIDE : INDIRECT EVIDENCE OF AN APOPROTEIN SITE FOR ONE OF THE TWO OXIDIZING EQUIVALENTS Francoise Unit6 Received

COURTIN, Jean-Luc MICHOT, Alain VIRION, Jacques POMMIER and Danielle DEME

de Recherche sur la Glande Thyroide INSERM, 78, rue du G&&al Leclerc, March

28,

et la RegulatiM Hormonale 94270 Bicetre (France)

1984

The tjtration by ferrocyanide and the localizationof the oxidizing equivalents of lactoperoxidase "compound II" were studied as a function of pH. It was demonstrated that 1) whatever the pH, the structure of lactoperoxidase "compound II" was compatible with a Fe IV R0 state, 2) at acidic pH, ferrocyanide preferentially reduced the oxidizing equivalent localized on the heme iron to give an Fe III R' compound, 3) at pH 4.2 only the Fe III R" form was obtained after reduction of lactoperoxidase "compound II" with one mole of ferrocyanide and whereas of both Fe III R" and Fe IV R forms was present, at pH > 4.2, a mixture 4) lowering the pH from 7.2 to 4.0 induced a transition of Fe IV R state but increasing the pH from 4.0 to 7.2 did not permit to Fe III RD state, the formation of Fe IV R compound from Fe III R" compound. Lactoperoxidase (Fe IV ?+ compound which

is

retains

shown

to retain

species

an oxidized

which

spontaneously

converts

similar,

(HRPO) compound

which

pHs (3).

and H202 form

I)

spectrally

peroxidase

(LPO)

on the

two oxidizing one oxidizing

the

apoprotein.

Abbreviations:

this

equivalents

However been

presented

per

its

located

the presence

presumed

II"

at neutral LPO-H 0 2 2

which

also

retains

was Fe IV R", and the other

of one oxidizing

equivalent

paper

this

show the

study

on

on the

(3).

titration

as a function

II

has been

on the heme iron

in this

LPO, Lactoperoxidase;

unit

structure

II"

to horseradish HRPO compound

this

c peroxidase,

shown in

II"

to the

between

clearly

of the LPO "compound

regions,

hematin

compound

an LPO "compound

LPO "compound

similarities

(4,5),

equivalent

has not

The data

equivalent,

ES of cytochrome

equivalents

i.e

In contrast

spectral

into

and visible

(1,2).

two oxidizing

and compound

equivalents

II

one oxidizing

Based

apoprotein

in Soret

addition

of the oxidizing

of pH. Whatever

HRPO, Horseradish

the

peroxidase

0006-291X/84 $1.50 463

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 121, No. 2, 1984 PH, this these

BIOCHEMICAL

compound

retained

two oxidizing obtained

was similar

to that

was located

on the

MATERIALS

AND METHODS

PRODUCTS

two oxidizing

equivalents

Moreover,we

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to ferrocyanide

a one-half of native

equivalents.

reduced

The reactivity

varied

LPO species

LPO, suggesting

with

whose

that

its

of

the PH. Soret

oxidizing

spectrum equivalent

apoprotein.

:

Potassium iodide was purchased from Prolabo , ferrocyanide from Merck ; Perhydrol 30 % H202 from Merck ; 3'5' diiodotyrosine and lactoperoxidase (absorbance ratio A412/A280 = 0.9) from Sigma ; pronase from Calbiochem ; Na [125I] from the Commissariat a 1'Energie Atomique. Poorly iodinated goiter thyroglobulin and [12'1] - labeled thyroqlobulin with low hormone content were prepared as previously described (6). ANALYTICAL

METHODS

:

H202 concentrations were determined spectrophotometrically from the LPO oxidation of iodide to iodine under the following conditions : 1 ml of 50 mM phosphate buffer, pH 7.4 contained x ul of H202, 5 ug LPO and 12 mM potassium iodide. The 13 formation was measured by O.D. at 353 nm (7). Under these conditions, a final concentration of 10 uM H202 corresponded to a 0.230 change of absorbance using a 1 cm o tical light path cuvette. Solutions of ferrocyanide (K4 [Fe (CN)6 s . 3H20) of known strength were made by careful weighing and volumetric dilution. The concentration of LPO was determined spectrophotometrically at 412 nm using a molar absorbance of 114 mM-l cm-1 (81. LPO was extensively dialyzed against 50 mM phosphate buffer, pH 7.4 or 50 mM acetate buffer, pH 4.0. Under these conditions, and with most commercial preparations, saturation with H202 was achieved with a molar ratio of s 1. The titration of LPO with H202 was done by measuring the changes in the absorbance at 430 run. Coupling experiments were performed in 50 mM phosphate buffer, pH 7.4 at room temperature. The detailed experimental conditions are described in the legend of each figure. At different time periods, aliquots of the incubation medium were removed and added to a solution of NaHS03 to stop the reaction. The content of iodoaminoacids was measured after pronase hydrolysis using paper chromatographic methods 161. AND DISCUSSION

RESULTS Titration prepared

of at

the

oxidizing

acidic

At pH values 11" by the

addition

The comparative followed

below

7.4,

native

of an equimolar titrations

to completely were

retained

on

the

LPO-H202

compound

PH.

of this

at 430 nm. Figure

was required two moles

equivalents

required

LPO was converted amount compound

1A shows that reduce

at pH 7.3.

of H202 with

only

the heme iron A close 464

into (fig.

LPO "compound lA,

insert).

ferrocyanide

were

one mole of ferrocyanide site

examination

at pH 4.2, of the

whereas influence

Vol.

121,

No.

0

BIOCHEMICAL

2, 1984

a5

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 1.5 K.I /Compound II

[Fe tCN)e]

,------4

2

5

7

6 PH

Fig. 1A : Titration of lactoperoxidase (LPO) "Compound II" with potassium ferrocyanide. The percent change in absorbance at 430 nm follcxving addition of ferrocyanide is plotted against the molar ratio of ferrocyanide added to "compound II". Titration was carried out at pH 4.2 (m) and at pH 5.3 (0) in 1 ml 50 mM acetate buffer and at pH 7.3 (0) in 1 ml 50 mM phosphate buffer with 10 nmol LPO. Insert represents the titration of WO(10 nmol)with hydrogen peroxide 5.3 (0) and 7.3 (0). at pH 4.2 (ml, Fig. mole

1B : Fe IV reduction of one mole of LPO-Ze IV R0 compound by one of ferrocyanide as a function of pIf. At different pHs, 10 nmol H202 was added to 10 nmol native LPO in 1 ml 50 mM acetate or phosphate buffer. The reduction was carried out with 10 nmol ferrocyanide. The decrease in absorbance at 430 nm was determined 5 min. after the addition of ferrocyanide. 100 % represents the differential absorbance between the native enzyme and the Fe IV R' compound spectra.

of

pH

fig.

on

the

1B.

10

nmol

An

increasing

cyanide. the

of

LPO

plus

the

heme

10 nmol

reduction

oxidizing were

H202 of

equivalent

the

heme

with

the

is

reduced

by

iron

site

presented

10 nmol

in

ferro-

was

observed

as

two

assumptions

pH decreased. These

at

reduction

data

acidic

compound native compound

pHs, I LPO

formed was

which

might in

the by

be

consistent

absence the

addition

spontaneously retained

of

a hydrogen of

one

converted only

one

following donor,

mole into

oxidizing

465

the

LPO-Fe

IV

of

H202

to

one

either

a)

an

LPO-Fe

equivalent,

i.e

mole

the

:

o+ n of IV

o+ 'TI

R

BIOCHEMICAL

Vol. 121, No. 2, 1984 radical

was lost

equivalents,

or b) an LPO-Fe

one as ferry1

protein

moiety

; this

reduced

by ferrocyanide

To investigate the

apoprotein,

one mole change

in

H202 to

IV R0 compound

at the heme iron

the number

species

In the added

as a free

would

in the

be preferentially

equivalent

of ferrocyanide

(fig.

radical

Reduction 2).

on

oxidized

by

was determined

After

we observed

moles

retained

the a rapid

addition

by the

of 10 nmol

increase

in the

absorbance.

first twice.

decrease

in the

complete

reduction

10 nmol

ferrocyanide,

of two experiments

(fig.

