Cu(II)-dependent inactivation of Mn-catalase by hydroxylamine

BIOCHEMICAL

Vol. 124, No. 1, 1984 October 15, 1984

CU(II)-DEPENDENT

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 75-79

INACTIVATION

OF MN-CATALASE BY HYDROXYLAMINE

Yasuhisa Department School of Received

August

SUMMARY:

17,

Kono

of Bacteriology, Medicine, Yonago,

Tottori Tottori

University

683, Japan

1984

Hydroxylamine

is a strong inhibitor of the Mn-catalase of in the presence of hydrogen peroxide [Kono, Y., and Fridovich, I‘. (1983) J. .Biol. Chem. 258, 13646-136481. In the presence of CuCl the Mn-catalase was rapidly inactivated by hydroxylamine without tie addition of hydrogen peroxide. FeSO and MnCl were approximately 10% and 4% as effective as was CuCl . Unde# anaerobic conditions, the inactivation did not occur. The chelatjng agents such as EDTA and histidine completely prevent the inactivation. These results indicate that the hydrogen peroxide produced during the autooxidation of hydroxylamine catalyzed by C&l2 participates in the CuC12-dependent inactivation by hydroxylamine. 0 1984 Academic press, he.

LactobaciZZus

plantam

In contrast recently atide

to heme-containing

isolated

plus

H202 (2,

LuctobaciZtus

from

and cyanide,

but

3).

is

We proposed

NH20H inhibits

Cu(I1)

(7),

papain

and O2 are

(8),

required

by chelators H202 during (16). the

for the

of

action

site

(3).

inhibited

when exposed

to NH20H

involve

the

as catalase

(4),

cytochrome

c oxidase

(6),

and alcohol

was by

such

inactivations

(13-15).

This

or by removal

metal-catalyzed

copper

not

of NH20H might

dehydrogenase

(10).

of ribonuclease

of NH20H is catalyzed

Cu(I1)

that

(5), (9)

the

is

which

(11)

by NH20H.

by Cu(I1)

I now find presence

for

(12)

The autooxidation profoundly

the

activities,

urease

Mn-catalase,

inactivated

carboxylase

and carboxyesterase

ions,

active

many enzymatic

ribulose-1,5-bisphosphate urocanase

that

the

pZantarum (l),

irreversibly

of Mn(IV) at the

production

catalase,

the ions

by trace

autooxidation

of oxygen.

autooxidation

Mn-catalase without

is the

amounts

of metal

is suppressed

The formation

of 0; and

of NH20H has been reported rapidly

addition

inactivated of

H202*

by NH20H in

In this

report

0006-291X/84 75

$1.50

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

Vol. 124, No. 1, 1984

BIOCHEMICAL

I describe

that

during

autooxidation

the

the

inactivation of

is

AND BIOPHYSICALRESEARCH

dependent

NH20H by copper

upon the

COMMUNICATIONS

H202 formed

ions.

MATERIALS AND METHODS The Mn-catalase was isolated from L. pZurtarwn and-jts-Concentration at 280 nm was estimated using an absorbance coefficient of 184 mM cm (1). The manganese-containing superoxide dismutase from Escherichia coZi was kindly provided by Dr. I. Fridovich (Duke University), Exposure of the Mn-catalase to NH OH plus CuCl was performed in test tubes at 25°C with continual shakin 6 at 100 stro ii es/min, while the incubation mixture was held to 0.1-0.2 ml to insure adequate aeration. Incubation mixture contained 10 mM Tris-HCl at pH 7.0, 0.256 uM Mn-catalase, 50 PM NH OH and 0.1 mM CuCl At intervals, 10 ul of the incubation mixture wag diluted 300-fol$'with the assay mixture of catalase. Catalase activity was measured as previously described (l)> using a Shimadzu Multipurpose MPS-2000 spectrophotometer at 25°C. NH OH.HCl was obtained from Nakarai Chemical Co. (Kyoto, Japan). Aqueous sglution of NH OH was prepared daily and kept in a tight stoppered test tube unti12use; RESULTS AND DISCUSSION When 0.256 and 0.1

uM Mn-catalase

mM Cu(I1)

time-dependent Mn-cata7ase

at pH 7,

manner was first

Mn-catalase.

