Plant Physiol. (1981) 67, 1190-1194
0032-0889/81/67/1 190/05/$00.50/0
Ferricyanide Reduction in Photosystem II of Spinach
Chloroplasts'
Received for publication June 24, 1980 and in revised form December 2, 1980
RITA BARR AND FREDERICK L. CRANE
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
the electron transport chain in place of DBMIB to prevent ferricyanide reduction at site 1. The two inhibitors used in this unique
Ferricyanide can be reduced in Photosystem II of spinach chloroplasts manner include bathophenanthroline, a chelator, which may inat 2 separate sites, both of which are sensitive to 3-(3,4dichlorophenyl)- hibit a nonheme iron located between plastoquinone A and cy1,1-dimethylurea, but only one of which is sensitive to dibromothymoqui- tochromef (3, 8), and DNP-INT, a compound shown by Trebst
none. Data presented in this paper emphasize ferricyanide site II of and associates (14) to inhibit the main electron transport chain of
Photosystem II, which is sensitive to thiol inhibition and may reflect a chloroplasts at the same site as DBMIB or between component B
cyclic pathway around Photosystem II. Ferricyanide reduction sites I and and plastoquinone A. The choice of these compounds in place of
2 also differ from each other in fractions isolated from discontinuous DBMIB to study a possible cyclic ferricyanide reduction pathway
sucrose gradients, from fragmented chloroplasts, and upon trypsin treat- in PSII offered the advantage of not acting as electron acceptors
ment. Sucrose density gradient centrifugation shows that ferricyanide themselves and having fewer effects on the chloroplast membranes
reduction site 1 activity at pH 6 decreases from 30 to 50% in various than DBMIB. We also show data from experiments designed to
isolated fractions, while the dibromothymoquinone-insensitive activity at induce a partial physical separation of the two PSII ferricyanide
pH 8 (site 2) is stimulated from 15 to 35%.
sites.
Fragmentation of chloroplasts also stimulates ferricyanide site II activity, but trypsin treatment destroys ferricyanide reduction site II completely
MATERIALS AND METHODS
in 6 minutes. Ferricyanide reduction site 1 still retains 50% activity at this
point. The meaning of these differences is discussed in terms of the physical
Spinach chloroplasts were prepared from market spinach (Spilocation of these two sites on the thylakoid membrane.
nacia oleracea) in 0.4 M sucrose: 0.5 M NaCl (SN chloroplasts) as
previously described (4). Grinding time in a Waring Blendor for
deveined, chilled spinach leaves was 15 s. The ground slurry was
filtered through eight layers of cheesecloth and a layer of Miracloth before centrifugation at 600g for 2 min to remove unground
debris and nuclei from the ruptured cells. The supernatant was
recentrifuged at 2,500g for 10 min to obtain a chloroplast pellet,
Ferricyanide reduction by isolated spinach chloroplasts is one but leave chloroplast fragments and mitochondria in the superof the early Hill reactions known since the 1950s and studied by natant. The isolated chloroplasts were suspended in SN. Chl was
too many investigators to mention here. Trebst et al. (13) first determined according to Arnon (1). Chloroplast stock solutions
showed that a portion of ferricyanide reduction was insensitive to always contained 1 mg Chl/ml.
inhibition by DBMIB.2 Thus there are 2 PSII ferricyanide reduc02 evolution or uptake was measured with a Clark-type 02
tion sites. A third site occurs in PSI in Izawa-type chloroplasts (7). electrode attached to a Yellow Springs Instrument Oxygen MonBanaszak et al. (2) first reported differences in chelator inhibition itor. A water-jacketed cell (24C) containing 1.5-ml reaction mixpatterns on the ferricyanide pathway according to pH of the ture was used. Reaction rates were recorded with a Sargent-Welch
medium. Studies by Reimer and Trebst (9) showed that DBMIB SRG recorder. Illumination for chloroplast assays was provided
inhibition of the H20-- NADP+pathway could be reversed by by a specially built light source, equipped with a GE quartzline
thiols. Sireci et al. (12) tested many thiols on ferricyanide reduction CBA lamp. A 250-ml round bottom flask, filled with saturated
and found a different explanation for the apparent reversal of CuS04 solution, located between the reaction vessel and the lamp,
DBMIB inhibition by thiols. It is possible that, when low concen- served as a heat shield. Reaction mixtures are given in figure
trations of thiols (1-100 ,UM range) are used to inhibit the DBMIB- legends. All reaction mixtures were incubated in the dark for 3
insensitive portion of ferricyanide reduction in PSII, a cyclic min before turning on the light.
