Vol. 79, No. 3, 1977

COKPETITIVE

BIOCHEMICAL

II'JHIBITION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF Cu,

En SUPEROXIDE

DISMJTASE

3Y

MONOVALENT ANIONS Adelio Institute Italy

Rigo, Roberto of Physical

Stevanato Chemistry,

and Paolo Viglino University of Venice,

Venice,

and Giuseppe Institute merino, lY Received

Rotilio of Biological Chemistry, and CNR Center for Italy, October

21,

University Molecular

of Camerino, 3iology, Rome,

CaIta

1977

SUKMARY Polarographic measurements showed that 1J- and halides in hibit the activity of bovine Cu, Zn superoxidg dismutase in a competitive fashion, as previously demonstrated for CN- and OH-. All anions increase the spin-lattice nuclear magnetic relaxation time (T1) of aqueous solutions of the enzyme as well, but the stability constants measured from T1 data are lower than those calculated from activity data. The results suggest that substrate and anionic inhibitors bind during the cataly tic action af the water coordination position of the enzyme may have a greater affinity copper t and that these inhibitors for the cuprous form of the enzyme which is generated in the catalytic cycle. Copper-zinc tion

dismutases

anions

by a mechanism

of superoxide

ion

at the

first

O;,

active giving

ach the

diffusion mechanistic

substrate vailable

obtained until

:fe have

and oxidation

these

enzymes

level,

mainly

by pulse

recently

for

shown that

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

in vrhich

by the

limit

the

reduced

and reoxidized

controlled of

catalyse

is alternatively

of reduction

Inhibition at the

site 02,

The rates

H2°2s

tion.

superoxide

the

-

copper

to Cu+ by

second are

dismuta

02,

equal

the

giving and appro -

(1). is

still

poorly

because

of the

low yield

of

radiolysis,

the only

technique

a-

mechanistic

studies

determination

of

characterized

of 0; dismuta catalytic constants

776 ISSN

0006-29IX

Vol. 79, No. 3, 1977

in

the

presence

be obtained ble

BIOCHEMICAL

of relatively

with

to measure

de dismutase

the

copper

site

the

All

and were

thus

copper-zinc (3).

superoxi

In this

the mechanism which

a-

report of

we

inhibi

-

are known to bind

to

enzyme.

to the

reagents

(Milan). rographic

High

of the

and equilibrated

The concentration

tions

was determined magnetic

of

(Amel, with

Twice

(2)

wa-

a Model

461

0.02

triphenylphosphine obtained

(T.,)

in (4).

of water

NMR apparatus

M

oxide

different

reported

SIO

by the pola-

at 25OC in

of superoxide

pulsed

(4).

distilled

with

with

time

blood

O2 and N2 were from

N2 - O2 mixtures

relaxation

red

and Fridovich

Milan)

as previously

a Varian

bovine

enzyme was measured

saturated

with

tent.

grade.

currents

apparatus pH 9.8

from

of McCord

mixtures

of catalytic

Amel polarographic

by

procedure

purity

method buffer

was prepared

were analytical

The activity

asured

the

cells

anions,

dismutase

was used.

nuclear

(2)

of 0; can

AND METHODS

according

borate

of

to investigate

of

Superoxide cells

concentrations method

%I and Vmax values of bovine red blood

of SOD by monovalent

MATERIALS

ter

high

a polarographic

have used this method tion

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

O2 conthese

condi -

Longitudinal protons

working

was me

at 16 MHz.

RESULTS Activitjr

measurtients Double

hibitor

reciprocal

concentration

F- are OH- (3)

competitive which

inhibition Similar

copper

of

l/V0

vs

showed (Fig. inhibitors,

however

constants

are reported bovine

plots

are

as already far

more from

first

entry.

valueswere superoxide

1,

found

the

when the

dismutase

777

Ni,

Cl-,

shown for

effective

calculated

in Table

that

1)

at constant

l&j

in

Br-

and

CN-

and

inhibitors. slopes

enzyme

was measured

The

of such plots, activity

of

at constant

the

BIOCHEMICAL

Vol. 79, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

/

. r

1

-2

I

I

2

4

la3

If’)

k

1 - Double

reciprocal plots of initial velocities emus 0; concentration. Enzyme concentration : 1x10 -3 M. 0 no irihibitor ; A .O.O8M F-; D 0.08Pi Cl- ; m 0.08M Sr- ; A 0.05!< N; . Ionic strenght:O.l (by addition of 2M NaC104)

Figure

fo;3 (air of

the

equilibrated inhibitory

anion

Increasing

ionic

anions

(Table

of all

had no effect 2,

solutions) (Table

and at five I,

second

strength

decreased

I,

entry).