The degree

inactivation

were

the

Pn-catalase

Inactivation

obtained

when'0.58

enzymatic

(Fig.

lA,

order

with

of

from

IO 20 MINUTES

of

was incubated

the

with

activity

lines

3 and 5).

respect

inactivation

evaluated

0

was incubated

to the and the

the

pseudo-first

10 uM or 50 pM NH20H was rapidly

lost

The inactivation residual

order

of the

activity

protection

against rate

in a

of the

constants

so

Mn-catalase

aerobically

by NH OH plus

(A) or a?iaerobically

CuCl

. The

PB) in 10 mM

and 50 mM (line 4 in A) Tris-HCl at pH 7 or 10 mM potassium phosphate at pH 7 (line 5 in A;O). Line 1 in A was obtained when CuCl was omitted. Line 2 in A was obtained when NH OH was omitted. Line 3 14 A was obtained when 10 PM NH OH was added. Line'5 in A (0) was control. Line 2 in B was

mM H202 was added. 76

B

Vol. 124, No. 1, 1984 for

the

inactivation.

NH20H nor

Under

Cu(II),

of the

enzymatic

enzyme

against

recovery

of

when present activity

the

min-');

the

Mn(I1)

were

results

are

Mn(II),

to catalyze

in accord

lA,

lines

of Tris

a slight

rates

the

the

The effect of

inactivation

1).

high

affinity

of 0.1

complex of the forms

with

a stable

not

2).

inactivation

lB,

the

enzyme

can

inhibit

(Fig.

mM histidine, (17),

(lines Cu(II),

was only

line

1).

the metal-catalyzed

inactivation

mM and 0.5

with

and

of

(Fig.

activity

The

Fe(I1)

of

ions

and

NH20H (14-16).

was investigated

complex

by Fe(I1)

N2. There

the

the

inactivation

(Fig. but

in any

induced

under

copper

enzymatic

Cu(II),

result

was incubated

suppressed

The addition

of

caused

02 in the

which

NH20H (13-16),

not

respectively of

of inactivated

also

rate

inactivation

for

mixture

5 mM EOTA completely

(line

and Mn(I1)

of

need

of chelators,

autooxidation

Fe(I1)

autooxidation the

pH 7 did

highest

decrease

Dialysis

at

ability

neither

any significant

buffer

the

the

conditions,

1 and 2).

by Cu(II),

the

reaction

and gradual

of

that

with

to verify

Mn-catalase,

Tris

caused

activity.

10% and 4% of

In order

that

alone,

(Fig.

enzymatic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

same experimental

by NH20H. CuC12 caused

(k app=0.15

protection

the

3000 volumes

inactivation

of

BIOCHEMICAL

resulted

3).

The addition

of Mn-catalase which

forms

a

in 69% and 98%

2 and 3).

It

has been reported

which

catalyzes

the

100A I 2

x) 8 c 5 ‘: :

IO

lIs!il IO 30 3

0zso

MIN”G

Inactivation of the Mn-catalase by NH20H induced by CuC12, Line 1; 0.1 mH NnC12. Line 2; 0.1 mM FeS04. Line 3; Effects of EDTA and-histidine on the inactivation of the F n-catalase by NH20H plus CuC12. Line 1; plus 5 mM EDTA. Line 2; 0.5 mll histidine. Line 3; plus 0.1 mM histidine. Line 4; control. 77

plus

Vol.

124,

No.

oxidation

of

buffer

BIOCHEMICAL

1, 1984

was

cystein used

and

(18).

In

almost

all

inactivation

rate

of

concentration

of

Tris

above

when

10

was

not

affected

place

of

10

catalyzed to

H202

The rate

mM Tris

of

below

the 4 min

Omission

of

production

The

and

(Fig.

4,

resulted

in

Mn-catalase

of

dismutase

).

Tris

but

the

without

The

inactivation was

not

enhanced

enzymatic

conditions not

then

NH20H

4, did

the

activity

(Figs.

of EDTA (Fig. of

sufficiently

circumstances.

of

did

in

Cu(II)-

mixture

loss

addition

used

inhibits

present

mixture

The

increasing

7)

incubation

anaerobic

Tris

with

apparently

the

inclusion

1B and affect

line not

4).

the

4).

The

cause

any

1).

superoxide

plus

buffer

incubation

line

by

0

a complete

the

4). (pH

in

and

line

COMMUNICATIONS

a Tris-complex.

phosphate

causing

H202

increase

~11 superoxide

(14),

as

‘IA,

to

the

a slight NH20H

NH20H

mixture,

decreased

mM H202

nor did

Cu(I1)

addition

5,

aerobic

from

rate, of

0.58

both

Cu(I1)

inactivation

1.6

of

under

line

rate-limiting

inactivation,

inactivation addition

lA, of

occurred

mM (Fig.

mM potassium

loss

addition

Cu(I1)

RESEARCH

incubation

however, 10

(Fig.

