Vol. 44, No.

6, 1971 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1
EFFECT OF ANIONS ON CHLOROPLAST PARTICLES

F.A. McEvoy and W.S. Lynn

Department of Biochemistry
Duke University Medical Center
Durham, North Carolina 27706

Received July 12, 1971

SUMMARY

As previously shown, monovalent anions, such as CI-, SCN ,

NO 3 , stimulate electron transport, with water as substrate, in
aged or EDTA-treated chloroplasts. The rate-limiting step of
electron transport in chloride-deficient chloroplast particles is
shown to be the oxidation of cytochrome 559 by exogenous electron
acceptors. Monovalent anions, such as Cl-, lower the apparent K m
of exogenous electron acceptors, apparently by facilitating fluxes
of the oxidized and reduced forms of the acceptors across the chloro-
plast membrane. Thus, a major function of anions in the water-
splitting steps of photosystem II, is to facilitate transport of
external oxidants.

INTRODUCTION

ChlOride has long been known to stimulate photosynthetic

1 i 2
electron transport. Bove et al. suggested that chloride specifically

affected the water-splitting system and this concept was extended by

other authors. 3'4 Izawa et al. 5 showed that up to 10-fold activation

of electron transport by chloride concentrations above 5 mM could be

reproducibly observed in EDTA-treated chloroplasts and postulated that

the site of chloride activation was on the oxidizing side of photosystem

II between the water-splitting enzyme and a proposed oxidant, "Y".

The results in this report show that CI- and other monovalent anions

lower the effective K of the various electron acceptors, i.e. facil-

m
itate transfer of electrons between bound reduced cytochrome and the

iThis investigation was supported by a grant from the National

Science Foundation, Grant GB-17440.

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exogenous acceptors by i n c r e a s i n g transport of the a c c e p t o r s

across the membranes.

METHODS

Electron transport to f e r r i c y a n i d e was m e a s u r e d titrimetric-

ally 6 w h i l e benzoquinone reduction was m e a s u r e d colorimetrically.

The EDTA p a r t i c l e s described here w e r e 0.3% d i g i t o n i n spinach sub-

chloroplast p a r t i c l e s 7 treated with E D T A as before. 8 Postillumin-

ation oxidation of c y t o c h r o m e 559 was m e a s u r e d as p r e v i o u s l y des-

cribed. 9 For s p e c t r o p h o t o m e t r i c studies the r e a c t i o n mixture con-

tained Mg SO 4 (2 X 10 -4 M), p h o s p h a t e (i X 10 -3 M), sucrose (0.1

M), PMS + (2 X 10 -6 M), azide (2 X 10 -4 M) and c h l o r o p l a s t particles

at a c h l o r o p h y l l concentration of 40 ~g p e r ml in a final v o l u m e

of 3 ml. Cytochrome 559 a b s o r b a n c e changes were measured both w i t h

538 and 578 nm as r e f e r e n c e wavelengths. Light-dark difference

spectra of these EDTA particles have revealed 8(525 nm) and e(559

nm) peaks, both of w h i c h exhibit light-dark difference spectra

compatible with oxidation and r e d u c t i o n of a b type of cytochrome.

Cytochromes f and b563 could not be d e t e c t e d in these p a r t i c l e s

either in l i g h t - d a r k difference spectra or in dark d i f f e r e n c e spectra

i0
using ascorbate, dithionite and ferricyanide. This is in agree-
12
ment with the o b s e r v a t i o n of B o a r d m a n and A n d e r s o n that cytochrome

559, but not the other cytochromes remains tightly bound to the

chloroplast particle during digitonin fractionation.

For e l e c t r o n transport measurements the r e a c t i o n mixture

contained chloroplast particles (I0 ~g per ml), either phosphate

(i X 10 -3 M) or Tris SO 4 (2 X 10 -4 M) as buffer, sucrose (0.i M)

and Mg SO 4 (2 X 10 -4 M) in a final volume of 15 ml for titri-

metric assays and 3 ml for c o l o r i m e t r i c assays. In all exper-

iments, the pH was 7.8.

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RESULTS AND DISCUSSION

AS p r e v i o u s l y observed by Izawa et al. 5 e l e c t r o n trans-

port in E D T A - t r e a t e d chloroplast particles is s t i m u l a t e d 2 to

6-fold by a d d i t i o n of c h l o r i d e at a final concentration of i0 m M

when either ferricyanide or i n d o p h e n o l s are used as e l e c t r o n

acceptors.

Fig. 1 shows the e f f e c t of c h l o r i d e ions on the p o s t i l l u m -

ination oxidation of c y t o c h r o m e 559. As p r e v i o u s l y shown, 9 cyto-

chrome 559 is 100% r e d u c e d during illumination with saturating

light and is o x i d i z e d in the p o s t i l l u m i n a t i o n phase. In the pre-

sence of an oxidant, such as f e r r i c y a n i d e , the c h r o m o p h o r e is

partly reduced in the light and is o x i d i z e d postillumination. Add-

ition of s o d i u m chloride at a final concentration of i0 m M d e c r e a s e s

the amount of c y t o c h r o m e which is r e d u c e d in the light and also

accelerates the rate of dark oxidation of the cytochrome. SCN- or

NO 3- are e q u a l l y effective, whereas sodium or p o t a s s i u m or m a g n e s -

ium sulfate or p h o s p h a t e are ineffective. Unlike ferricyanide,

Light Lighl Lighl

CI-or SCN-'