third

on superoxide

concentrations

entry). the

inhibition

Citrate,

dismutase

power

CNST

and &JO;

activity.

measurements The longitudinal

water

protons

measured tration ( l/T

IP

nuclear of aqueous

at different

magnetic solutions

anion

of the paramagnetic ) with CN-, 5, Br-,

relaxation of superoxide

concentrations contribution Cl-

time

to

of

dismutase

at pH 9.8. in

was

The ti-

the relaxivity

and F- is reported

778

(T,)

Figs.2

BIOCHEMICAL

Vol. 79, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE Stability constants (M-l) and T, measurements: 0.02 Different ionic strengths

Anion

from

activity

of superoxide dismutase from activity M borate buffer, pH 9.8, T= 25OC. were obtained by addition of 2M NaClO

measurements

pFo,Jca) CN-

I

2.9x105

from T, measurement at pk fJ,l2(')

j~rJ.l(~)

)y~O,2(~)

2.4~10~

2.3x1C5

9x1 o4 58

N;

83

87

68

Br-

28.5

29

21.5

Cl-

?I

13.5

9.5

1.6

3.2

2.0

1

F-

3.1

4'

0

a) The constant was measured from Lineweaver-Burk plots such as those of Fig.1 b) The constant was measured from experiments at [O;] 2 ~xIO-~N and at different anion concentrations. c) The measurements were made on enzyme solutions containing the same components as the solutions used in polarographic measurement. Ionic strength has no effect on water relaxation of enzyme solutions with or without coordinating ligands.

and 3, while

the

enzyme-anion

complexes

entry

of

Table

ted

the relaxivity

their

effects

laxivity CR-,

I.

stability

constants by this

It

appears

method that

to different on activity.

3 shows that

the

However

different

Br-

as high

2 moles

of

for

are reported

extents,

even at concentrations Fig.

measured

the various in anions

roughly

the

affec

paralleling

showed no effect as IN.

CN- ions

are

last

on re-

In the

case of

bound

per mole

of protein. DISCUSSION The interaction

of the

copper

779

binding

site

of copper,

zinc

-

Vol. 79, No. 3, 1977

BIOCHEMICAL

2 - Paramagnetic ar magnetic 16 KHz of a presence of Sk Br-; A 0.02X borate concentration:

Figure

superoxide

dismutases

gated

by magnetic

ports

on this

affinity

toward

the

molecule

of all

the

tested

version

of

rhombic

to axial

reaction between feet

in

of

This the

CN- and other

polarography the

spite

enzyme

netic scopic

data

Ni,

shape

(6,7).

pointed

is roughly

the

780

to repla-

The reaction

brought

about mechanism

of stability

by pulse

to that

The availability

far

to a similar

CN- and Ni are inhibition constant

comparable

rehighest

copper

of bovine either

early

enzyme-bound

showed that

and that

measurements.

OH-)

ion.

and a conflicting

measurements (3)

showed the

difference

properties

in

was suggested

F-,

of the

great

anions

which

to the metal

(CN’,

has been investi-

techniques

copper

coordinated

EPR line

Activity

CN-,

enzyme-bound

anions

anions

and optical

(5,6,7).

of Ni on magnetic

se (5).

monovalent

resonance

ce a water

the

contribution to the longitu&inal nuclerelaxation rate of water protons, at solution of superoxide dismutase in the different anion concentrations. F-; 8 Cl-; 0 Kbuffer, ~g 9.8. &peroxide dismutase 2.1x10 FI.

with

enzyme

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a con from of

constants

report superoxide

on the dismuta-

radiolysis

of a kinetic

from

or

(1)

also inhibitors calculated from

measured

ef

of ki-

spectro-

method

wor-

Vol. 79, No. 3, 1977

BIOCHEMICAL

I

Figure

3

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

1

I

- Relaxivity of water protons at 15 XHz as the func :;lzjof the molar ratio LCNJ / [superoxide dismu0.02X borate concentration:

bufferL4p!I 9.S. Superoxide 9x10 I.1

dismutase

led us to study the reaction of king at high 0; concentrations the enzyme with monovalent anions in more detail, and, in parti cular, to compare the activity and equilibrium data in a more precise way. A competitive pattern of inhibition was observed with anions having lower affinity toward enzyme (Ni and halides similarly to that observed with CN- and OH- (4) and this confirms that an identical mechanism can be suggested for all aniNi behaved as the other anions, at variance ons. In paticular, with previous suggestions by Fee and Gaber (5) and by Hodgson and Fridovich (8). The former authors proposed another Ni-bin ding site with higher affinity than copper because of the lack of T, effects in the presence of low Ni concentrations which produced an altered EPR spectrum. The latter authors suggested that Ni binds to copper at a different position of that normal781

