BIOCHEMICAL

Vol. 64, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

J. Lumsdenand D.C. Hall University

Received

of London King's College, Department of Plant Sciences, 68 Half Moon Lane, London SE24 9JF U.K.

March

20,1975

SUEi4.iU Perticles prepared from spinach chloroplast membraneswith Briton X-100 inhibited the superoxide-mediated reduction of nitro-blue tetrazolium by riboflavin. This superoxide dismutase-like activity was of two kinds, one inactivated by heating and inhibited by HsC, and the other insensitive to both of these treatments; both activities were destroyed by washing with concentrated Tris buffer or with EDTA. Attempts at reconstitution with transition metal ions suggested that two different forms of bound manganesemay be responsible and it is proposed that the inhibition by H,O, is indicative of three different oxidation states of particle-bound manganese. The possibility that the photosynthetic water-splitting system and superoxide dismutase have evolved from a single precursor is discussed.

INTBODUCTION It important,

is well established that membrane-boundmanganeseplays an

if undefined, role in photosynthetic

and algae 192. Currently,

most studies of chloroplast

ments of whole chloroplasts heating4,

Pb involve

or conditions necessary for reconstitution

illuminated

treat-

pools of En6 - one pool, removable by bound,

electrons to the primary donor of the photoact.

chloroplast

In

systems, it has been shown that Mn2' can stimulate

the oxygen uptake mediated by methyl viologen in a manner similar which functions

either

5. By such methods,

which can accept electrons from water and a second, tightly

pool which transfers

by plants

which remove the Mn such as Tris-washing 3 or

evidence was found for two distinct Tris,

oxygen evolution

by the reduction of superoxide (0;).

to ascorbate,

However, no suggestion

was madeas to the possible fate of the resultant En>+, an unstable ion in a aqueous systems . In similar experiments, Walker --et al9 observed an oxygen

Abbreviations:

Copyright AU rights

SOD- superoxide dismutase E.C. 1.15.1.1 ; SDS - sodiwp dodecyl sulphate ; NBT - nitro-blue tetrazolium

o I9 75 by Academic Press. Inc. in aiz.v form reserved.

of reproduction

595

Vol. 64, No. 2, 1975

BIOCHEMICAL

uptake in a dark period following reaction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

illumination

which they attributed

of Mn3+ with H,O, thus: 2Mn3+ + H,O, ->

to the

2Mn2++ 0, + 2II+.

Free Mn2+ has long been known to be a potent inhibitor of lipid perox10 idation in biological membranes and recently it was shown that 1mMIJii2+ inhibited

the superoxide-mediated reduction of cytochrome c by xanthine

oxidasell.

Similarly,

micromolar concentrations

of Cu2*, from boiled erythro12 were found to inhibit lipid peroxidation .

cuprein (Cu/Zn SOD) or as C&l,, From pulse radiolysis

studies it was discovered that free Cu2+ could catalyse

the dismutation of 0; at pH 7.513; these workers also demonstrated that a number of complexes of Cus* with simple oligopeptides constants for 0; erythrocuprein

dismutation within

had 2nd order rate

the sameorder of magnitude as

and they commentedthat similar effects

other transition

might be expected for

metal ions.

As mentioned in our previous paper 14, the chloroplast

stroma contains a

soluble Cu/Zn SOD; Asada -et all5 had shown that about one third cyanide-sensitive

SODremains associated with the chloroplast

repeated hypotonic washes. Although intact SODactivity

chloroplast

in the presence of cyanide (B. Halliwell

of this

lamellae after

lamellae exhibit

- personal communication)

we have presented evidence for a cyanide insensitive

SOD-like activity

associated with subchloroplast particles.

activity

present in a soluble form after

A similar

no

appeared to be

treatment of lamellae with anionic detergents 14 ,

but we have now shown that this was an artefact.

However, in the present

paper, we reaffirm

that the SOD-like activity

associated with subchloroplast

particles,prepa.red

using the non-ionic detergent Triton X-100, is more than

a simple artefact,

though its precise physiological

significance

remains to

be clarified. MAT&&=

ANDMEThODS The Triton

of the method of ref. mg chlorophyll/ml.

14.

particles

were prepared by a modification

To a suspension of washed ohloroplast

was added an equal volume of lO$ (v/v)

in 50 mMpotassium phosphate pH 7.8, 1.0 M sucrose.

596

After

lamellae at 2.5

Triton X-100 (Sigma) stirring

for about

BIOCHEMICAL

Vol. 64, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5 min, the suspension was centrifuged stirred

briefly

into 2 volumes of cold polypropylene glycol

2025 (BDH). The turbid

sucrose and centrifuged

suspension was layered over 5C$ ("/w) rotor

at low speed and the supernatant

for 1 hr. at 50,000 x g. The pale-green pellet

beneath the sucrose layer

was resuspended in 50 mNpotassium phosphate pH 7.8,2C& stored in liquid

method16. Gel electrophoresis

in SDSwas by the method of ref.

RESULTS The red colour produced in assays of the'boluble of ref.

