576th MEETING, LONDON
909
New Insights on the Primary Electron-Acceptor Complex of
Photosystem II
RICHARD MALKIN* and JIM BARBER?
Department of Cell Physiology, University of California, Berkeley, CA, U.S.A., and
f Department of Botany, Imperial College, London S.W.7, U.K.
Studies of the oxidation-reduction potential dependence of the electrochromic
absorbance change at 518 nm and of the fluorescence yield change have been carried out
with chloroplasts. The contribution of several Photosystem-I1 components associated
with the primary electron acceptor has led to a new hypothesis describing the reactions
at the reducing end of the Photosystem-I1 reaction centre.
Flash-induced electrochromic absorbance change at 518nm
Absorbance changes in the 520nm region have been interpreted as indicating an
electrochromic response of pigment molecules to the electric field formed across the
membrane by the primary charge separation of the two chloroplast photoreactions,
Photosystem I and I1 (Junge & Witt, 1968; Wolff et al., 1969). We have measured the
flash-induced 518nm absorbance change in chloroplasts as a function of defined
oxidation-reduction potential in order to characterize more fully the contributing
components.
In experiments at pH7.6, three components were observed in the potential range from
+500mV to -350mV. A high-potential component (Component 1: Em at pH7.6 =
+385mV) was associated with Photosystem I on the basis of its response to far-red flash
activation and to the inhibitors of Photosystem 11, 3-(3,4-dichlorophenyl)-1,1dimethylurea and o-phenanthroline. This component is most likely associated with
pigment P700, the Photosystem-I reaction-centre chlorophyll. Two additional
components with more electronegative midpoint potentials were also observed.
One (Component 2) had Em=+35mV, whereas the other (Component 3) had
Em = -250mV. Components 2 and 3 were found to be associated with the Photosystem-I1
light reaction by using the above-described procedures to distinguish the two chloroplast
photoreactions.
In agreement with previous results, which indicated an equal contribution of
Photosystems I and I1 to the electric potential difference across the membrane
(Schliephake et al., 1968; Amesz & de Grooth, 1975), Components 2 and 3 from
Photosystem I1 contributed an equal absorbance change to that of Component 1
from Photosystem I. In addition, the relative contribution of Components 2 and 3 was
about equal.
The midpoint oxidation-reduction potentials of Components 2 and 3 were found
to be dependent on pH and showed a -60mV per pH unit dependence in the pH range
from 6.5 to 8.5. In contrast, the E,,, of Component 1 was independent of pH in this
pH range.
Studies of fluorescence yield changes
The fluorescence yield changes in chloroplasts have been interpreted on the basis of
a quenching component, Q, which has been proposed to act as the primary electron
acceptor of Photosystem I1 (Duysens & Sweers, 1963). According to the most widely
accepted model, the oxidized state of component Q quenches fluorescence by acting
as a trap, whereas the reduced state of component Q results in a closed Photosystem-I1
reaction centre so that the fluorescence yield is higher in this state. This simple model has
been used to explain much of the fluorescence yield data observed with chloroplasts,
but some anomalies still exist.
Cramer & Butler (1969), in studies of the oxidation-reduction-potential-dependence
of the fluorescence yield in chloroplasts, found that two quenching components were
present. We have been able to obtain a titration curve similar to that of Cramer &
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BIOCHEMICAL SOCIETY TRANSACTIONS
I
I
+300 +ZOO +lo0
1
0
I
I
I
I
-100 -200 -300 -400
Eh (mV)
Fig. 1.Oxidation-reduction potential dependence of the fluorescence yield of chloroplasts
The reaction mixture contained 0.33~-sorbitol,lorn-Hepes [4-(2-hydroxyethyl)-lpiperazine-ethanesulphonicacid] buffer (pH 7.6), Sm-MgCI,, chloroplasts (chlorophyll
concentration of lOpg/ml) and the following redox mediators at the indicated
concentration : anthraquinone-2-sulphonate (~OPM), 2-hydroxy-1,4-naphthoquinone
(~OPM),indigotetrasulphonate ( l O p ~ ) , 5-hydroxy-1,Cnaphthoquinone ( 3 0 ~ ~
and
)
1,4naphthoquinone ( 3 0 ~ ~Titrations
).
were performed under anaerobic conditions in
a cuvette described by Dutton (1978) by using 0.1 M-sodium dithionite (in 0.5~-Tris/HCI
buffer, pH8.0) for reductive titrations and 0.1 M-potassium ferricyanide for oxidative
titrations. Fluorescence was measured with a weak (10J.m-2*s-1), chopped (1 10Hz)
beam (filtered with Schott BG18 and BG38 filters) and the emission at wavelengths
longer than 685nm measured with an EM1 9558B photomultiplier (blocked with a
Schott RG685 filter and a Balzer's 695nm interference filter), connected to an Ortec
lock-in amplifier.