IOO

CONTROL 75 ~
+ BENZOQUINONE

25 ~
+ FERRICYANIDE
\
Fig. i. P o s t i l l u m i n a t i o n changes in a b s o r b a n c e of E D T A p a r t i c l e s
at 559 n m in the p r e s e n c ~ and absence of f e r r i c y a n i d e (2 X 10 -4 M)
or b e n z o q u i n o n e (2 X 10 -~ M). S o d i u m c h l o r i d e or t h i o c y a n a t e (2 X
10 -2 M) was added to each w h e r e indicated; b o t h gave i d e n t i c a l
results. The p a r t i c l e s w e r e i l l u m i n a t e d w i t h s a t u r a t i n g w h i t e light
for i0 sec w h e r e i n d i c a t e d and a b s o r b a n c e m e a s u r e m e n t s w e r e b e g u n
~0.5 sec after the e x c i t i n g light was s w i t c h e d off.

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benzoquinone does not completely oxidize the cytochrome 559, nor

can the cytochrome be reduced by brief illumination when excess

benzoquinone is present. However, in the presence of sodium chlor-

ide, cytochrome 559 is partly oxidized during illumination and is

slowly reduced in the postillumination phase (Fig. i). Thus, with

either benzoquinone or ferricyanide as acceptor the effect of chlor-

ide is to increase the steady state level of oxidation of cyto-

chrome 559 during illumination. Chloride has no effect on the

postillumination changes in absorbance of cytochrome 559 in the

absence of exogenous electron acceptors. These results clearly

indicate that oxidation of cytochrome 559 by the exogenous elect-

ron acceptor is a rate-limiting step of electron transport in

chloride-deficient particles.

As shown in Fig. 2, the apparent Km for ferricyanide in

salt-deficient EDTA particles is 100-fold higher than in the in-

tact particles. Addition of sodium chloride to EDTA particles

decreases the apparent Km for ferricyanide 10-fold. Since ferri-

-3
cyanide concentrations above 1 X 10 M cause inhibition of

5 0 0 --
"C

IX io-'r IX;TO- 6 IXlO-S IXlO-4 IXlO-3

FERRICYANIDE

Fig. 2. Electron transport measured at various ferricyanide concent-

rations in digitonin-particles in the presence of sucrose and NaCl
(: ~, in EDTA particles in the presence of sucrose and NaCl ~ ~,
EDTA particles in the presence of sucrose but without NaCl ~---~ and
EDTA particles in the absence of either sucrose or NaCl @ W. Where
added, sucrose and NaCI concentrations were 0.i M and .01 M respect-
ively.

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electron transport both in the presence and absence of salt, the

anionic properties of ferricyanide, at i0 mM, cannot be assessed.

When electron transport is measured in the absence of either salt

or sucrose, still lower levels are observed, and the apparent Km

for ferricyanide is further increased (Fig. 2).

The data in Fig. 2 shows that the affinity of the electron

acceptor for the photosynthetic reductants is g r e a t l y enhanced by

the monovalent anions. Since sucrose also partially activates

electron transport, it appears that the effect of monovalent anions

is partly osmotic. But the anions also have a further specific

effect, which is interpreted as an alteration of the internal concen-

tration of oxidant by facilitating the flux of the reactants, ferri-

cyanide and benzoquinone, together with their reduced products, in

and out of the chloroplast membrane. The apparent specificity of

the anion effect for the rate of electron transport through photo-

system II is due to the fact that reduction of the terminal electron

acceptors of phot0system I reactions, such as NADP + is generally

ii
limited by the enzymic reaction at the terminal steps, while ox-

idation of cytoehromes etc. by ferricyanide or benzoquinone is lim-

ited only by the internal oxidant concentration.

While these results clearly show that a major role of chlor-

ide or other anions in photosynthetic electron transport is to fac-

ilitate influx of terminal acceptors, a specific effect of anions

on the reactions which transfer electrons from water to the photo-

synthetic reaction center 2-5 may also exist. However, the high

ionic concentrations required (>I X 10 -2 M) and the lack of spec-

ificity for any particular anion make the above possibility un-

likely.

ACKNOWLEDGEMENTS

We are indebted to Mary Lynn for technical assistance.

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REFERENCES

i. Warburg, O., and Luttgens, W., Biokhimiya ii, 303 (1946).

2. BoyS, J.M., Bove, C., Whatley, F.R., and Ar--non, D.I., ~.
Naturforsch. 18b, 683 (1963).
3. Hind, G., Nakatani, H.Y., and Izawa, S., Biochim. Bi0phys.
Acta 172, 277 (1969).
4. Heath, R.L., and Hind, G0, Biochim. Biophys. Acta 172, 290 (1969).
5. Izawa, S., Heath, R.L., and Hind, G., Biochim. Biophys. Acta
180, 388 (1969).
6. Lynn, W.S., and Brown, R.H., J.Biol. Chem. 242, 418 (1967).
7. McEvoy, F.A., and Lynn, W.S., FEBS Letters iO, 299 (1970).
8. Lynn, W.S., Biochemistry 2, 4789 (1969).
9. Lynn, W.S., Biochemistry ! ~, 3811 (1968).
i0. McEvoy, F.A., and Lynn, W.S., manuscript in preparation.
ii. Boardman, N.K., Adv. Enzymol. 32, 1 (1968).
12. Boardman, N.K., and Anderson, J.----M.,Biochim. Biophys. Acta
143, 187 (1967).

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