17.

preparation"

14 is due to cholate while SDS,at > 50 @g/ml in the assay, reduces

the118560

tion"

sucrose and

(“/w)

use. SODwas assayed by a photochemical

until

nitrogen

in a swing-out

to 3ff$ of the control.

Acetone extraction

of the "soluble prepara-

showed that it contained enough residual SDS to account for its

activity.

Simple salts of the four metal ions known to occur in SCDwere

tested for their

effect

on the assay. All excrpt Zn2', which does not undergo

redox reactions,

inhibited

KST reduction,

cu2+, 0.82@ Mn2+ and 40@ Fe>' ; of.MlnM have routinely

used 4@ riboflavin

increases linearly with riboflavin

ssed NBTreduction,

to rationalise.

did not alter

level at O.lZ@l + 16. As we for erythrocuprein-Cc2

in this assay and the reaction rate

from 2 to 5@l riboflavin,

is difficult

with 50; of control

inhibition

by complex formation

3.3pM Mn2+, which totally

suppre-

the amount of H202 produced in the assay

with NBT omitted. Triton particles multiple

If particles

green bands were visible,

were subjected to SDS gel electrophoresis, just behind the free carotene and chlorophyll

bands. Staining for protein with Coomassieblue confirmed the absence of the high molecular weight chlorophyll-protein particles.

The dithionite

band characteristic

(reduced) minus ferricyanide

recorded at 77”K, exhibited

of photosystem I

(oxidized)

a single band at 557 nm indicative

spectrum,

of the presence

of cytochrome b 559. The inhibitory

effect

of the particles

and these could be differentiated A fairly

constant proportion

on the SODassay was of two kinds

by heating at 85°C or by addition

(about 69;) of the activity

597

of H202,

was inactivated

by

Vol. 64, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

heating for 3 min. at 85OC; the remainder was insensitive and if the perticles

were rapidly

centrifuged

down after

to prolonged heating, heating,

remained in

the supernatant and was probably due to Mns' or Cu@, both of which are released from chloroplasts

on detergent treatment,

Neither activity

impaired by heating for 15 min. at 50°C. This is in contrast evolution

was

to oxygen

in whole chloroplasts,

subchloroplast Unexpectedly,

particles

but an increased resistance of Mn in 18 to removal by heating has been reported .

it was found that added H202 had no effect

at concentration

of 2.M- it is difficult

to reconcile

on the assay, even

this with the accepted

mechanismof this reaction 16. In the presence of 1mMKCN,to inhibit catalase or peroxidase activity, the perticles

H202 appeared to suppress the activity

and increase NDT reduction

rather than an inactivation

(Fig.

which is not

with l&l

were then centrifuged

inhibited

KCN

and

out, washed

was found to be the sameas a control

incubated with KCN alone. This is the opposite situation erythrocuprein

of

1). That this was an inhibition

was shown by incubating particles

24 ti Ha02 for 2 hrs at 25'C. The particles and assayed and the activity

any

sample

to that found for

by H202 but is inactivated

by the

. Untreated

1 2L

I 1.6 HYDROGEN

I 72

I 96 PEROXIDE

I 120

1 1u

IJJMI

Figure 1. Zffect of hydrogen peroxide on SODactivity of Triton subchloroplast particles (175pg protein). All assays in presence of 1mMKCN.

598

BIOCHEMICAL

Vol. 64, No. 2, 1975

destruction

of a histidine

inhibition

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

site 19. It was noted that

residue at the active

by H,O, did not reduce the activity

of the particles

below the level

which was heat-insensitive. The effects

of a number of reagents on the activity

of the particles

were

investigated

by incubating the particles

centrifuging

down and uashing with buffer before assaying. A number of potenti-

ally

informative

in each reagent for 1 hr. at 4'C then

treatments (e.g. hydroxylamine)

absorption of the reagent by the particles

were rendered unusable by

with resulting

ambiguities in the

assay. Although M$+ at 0.2M has been reported to remove Mn from chloroplast photosystem II 20 , it had no effect on the particles; however both l.OM !lkis-Cl pH 8.4 and 2mMHDTAproduced complete inactivation

(Fig. 2). Inactivation

SDTA suggested that metal ions could be responsible for the activity particles

end attempts were made to reactivate

by

of the

Trig-and EDTA-washedparticles

by incubation with O.l.nM Mn SO, or CuSO, followed by careful washing and assaying. Both metal ions restored a SOD-like activity Cu2+ - reconstituted reconstituted

particles

particles,

were insensitive

to the particles.

to H,O, but Mns+ -

although only 2C$ as active as Cue+-reconstituted

*Controlpolilcles Q m- ‘JCOntrOl +O.ZLmM HZ02 n

Tris

lor EDTA)washed 1 I 20 LO PARTICLES

1 60

1 80

1 100

IyL)

Figure 2. Effect of washing with Trig-Cl (or with HDTA) on SODactivity of Triton subchloroplast particles. All assays in presence of 1mMXCN.

