The activity of the horseradish peroxidase Compound III

Vol. 18, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Ken-noeuke Yokota and Ieao Yenseki Biophyeice

Divieion,

Reeearoh Iuetitute

Hokkaido University,

of Applied

Kleotricity

Sapporo, Japan

Received October 16, 1964 Of the three well-known oxide,

peroxidaae

Compounds I, II aud III,

with reapeot reported

to oxidation

that

the last

level

Compound III.is

Chauce (1951) oonoluded

that

compound accumulater

the ueual experimental

one ha8 been the moat ambiguoue

and reaotivity.

Compouud III

reaots

(Yheorell

to inhibit

Compound III

Compound III

termediate

of thie

on the reaotivity the reactions Fig.

the reactivity

1963) that

after

DliP disappears!

In apite

because

peroxidaee the half

solution

Mason (1956)

of these suggestions results

reagenta.

inby

have been reported

of the difficulty

a suitable

of Compound III.

a stoiohiometrio

ia needed to convert

reaction,

with various

1, the DKF system represents

eatiaating et al --9

of co-pound III

of

Because of the appearance of

and quantitative

of Cowound III

ie formed not only iu

oompound aa an oxygen aotivating

iu the oxidaee xeaction.

Chauce aud Raeon, no direot

under

but aleo upon the addition

the peroxidaae-oxidaee

suggested the participation

with donors to

of peroxidase

to the aerobic peroxidaee

1940 ; Chanoe, 1952).

during

xapidly

the aotivity

condition@.

(DRF) directly

et al, --

end Mauri (1936)

ae a consequenoe no measurable amouut of

the preeenoe of exoeea hydrogen peroxide dihydrowfumarate

Keilin

decomposed in the presence of donors and

give the free enzyme and that this

oorpouude with hydrogen per-

in iaolatiug

Ae CM be men in

experimental

condition

for

It hae been found (Ymzaki

amount of DKF and hydrogen peroxide to Compound III,

which deoaya mually

time for decay ie eeve.ral minutes.

48

HO

BIOCHEMICAL

Vol. 18, No. 1, 1965

AND BIOPHY$ICAL

RESEARCH

Effect of electron donors on the stability of Compound III. Fig. 1. 0.05 hl acetate, pH 5.0, 25’. Compound III was produced by the addition of 0.2 mM dihydroxy-fumarate (DHF) and 10 @ hydrogen peroxide to the aerobic solution of horse radish peroxidase (9.5 jak!). The value E403 107.7 mM-Ll -1 for horse r8dish peroxidase was used according to Paul’s data (1963). The ratio of E403 to E278 for this peroxidase was 3.0. 0.1 mM indoleaoetic aoid (solid line) and 0.1 IN hydroquinone (dotted In the oontrol experiment DHF disappeared line) were added at arrows. completely 4 minutes after DHF and hydrogen peroxide were added. Hydroquinone reduced Compound III to free peroxidase but indoleacetic acid converted the enzyme to a compound similar to Compound IV vi8 free enzyme. The latter is a exceptional case and the details will be reported elsewhere. intermediates

such as Compound II

Compound III result

to free peroxidase.

(1955),

catalase

brings

had no effect

perimental

conditions

The addition

tion

on the decomposition shown in Fig.

of indoleaoetic

of Compound III

III

thst

yielding

Moreover,

Compound 0.2 @

under the ex-

1. aoid greatly

accelerates (Fig.

1).

the decompoSuch accelera--

was observed in the presenoe not only of elec-

tron donors but aleo of electron

oonoluded

of

with George’s

of Compound III

to give free peroxidase

of the deoomposition

dicrtes of peroxidase

of Compound III,

time of about 36 seconds.

half

the conversion

remov8l of the excess H202 by

about a decomposition

oatalase

during

This is inoonsistent

in which he showed that

II with a reaction

sition

are detected

aooeptors

oan be detected

the rate limiting

with the donors or acceptors

during

(Fig.

49

Sinoe no interme-

the deoomposition

step is the first themselves.

2).

reaction

The rate

it

may be

of Compound

constants

for the

Vol. 18, No. 1, 1965

BIOCHEMICAL

0.4'

I

AND BIOI’HYSICAL

1 2

0

I

I 4

RESEARCH COMMUNICATIONS

I

I 6

I

MIN.

Effect of electron aooeptore on the stability of Compound III. ~kkzntal conditione were eeme ee thoee deeoribed in Pig. 1. 0.1 nduI p-benzoquinone and 0.1 myI Cu*+ were added at arrowe.

zwcrtipnr, kinetic

vhioh oau nov be estimated, experiments

upon

Compound II Table I.

rate oonetents

and many electron There is no linear

but Compound II

reaotlone

peroxidaee

(1951, 1953), vho entlmsted

donors.

is very roughly

indoleaoetio

pound III. tione

will

between

J%8OtiOnIJ

between the two rate oonetants

It oen be easily

than Compound III seen from Table I

relative

high

affinity

for Com-

aoid end peroxidaee, of indoleecetic

may imply 8 role

acid

tiesue.

in plant

of perThis

be diecueeed elsewhere. Peroxidaee

myoglobin

Compound III

type etruoture

hae eometinee been thought

end Mason (1958) tied

ohaniem for the peroxidaee-oxidaee was recently

presented

for Compound III aible

out by Chance

vhioh hae aleo been found in other reao-

between indoleaoetio

oxidaee in the metaboliem

for the

103 times more aotive

acid haz a naerkably

This epeoifioity,

were oarried

Extenzive

Some of these are aleo ehovn in

relationehip

toward the ueual hydrogen donore. that

are ehown in Table I.

role

reaction.

thie

to

theory

have

in hir me-

Some more direot

by ua to make the ferroperoxidaae-oxygen

plausible

of Compound III

(Yamaaaki --9 et al in the

1963) and to predict

peroxidase-oxidase

50

reaction

an o~y-

evidenoe etructure a pos(Yamazaki

Vol. 18, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table I. Rate constants for the reaction of peroxidase derivatives with reductants and oxidants. pH 7.0 for Compound II (except a8 noted) and pH 5.0, 250 for Compound III.