The first

of

addition

absorbance

10 nmol

at

430 run to its

last

10 nmol

z 8 E

cl3-

Q

O.l-

2A),

level,

site.

which

After

of H202 were

maximum value.

ferrocyanide

10 nmol

ferrocyanide

10 nmol was accompanied

to the basal

of the heme iron

absorbance added

two oxidizing

site.

at pH 4.2.

lactoperoxidase,

band

retaining

of one oxidizing

the 430 nm absorption

430 nm Soret

were

LPO-Fe

we measured

10 nmol

IV R" compound

Fe IV and the other

the presence

of LPO-H202

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

corresponded second

sufficient This

to the

addition

to increase

demonstrated

had been previously

by a

of the

that

the

oxidized.

g Q2B

a

I

I

I

I

I

0

1

2

3 Time

I

I

I

I

1

0

1

2

3

,

lmin)

Fig. 2 : Titration of the oxidizing equivalents carried on LEV-HZ02 species measure of the number of moles of ferrocyanide oxidized per mole of LPOH202 species. At pH 4.2, 10 nmol H202 was added to 10 nmol native LPO in 1 ml 50 mM acetate buffer. The LPO-H202 species (Fe IV R" compound) was reduced with 10 nmol ferrocyanide. After a second addition of 10 nmol ferrocyanide (A) or 30 nmol ferrocyanide (B), 10 nmol H202 (A) or 2 additions of 10 nmol Hz02 (B) were added. (Numbers associated with arrows represent molar proportions between products added and LPO).

466

:

Vol. 121, No. 2, 1984

BIOCHEMICAL

In a second

experiment

(fig.

10 nmol and 30 nmol

ferrocyanide,

sufficient

obtain

to again

These

experiments

ferrocyanide pound

II",

another

species

whose

support

the

A similar thyroid are 0.85

mole

required

to completely

is

reached

catalysis

(fig.

to form

compound

stoichiometrically

two oxidizing

equivalents

Moreover,

the

data

with

Soret

of this

on a residue

compound

3 :

Kinetics performed

of

thyroid

(9,10),

figures

of

of oxidizing

the

lA,

equivalent at acidic

synthesis

5 hormone

2A and

state

equivalents of 0.8

of LPO-H202

the presence

R".

on

oxidizing

an

At neutral

10 TIME

2B clearly

of LPO-H202

pHs : the

apoprotein,

Fe III

12 Fig. species

two moles

stoichiometry

of

compound. in

of the

is probably

apoprotein.

at pH 4, implies

of a one oxidizing

was located

on the

preformed

reported

spectrum

of the

LPO results

at pH 7.4 by one mole

on this

of

LPO. These

located

studies

Since

one mole

by this

catalyzed

the existence a native

of native

equivalent

one mole of hormone

mole of iodothyronines

be oxidized

to that

3).

only

the heme of the LPO "com-

could

by the

of

value.

pH, although reduce

of ferrocyanide

additions

of 10 nmol H202 were

absorbance

of an oxidizing

conclusion

two successive

at acidic

was identical

existence

after

maximal

show that

spectrum

hormone

2B),

2 additions

the

was required

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

L

15

20

compound

equivalent

"R site". pHs,

show

our

The structure results

IminI

formatian

catalyzed

by

LPO-H202

at different pHs. LPO-Fe IV RD compound (5 nmol native ISO and 5 nmol H202) was prepared at pH 7.4 (0) or at pH 4.1 (0) and then assayed for the couplin reaction in 1 ml 50 mM phosphate buffer, pH 7.4, in presence 5 mm1 t 1251-j thyroglobulin (30 iodine atoms/molecule) and 5 nmol free diiodotyrosine.