599

Vol. 64, No. 2, 1975

particles,

did exhibit

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sensitivity

to H,O, which is not shown by free Mne+

(Fig. 3). Surprisingly, greatly effect

incubation with l,Or@l Zn2+ in addition

increased the recovery

of activity

(Fig.

3). As Zns+ alone had no

it may act by competing with Mn2+ for unreactive

making Mne+ more available MS+ - reconstituted

to the active

particles

sites.

to 0.1~84Mnz'

It

binding sites,

becameapparent that the

were not to be equated with untreated particles-

on heating at 85°C the reconstituted

particles

remained as a fine suspension

and did not clump together as the untreated ones did; more significantly, the SOD-like activity, unaffected

including

the peroxide-sensitive

part,

was completely

by heating.

DISCUSSION The discovery that certain Mn-containing proteins are superoxide dismutases21'22'23, suggested the possibility exhibit

a similar activity

that chloroplast

24, so that the assay system for this enzyme might

be used as a probe of the state a& chloroplast

Mn. If it is assumedthat

bound En is responsible for the SOD-like activity particles,

EiLrmight

of the Triton-subchloroplast

it appears that it is no longer in the "native"

site,

since it can

be displaced by ZETA but not by IQ$+ ; nor is it in the site described by Gross25

PARTICLES

Figure 3. with M&O,.

iyL.1

Reconstitution of SOD-like activity All assays in presence of 1mBlKCN-

600

by incubation of particles 0.24mM He02.

BIOCHEMICAL

Vol. 64, No. 2, 1975

as being present in whole lsmellae, competitively

AND BIOPHYSICAL

since neither Mge+ nor Zn2+ appear to bind

with Mns+.

The peroxide inhibition

might be explained in terms of three different

redox states of bound Mn with the Mn (IV)/Mn(III) removal of 0; more effectively for the -E.coli

RESEARCH COMMUNICATIONS

couple catalysing

than the Mn(III)/tuln(II)

the

couple, as proposed

enzyme by Pick --et al 26 e.g. fast (heat-sensitive

bound Mn)

peroxide inhibition

bound IQ

Such higher oxidation splitting

reaction

states of manganesehave been implicated in the water-

of chloroplasts 27.

Someof this work may be of relevance to the problem of how the ancestors of to-day% blue-green algae were able to develop a water-splitting capability life

with comcomitant 0, evolution

when they, in commonwith all

forms at that time,had not previously

been exposed to toxic

trations2S . One possible explanation is that a single primitive protein

or peptide carried out both the functions

splitting,

with a subsequent independent evolution

f'Hill-factor*'

may be a retained primitive

of the two functions. of a Mn-

site on the lamellae. This

characteristic

to determine whether it has SODactivity

and it would be of interest

comparable to that reported for

simple Cue+ complexes13. It has been pointed out?' that, reducing atmosphere, copper would have been largely water concentration

mangano-

peptide from the blue-green alga Phormidium luridum 29,

which appears to act near the water-splitting factor

0, concen-

of 0; dismutation and water-

Possibly in support of this hypothesis is the recent isolation containing

other

under the primitive

insoluble while the sea-

of Mne+ would have been very high - thus simple Mn

complexes, but not copper ones, would have been available.

601

Vol. 64, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgements Wewish to thank Drs. R. Cammack and J.R. Bolton and Mrs. Ana Montes for useful discussions and Dr. K. Kahn of Liverpool Polytechnic for the loan of a rotor. References 1. Cheniae, G.M. (1968) Ann. Rev. Plant Physiol. 2l, 467-498. 2. Heath, R.L. (1973) ht. Rev. Cytol. 2, 49-101. 3. Yamashita, T, and Butler, W.L. 1969 Plant Physiol. 44, 435-438. 4. Yemashita, T. and Butler, W.L. t 1968 Plant Physiol. u, 2037-2040. 167-181. 5. Cheniae, G.M. and Martin, I.F. (1971 Biochim. Biophys. Acta a, 6. Cheniae, G.M. and Martin, I.F. (1970 i Biochim. Biophys. Acti=, 219-239. 7. Epel, B.L. and Neumann,J. (1973) Bioohim. Biophys. Acta. m, 520-529. 86-94. 8. Ben-Hayyim, G. and Amon, M. (1970) Biochim. Biophys. Acta 2, 9. Walker, D.A., Ludwig, L.J. and Whitehouse, D.G. (1970) FEBSLetters 6, 281-284. 10, Thiele, E.H. and Huff, J.W. (1960) Arch. Biochem. Biophys. 88, 203-207. 11. Fong, K-L., McCay, P.B., Poyer, J.L., Keele, B.B. and Misra, H. (1973). J. Biol. Chem.3, 7792-7797. 12. Pederson, T.C. and Bust, S.P. (1973) Biochem. Biophys. Res. Commun.& 1071-1078. 13. Brigelius, R., Spottl, R., Bors, W., Leng-felder, E., Saran, M. and Weser, U. (1974) FEBS Letters & 72-75. 14. Lumsden, J. and Hall, D.O. (1974) Biochem. Biophya. Res. Commun.2,

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