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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