467

of

Vol. 121, No. 2, 1984 indicate

that

BIOCHEMICAL

both

forms

of LPO-H 0 22

compounds

coexist.

reduction

and Fe IV R compound

Changes

in

Therefore

oxidizing

one oxidizing

equivalent

be proposed

pendent is

postulated

site

first

reduced

alkaline

pH).

between

the

establish

second

the

III

transfer

experiments

involving

First,

(figure

species

into

in pH were

this

problem, out

at 430 nm was decreased

The pH was then of the

raised

absorbance

10 nmol

was observed.

ferrocyanide,

absorbance

not

on the

oxidizing

"R site".

by 10 nmol

by change

equivalent

Second,

(fig.

addition

However,

from 4B),

ferrocyanide

in the

spectrum,

the

after

"R site" the

at pH 7.4,

"R site"

to

two types

of

4A and 48).

by 10 nmol

to the basal

level).

addition

to increase

by the

the

oxidizing

in pH from

i.e

to the

by a transfer

of the

second

mole

equivalent

4 to 7.4,

was

of the

"Fe site".

of 10 nmol Fe IV R" compound

the pH was lowered 468

the

the

R" compound

Consequently,

change

reduction

The preparation

described,

a second

oxidized the

of

previously

sufficient

Moreover,

way to

of NaOH. No modification

after

maximum value.

had been previously

accompanied

to a Fe III

10 run01 H202 were

at 430 nm to its

of ferrocyanide carried

to 7.5 by the

direct

(fig.

conditions

ferrocyanide,

(absorbance

reduced

at

to examine

from

were

which

a transition

possible

10 nmol Fe IV R0 compound

site

equilibrium

equivalent

4A) at pH 4 and under

"inde-

pH and "R site"

the other.

carried

can

of the

The

be to demonstrate

To investigate

changes

curves

The most

at pH 4.2 made it

versa.

titration

species.

would

two mechanisms

a pa-dependent

of the oxidizing

and vice

(51,

at acidic

postulates

enzymatic

retaining

of the oxidizing

site"

enzymatic

R" compound

of the

"Fe site*

("Fe

mechanism

compounds

by ferrocyanide.

the nature

mechanism

one of the half-reduced of an LPO-Fe

et al.

of pH on the

by ferrocyanide

two half-reduced

the

problem

effects

specifies

A second

"RO site"

of PH.

IV R" compound

mechanism"

the

on LPO-H202

by Coulson

c peroxidase-Fe

R" and Fe IV R

formation. location

the

Fe III

the pH favors

as a function

to explain

cytochrome

species,

increasing

equivalent

As previously

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to 4.0 by the

Vol. 121, No. 2, 1984

BIOCHEMICAL

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I ,/ I

0

1

”

I 1111 I

5

Time

0 0.5 ” cmim

5

I

6

7

Fig. 4 : Changes in oxidizing localization in half-reduced LPO compounds as a functica of PH. pli 4, 10 nmol LPO-Fe IV R" (A) : in 1 ml 50 mM acetate buffer, compound was reduced with 10 nmol ferrocyanide. The pH was shifted to 7.5 by addition of NaOli. 10 nmol ferrocyanide was added before addition of 10 nmol H202 required to again obtain the Fe IV R" compound. 1 ml 50 mM phosphate buffer, pH 7.4, 10 nmol LPO-Fe IV R" (B) : in compound was reduced with 10 nmol ferrocyanide. The pH was shifted to

4.0 by addition of HCl. 10 nmol ferrocyanide was added before addition of 10 nmol Hz02 required to again form the Fe IV Ra compound. addition

of HCl.

absorbance 10 nmol

The decrease

at 430 nm to its ferrocyanide,

addition

of H202.