Butler (1969), as shown in Fig. 1. The experimental curve can be fitted by assuming that
two quenching components are present in approximately equal concentrations. One component has Em = +25mV, whereas the second has Em = -270mV (at pH7.6). Both
components function as one-electron carriers (n = 1 in the Nernst equation). It should
be noted that the two fluorescence quenching components have almost identical
midpoint potentials to Components 2 and 3 observed in the titration of the flashinduced absorbance change at 518nm. The pH-dependence of Components 2 and 3 in
our experiments is also the same as that of the fluorescence quenchers.
The properties of Components 2 and 3 in the 518nm electrochromic absorbance
change suggests both components undergo photoreduction in light-driven electrontransport reactions. We have investigated the role of these components in more detail
in studies of light-induced fluorescence yield changes at defined oxidation-reduction
potential established before actinic illumination. As shown in Fig. 2(a), the weak measurand strong actinic illumination increases
ingbeam takes the fluorescenceto a low level (Fo)
the yield to its maximal value (F,,,). An approx. 4-fold increase in fluorescence yield
(F,/Fo)is obtained in a chloroplast sample adjusted to Eh= +250mV before illumination.
This sample contains the redox mediator, 2-hydroxy-l,4naphthoquinone,and this
compound, by acting either as a fluorescence quencher itself or as an electron acceptor,
causes an approx. 20% decrease in yield as compared with a sample with no mediator
present. Thus we routinely observe a &fold increase in fluorescence yield at a potential
of +250mV whether the redox mediator is present or absent. At a redox potential of
1978
576th MEETING, LONDON
911
Fig. 2. Light-induced fluorescence-yield changes at defined oxidation-reduction potential
The reaction mixture was as in Fig. 1 except that only one redox mediator was
included (2-hydroxy-l,4-naphthoquinoneat a concentration of 3 0 , ~ ~The
) . redox
potentials in (6) and (c)were adjusted with sodium dithionite and in ( d )with potassium
ferricyanide to the indicated E,,. Fluorescence was monitored as described in Fig. 1,
except that strong actinic illumination of the sample was also used (Schott BG18 and
A , Measuring light on; v, measuring
BG38 filters intensity of 4 . 0 10SJ.m-2.s-').
~
light off; t, actinic light on; 4,actinic light off. The measurements were all done on one
sample in the indicated sequence.
-90mV (Fig. 26), the Fo value has increased by a factor of 2 (owing to dark reduction
of the first quenching component), but strong actinic illumination still is able to increase
the fluorescence yield approx. 2-fold. On lowering the redox potential even further to
-330mV (Fig. 2c), a different pattern is observed. The Fo value is now as high as that
obtained by strong illumination because both quenching components are reduced
in the dark. Strong actinic illumination now results in a slow decrease of the fluorescence
yield to a value approximately half of that observed in weak light. A second illumination
with strong light causes no further changes in yield. To determine if any irreversible
photodestruction had occurred during the illumination, resulting in the yield decrease,
the redox potential of the sample was re-adjusted to a more positive value (Eh = -190mV
in Fig. 2d), and the changes observed were similar to those in Fig. 2(6), indicating that
the system was reversible.
Control experiments demonstrated that the pattern of fluorescence-yield changes
shown in Fig. 2 were not dependent on any redox mediator. Thus similar results were
obtained with indigotetrasulphonate (Em= -46mV), anthraquinone disulphonate (En,=
-185mV), 2-hydroxy-l,4-naphthoquinone(Em= -145mV) or with no added mediator.
The inclusion of the mediator allowed for proper redox poising of the sample in a
condition where one of the two fluorescence quenchers was reduced in the dark
before strong illumination and allowed for a more precise study of effects associated
solely with photoreactions of the low-potential quencher.
Hypothesis on the nature of rhe Photosystem ZZ primary electron acceptor complex
Central to our hypothesis on the functioning of the primary electron-acceptor complex
in Photosystem I1 are two new observations which have been made in this study.
(1) The low-potential fluorescence quencher (Emof approx. -270mV at pH 7.6),
previously detected only in potentiometric titrations, can undergo photoreduction.