Compound II (M-l x sec. -l)

Reductente

(Chance,

Compound III (M-l x 880. -l:

1951and 1953)

3 x lo6 2 x lo6 3 x 105

Hydroquinone Catechol Reeorcinol p-Wee01

3x 4x 3x 5x 3x

Pyrogallol Phenol Guaiacol o-Phenylenediamine p-Phenylenediamine

5.3 x lo2 4.1 x lo2 2.1

7.0 x 5.5 x 6.7 9.0 x 1.9 x 1.7 x 7.8 x 3.4 x

105 105 105 107

107

Ferrocyanide Reductone Dihydroxyfumaric

1 x 106(pH 4.2) 1 x 104(pH 4.6)

acid

very

2 x 104(pE4.6) 1.8 x lo4 * (pH 5.0)

Ascorbic acid Indoleacetic acid

4.6

x

10 lo2 lo2

103 104 10 105

slow 10

2.2 x 105

Oxidants

Ferricyanide

1.3 x lo2 3.3 x 10

cu*

1.2 x 105

p-Benzoquinone

This was estimated from the velooity of iron'reduation aaoompanying the peroxidetic oxidation of indoleacefic acid under anaerobic oonditions (Yamazaki --9 et al 1960)

et al --9

1964). Consideration

may now be given to the problem whether

Compound III

accepts only one electron

in the first

reaation

or two electrons

with donor molecules. 51

Although

simultaneously it

is not easy to

Vol. 18, No. 1, 1965

BIOCHEMICAL

reach a firm

conclusion,

compound III

decomposition

duction

it may be safely probably

because one-electron

can react with Compound III Table I).

AND BIOPHYSICAL

the following

of Compound III

corresponds

the first

donors such as p-ores01 as well ae two-electron

Peroxidase

(+=+I

and ferrocyanide donors (as seen in

- e1ectmn,

for Com-

+ H202 (-’

respectively.

Compound I)

(1)

Peroxidaee

+ 02

(Fe:)

The rapid conversion ferricyanide

of oxymyoglobin

or quinones

might suggest a hybrid ferri-perhydroxyl

to metmyoglobin

seema to be analogous

structure

in the presence of

to Reaction

(2).

This

for Compound III

of ferro-oxygen

and

intermediate

in the hydroxylation

complexes.

Compound III

is not an active

acid catalyzed on the stability conclusion

by peroxidase

because salioylic

of compound III.

This ie consistent

might be perhydroxyl

of

acid has no with the

by Maeon and Ruhler (1961) that an active

mediate for the hydroxylation worthwhile

structure

(Fe?)

(Fe?,)

salicylic

re-

meohaniams may be suggested for the decomposition

+ electron,

Compound III

step of

to a one equivalent

in the presence of donors and acceptore,

Compound III

previous

said that

If one assumes the ferroperoxidase-oxygen

pound III

effect

RESEARCH COMMUNICATIONS

inter-

It might be

ion.

to note here that DHP ie a very poor donor for Compound III,

as reported

by Chance (1952) and ourselves

why Compound III

accumulates

phane, tyrosine,

uric

decomposition

acid and xanthine

of Compound III.

firmed by observing

effectively

(1963).

in the DHP reaction. have no detectable

These negative

the absorption

spectra 52

This ie the reason

results

Tryptoeffect

on the

have been con-

in the visible

region.

Vol. 18, No. 1, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDOBfENTS We wish to thank Dr. P. Nicholls of State University of New York for his helpful comments. This investigation has been supported by research grant from the National Institute of Health (AM 06518) and the Scientific Research Pund of Ministry of Eduoation of Japan.

2, 424 (1961) Buhler, D. R. and Mason, H. S., Aroh. Chanoe, B., Advances in Enzymology, Chanoe, B., Aroh. Biochem. Biophys., Chance, B., Teohniques of Organio Chemistry, VIII, 627 (1953) George, P., J. Biol. Chem., 3, 427 (1953) Keilin, D. and Mann, T., Proc. Roy. Sot. London, B 122, 119 (1936) Mason, H. S., &oc. Intern. Symposium on Enzyme Chemistry, Tokyo and Kyo$oSKP ;20 (1958) 9.) 0, The Bhzymee (ed. Royer, P. D., Lardy, H., mbaok, 2nd.'e& ., Vol. 8, p 227, Academic press 1963 Theorell, H. and Swedin, B., Nature, x, 71 (1940) Yamazaki, I., and Souzu, H., Arch. Biochem. Biophye., 86, 294 (1960) Yaplszaki, I. and Piette, L. H., Bioohim. Biophye. A&a., n, 47 (1963) Yamazaki, I., Yokota, K. and Nakajima, R.., Intern. Sympoeium on Oxidaeee and Related Oxidation-Reduction Systems, Amherst 1964

53