BIOPHYSICAL

present

Mn-catalase,

oxidative

make

the

AND

dismutase in

Cu(I1) enhanced

the

rate

[Fig.

5, the

to

the

incubation

of

the

inactivation

lines rate

2 and of

3);

mixture of 0.8

inactivation

pM

and

by

1.6-

4. Effect of hydrogen peroxide on the inactivation of the blase %&he by NH OH plus CuCl Line 1; plus 0.58 mM H 0 without NH OH. Line 2; control. zinc 3; plus 6158 mM H202. Line 4; plug a.58 mM H20Z2and 5 mM EDTA. Fig. 5. Mn-catalase superoxide

Effect of superoxide dismutase on the inactivation of by NH OH plus CuCl . Line 1; control. Line 2; plus dismutgse. Line 3; Flus 1.6 IBM superoxide dismutase.

78

the 0.8

PM

Vol. 124, No. 1, 1984

and 2.3-fold.

Thus,

reaction

and that

increase

in the

dismutation All

0; does the

state

of 0; , which of

evidence

not

rate

is

formed

obtained

here

is

in the

of

inactivation

concentration

inactivation

NH2OH solution

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

participate

increased

steady

Cu(II)-dependent aerobic

BIOCHEMICAL

of

during

the

indicates

a continuous

is

due to the

H202 produced

via

enzymatic

autooxidation

of

NH2OH (16).

the

of Mn-catalase

inactivation

involvement

of H202 in

by NH20H. Therefore,

source

of H202 in the

presence

of

Cu(I1). ACKNOWLEDGEMENTS I would helpful

like

discussions

to thank and for

Drs. their

K. Asada

and I.

constructive

Fridovich

for

comments

on the

many manuscript.

REFERENCES (1) (2) (3) (4)

Kono, Y., and Fridovich, I. (1983) J. Biol. Chem. 258, 6015-6019. Kono, Y., and Fridovich, I. (1983) J. Bacterial. 155, 742-746. Kono, Y., and Fridovich, I. (1983) J. Biol. Chem. 258, 13646-13648. Nicholls, P., and Schonbaum, G. R. (1963) Enzymes, 2nd edition, (eds. by Boyer, P. D., Lardy, H., and Myrblck, K.) Vol. 8, pp. 147-225, Academic Press, New York. (5) Ehagwat, A. S., Ramakrishana, J., and Sane, P. V. (1978) Biochem. Biophys. Res. Corrunun. 83, 954-962. Y. (1970) J. Biochem. 68, 145- 156. (6) Yoshikawa, S., and Orii, (7) Phillips, A. T., LaJohn, L., and Lewis, B. (1977) Arch. Biochem. Biophys. 184, 215-221. (8) Masuda, T. (1959) J. Biochem. 46, 1489-1496. (9) Fishbein, W. N., and Carbone, P. P. (1965) J. Biol. Chem. 240, 2407-2414. M. M. (1953) J. Biol. Chem. 201, 785-794. (10) Kaplan, N. O., and Ciotti, (11) Lin, Y. C. (1972) Biochim. Biophys. Acta 263, 680-682. (12) Runnegar, M. T. C., Blakeley, R. L., Fernley, R. T., Webb, E. C., and Zerner, 9. (1968) Biochim. Biophys. Acta 167, 632-634. (13) Anderson, J. H. (1966) Analyst 91, 532-535. (14) Anderson, J. H. (1964) Analyst 89, 357-362. (15) Moews, P. C. Jr., and Audrieth, L. F. (1959) J. Inorg. Nucl. Chem. 11, 242-246. (16) Kono, Y. (1978) Arch. Biochem. Biophys. 186, 189-195. (17) Peisach, J., Aisen, P., and Blumberg, W. E. (eds.) (1966) The Biochemistry of Copper, pp. l-209, Academic Press, New York. (18) Takahashi, M.,Takano, T., and Asada, K. (1981) J. Biochem. 90, 87-94. (19) Shinar, E., Navock, T., and Chevion, M. (1983) J. Biol. Chem. 258, 14778-14783. (20) Renaux, S., Louro, W., and Bemski, G. (1977) Magn. Reson. 28, 427-431. (21) Piskart, L., Freeman, J. M., Laker, W. J., Peisach, J., Perkins, 9. (1980) Nature 288, C5Mji7Stenkamp, R. E., and Weinstein, .

79