This photoreduction has been directly demonstrated under conditions of steady-state
illumination and is inferred from the results on the redox potential dependence of the
flash-induced A S I Schange.
(2) Under conditions where the low-potential fluorescence quencher is chemically
reduced in the dark, subsequent illumination causes a decrease in the fluorescence yield,
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BIOCHEMICAL SOCIETY TRANSACTIONS
and a new state of lower yield accumulates. This state appears to be a photochemically
inactive one in that no further fluorescence yield changes can be photoinduced.
To accommodate these findings, we propose that the primary electron-acceptor complex of Photosystem I1 contains two different electron accepting groups. The two
components observed in the titration curves of fluorescence yield and the 518nm electrochromic shift are considered to arise from two quenching states, Q- and Q2-. Both Qand Q2- are produced as a result of light-driven electron transfer reactions, and we would
suggest that component Q undergoes a stepwise reduction leading to the fully reduced
state, with the singly reduced species being first formed. In terms of the role of
plastoquinone as the primary electron acceptor of Photosystem I1 (Van Gorkom, 1974;
Knaff et al., 1977), this would suggest a role for the hydroquinone as well as for the
semiquinone, the latter being a species that has been spectrally observed.
We interpret the decrease in fluorescence yield obtained during illumination of
samples in which component Q is previously reduced to arise from a lower
fluorescing state in which an additional electron acceptor is reduced. This acceptor
would function before the primary electron acceptor plastoquinone, and accumulation
of this first acceptor in the reduced state results in a photochemically inactive
Photosystem-I1 reaction centre (pigment P680+ is presumed to be reduced after the
initial charge separation by rapid transfer of electrons from reduced donors).
Evidence for an intermediate functioning before component Q was first obtained by
Van Best & Duysens (1977), and more recently, Klimov et al. (1977) demonstrated the
photoreduction of a pheophytin molecule by Photosystem I1 when component Q was
in the reduced state. In agreement with the results of Klimov et al. (1977), we observe
a decrease in fluorescence yield under conditions where the Photosystem-I1 reaction
centre accumulates in a state in which the intermediate is reduced in addition to having
component Q fully reduced. These states are summarized in the equations shown below:
P680.I.Q 5 P680.I.Q’Low fluorescence High fluorescence
state
state
P680-1-Q’- Lllht\ P680-I--Q2Low fluorescence
High fluorescence
state
state
To summarize our concepts, we would suggest that the primary electron-acceptor
complex in Photosystem I1 contains an additional electron acceptor, possibly a
pheophytin, besides a special plastoquinone molecule. The photoreduction of the
plastoquinone molecule occurs through two redox states, the semiquinone and the hydroquinone, and these states result in an increase in fluorescence yield. The photoreduction
of the additional acceptor, producing a complex in which all the electron acceptors are
fully reduced, results in a state with an intermediate yield of fluorescence. This model is
still considered as a working hypothesis, however, and further spectroscopic
characterization of these predicted states is required to test their proposed roles in the
primary electron-acceptor complex.
Amesz, J. & de Grooth, B. G. (1975) Biochim. Biophys. Acta 376, 298-307
Cramer, W.A. & Butler, W. L. (1969) Biochim. Biophys. Acta 172, 503-510
Dutton, P. L. (1978) in Methodr in Enzymology, Biomembranes,Part C (Fleischer, S . & Packer,
L., eds.), Academic Press, New York, in the press
Duysens, L.N. M. & Sweers, H.E. (1963) in Studies onMicroalgaeandPhotosynthetic Bacteria,
pp. 353-372, University of Tokyo, Tokyo
Junge, W. & Witt, H. T.(1968) Z . Naturforsch. 23b. 244-254
Klimov. V. V., Klevanik, A. V., Shuvalov, V. A. & Krasnovsky, A. A. (1977) FEBS Lett. 82,
183-186
Knaff, D. B., Malkin, R.,Myron, J. C. & Stoller, M. (1977) Biochim. Biophys. Acta 459,
4 0 2 4 11
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Schliephake, W., Junge, W. & Witt, H. T. (1968) Z . Nuturforsch. 23b, 1571-1578
Van Best, J. A. & Duysens, L. N. M . (1977) Biochim. Biophys. Actu 459, 187-206
Van Gorkorn, H. J. (1 974) Eiochim. Eiophys. Actu 3 4 7 , 4 3 9 4 4 2
Wolff, C., Buchwald, H. E., Riippel, H., Witt, K. & Witt, H. T. (1969) 2. Nuturforsch. U b ,
1038-1 041
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