7 to 4.2 permits

"Fe site"

to the

of the oxidizing the pH (fig.

basal

formation

of 10 nmol

pH from

in pH was accompanied

This

4A).

which

integrates

site"

mechanism.

a second

the

"R site"

we propose

transition

only

not

decreasing

observe

to the

a sequence

Fe IV R + Fe III

of

required

equivalent

we could

in

addition

shows that

of the oxidizing

from

Therefore, the

experiment

In contrast,

equivalent

After

of Fe IV R" compound

shifting

"R site".

level.

by a decrease

"Fe site"

of reactions R" into

the

the

from

the

any transfer by increasing (scheme

1)

"independent

Fe IV R Fe III

H2°2 R -Fe

IV o+ n -C

Fe IV Ra

>+:I Fe III

Native reduction reduction

LPO

2

at alkaline at acidic

ox.

eq. LPO

1 ox.

pHs (----) pHs (-) Scheme 1 469

eq.

Fe III

R

R' LPO

Native

LPO

Vol. 121, No. 2, 1984 This is

the

be the

BIOCHEMICAL

scheme implies

"R site"

the

"R site"

the

"Fe site"

We should

followed

Fe IV R0 and LPO-Fe

LPO-Fe not

Fe IV R compound

(5).

an Fe IV heme but implying

stoichiometric

located

on the

of the

"Fe site"

first

first site

exclude

of the

ferrocyanide

In contrast,

site

to be reduced

to be reduced

a first

oxidizing

reductions clearly

could

reduction

of

equivalent

from

at the

Recently,

same pH, Coulson

a cytochrome

c peroxidase

has been obtained of the

of hydrogen of peroxidases site

ferrous

studies.

R0 compound

R" compound

the oxidation

and "R site"

III

c peroxidase-

in these

III

radical

same oxidation

of cytochrome

appeared

an LPO-Fe

c peroxidase-Fe

no free

amount

The preparation

the

however,

at pH 4, to obtain

a cytochrome

procedure

not,

IV R0 compounds

IV R compound.

obtain

pHs,

the

"R site".

between

was possible,

pHs,

by displacement

to the

Differences

It

1) at alkaline

and 2) at acidic

"Fe site".

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

free

et al.

free

could

of the

species

by Ho et al.

(Fe II)

of

peroxidase

carrying with

a

by a

peroxide. containing makes it

and the

one oxidizing possible

functional

equivalent

to examine

relationships

the properties between

them.

ACKNOWLEDGMENTS This work was supported in part by Research grant na 858 from the Unite de Recherche et d'Enseignement de Kremlin-Bicetre (France). The authors wish to thank Mrs A. Guedec and C. Sais and Mr M. Balhoul for preparation of manuscript. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Chance, B. (1949) Science 109, 204-248, Wash DC. Maguire, R.J., Dunford, H.B. and Morrison, M. (1971) Can. J. Biochem. 49, 1165-1171. Courtin, F., D&me, D., Virion, A., Michot, J.L., Pommier, J. and Nunez, J. (1982) Eur. J. Biochem. 124, 603-609. Yonetani, T. (1966) J. Biol. Chem. 241, 2562-2671. Coulson, A.F.N., Erman, J.E. and Yonetani, T. (1971) J. Biol. Chem. 246, 917-924. Virion, A., Deme, D., Pommier, J. and Nunez, J. (1981) Eur. J. Biochem. 118, 239-245. Alexander, N.M. (1962) Anal. Biochem. 4, 341-345. Morrison, M., Hamilton, H.B. and Stotz, E. (1957) J. Biol. Chem. 228, 767-776. Virion, A., Pommier, J., D&me, D. and Nunez, J. (1981) Eur. J. Biochem. 117, 103-109. Gavaret, J.M., Cahnmann, H.J. and Nunez, J. (1981) J. Biol. Chem. 256, 9167-9173. Ho, P.S., Hoffman, B.M., Kang, C.H. and Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363. 470