Vol. 79, No. 3, 1977

BIOCHEMICAL

ly

as they

occupied

by H20,

de dismutase were not

activity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

observed

by 0.01

reproduced

in

M Ni.

other

to the

relatively

(2 g. Therefore,

on the

basis

strength

-substrate

the

the

and that

same,

general

i.e. effects

of the water Table

plexes

it

of

I is

the

the

I,

Possible

formed

are:

the

sites

during

turnover;

of

some anions

As regards

point

a) the

titration

the

-

in

en-

the

time

for

reported of corn

proton

relaxation

on the

enzyme;

the

enzyme

data

constants

binding

states

enzyme

as shown by

stability

explanations

(Cu+)

effect

relaxation

of

and water

and reduced

the

2 and 3).

polarography

constants

CN-(Fig.3)

data used

that

spin-lattice

feature

the binding

with

latter

position,

between

of

results

of 0; and inhibitors

and Figs

4 0; and H20 bind at different b) conformational changes occur c)

position

interesting

from

measurements.

anionson

differences

calculated

the

are

coordination

(Table

the most

(9)

can be proposed

binding

water

protons

However, in

the

former

including

complexes

the

the

of our data,

I),

of superoxi

low Ni concentration

and enzyme-inhibitor

zyme reaction is

(Table

Uhile

laboratories

may be imputable of ionic

no inhibition

oxidized

copper

are

of superoxide

(Cu++)

different. dismutase

shows that

in the oxidized form of the enzyme, ++ which has two equivalent Cu ions, one in each of two identical +-Isubunits (I), 1 CN- binds to each Cu stoichiometrically and the re is no UN- binding obtained

from

and 0; for

activity

the

ce in behaviour lated

to the It

tions

sites

with

experiments.

copper

ions

catalytic

whether

formation

during

constant

of the

latter

hypothesis

bility

constants

easy the

of

to decide observed

two

Since

and working

action

enzyme

the

from

oxidation

dismutase enzyme

is

states

of CN- and halide

782

with

the

appears

to that H20

differen to be re-

enzyme.

of

the

on account ions

in

turnover

due to a change

or to different

plausible

equal

CN- competes

experiments

behavior

action

is more

constant

of superoxide

of resting

is not

stability

with

values enzyme of the

condi of con -

of binding copper.

the higher Cu+ (10).

The

sta CN-

Vol. 79, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and Cl- have been demonstrated to bind the cuprous form of the enzyme by 35 Cl line width measurements (11). On this line it is interesting to notice that, while for the Cu+ free ion the stabi lity constant decreases in the order I- -) Br- 2 Cl- (16)f.n the case of superoxide dismutase we found Br- > Cl- and I- N 0. This result can be interpreted in terms of non accessibility of I- to the active site pocket of the reduced form of bovine superoxide dismutase. If this assumption is correct the active site should have dimensions between the ionic radius of Br- and I- which are 1.87 and 2.12 i respectively (12). Lastly it should be pointed out that, in spite of the low value of its stability constant, Cl- appears to be an effective in hibitor of red cell superoxide dismutase at physiological concen trations. REFERENCES 1) Fielden,E.M.,Roberts,P.B., Bray,R.C., Mautner,G.N., Rotilio,G. and Calabrese,L. (1974) Biochem.J.,199, 49-60. 2) Rigo,A., Viglino,P. and Rotilio,G. (1975) Anal.Biochem.,68, 1-8. 3) Rigo,A., Viglino,P. and Rotilio,G. (1975)Biochem.Biophys. Res.Commun.,63, 1013-1018. 4) McCord,J.M. and Fridovich,I. (1969) J.Biol.Chem.,244,60496055.

Fee, J.A. and Gaber,B.P. (1972) J.Biol.Chem.,247, 60-65. Finazzi-Agro A., Calabrese,L., Bossa,F., Guer6) Rotilio,G., rieri,P. and Mondovi,B. (1971) Biochemistry 10,616-620. 7) Rotilio,G., Morpurgo,L., Giovagnoli,G., Calabrese,L. and 2182-2187. Mondovi,B. (1972) Biochemistry,ll, (1975) Biochemistry,l4, 52148) Hodgson,E.K. and Fridovich,I.

5)

5299.

9) Rotilio,G., Calabrese,L., Mondovi,3. and Blumberg,W.E. (1974) J.Biol.Chem., 249, 3157-3160. "L'analyse qualitative et les reactions IO) Charlot,G. (1963) en solution", Masson, Paris, 274. and Uard,R.L. (1976) Biochem.Biophys.Res.Commun. 11) Fee,J.A. 71, 12)

427437.

(1972) "Advanced Inorganic Cotton,F.A. and Wilkinson,C. mistry" Interscience, New York, 458.

783

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