pathway around PSII becomes inhibited in the light, resulting in
Separation of various chloroplast fractions on discontinuous
increased electron transport rates on the main pathway to fern- sucrose gradients was performed with 50, 60, 65, 70, and 80%
cyanide reduction. In this study, we have chosen to compare the sucrose, buffered with 50 mm Tris-Mes (pH 7), which contained
DBMIB-insensitive portion of ferricyanide reduction in PSII, 5 mM MgCl2. Centrifugation time was at 82,500g for 90 min in a
which is sensitive to thiol inhibition, with two other inhibitors of Beckman SW 27 rotor. The top layer found in 50% sucrose
contained small chloroplast fragments, the band between 60 to
'Supported by National Science Foundation Grant PCM 7820458.
65% sucrose was the main chloroplast band, consisting of damaged
2
Abbreviations: DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzo- chloroplasts. The small band immediately following 65% sucrose
quinone (dibromothymoquinone); DNP-INT, 2,4-dinitrophenylether of contained mainly stripped chloroplasts, while in the bottom pellet
iodonitrothymol; BP, bathophenanthroline; MV, methylviologen; SM, sil- the chief constituents were presumed aggregates of damaged
icomolybdic acid; DMBQ, 2,5-dimethylbenzoquinone; DCIP, 2,6-dichlo- chloroplasts.
roindophenol; FeCN, potassium ferricyanide; TSA, thiosalicyclic acid;
Mechanical chloroplast breakage was accomplished by the techSN-0.4 M sucrose with 0.05 M NaCl.
nique of fragmentation. This consisted of drawing a chloroplast
1190
ABSTRACT
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1981 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 67, 1981
FERRICYANIDE REDUCTION IN PHOTOSYSTEM II
suspension into a l-ml Hamilton syringe with a 26-gauge needle
attached, and expelling the suspension as many times as possible
during a certain time period, such as 5 min. Monitoring breakage
on sucrose gradients as above showed that, after 5 min of fragmentation, most of the material put on the discontinuous sucrose
gradients stayed in the top band of 50%o sucrose, indicating that
this material was mainly chloroplast fragments.
Trypsin treatment of water-shocked chloroplasts consisted of
incubating 1 mg trypsin/mg Chl on ice for 2 to 8 min. At the end
of each incubation period, trypsin inhibitor was added to a final
concentration of 2 mg/mg Chl. The treated samples were then
diluted 10 times with fresh SN and centrifuged for 10 min at
7,500g. The pellet was suspended in SN to give a final concentration of I mg Chl/ml for assaying chloroplast activity.
RESULTS AND DISCUSSION
1191
C
O
4-
0
c
0N
0
CY
0
4-
0
After Trebst et al. (13) showed in 1970 that even high concentrations of DBMIB (20 pM) inhibited ferricyanide reduction in
chloroplasts no more than 60%o, it became obvious that ferricyanide could accept electrons at at least two sites in the electron
transport chain. Banaszak et al. (2) then showed that, depending
on pH, the DBMIB-insensitive ferricyanide reduction in PSII
could be differentially sensitive to orthophenanthroline inhibition.
These studies led Sireci et al. (12) to test other inhibitors on
ferricyanide reduction in PSII. It was found that the DBMIBinsensitive ferricyanide reduction (site 2) was selectively inhibited
by various thiols, whereas ferricyanide reduction on the main
c
._
0
-
o
0-
c
._
._r-
a
DNP-INT (pM)
FIG. 2. The effect of DNP-INT on various electron transport reactions
e+50
in isolated spinach chloroplasts. Reaction mixtures for each reaction as in
Figure 1, except 2.5 pM DBMIB added to the following reactions: H20
-- DCIP, H20 -- FeCN (pH 8), and H20-- DMBQ. Control rates for
each reaction shown in the figure, from top to bottom, left column first,
then the right one, were as follows: 226, 124, 197, 338, 203, 474, 226, 694,
508 ,ueq/mg Chl.h. +, Stimulation; -, inhibition of rate in relation to'
c
control.
0
C.)
0
0
CY
0_
0
0
C
0
-
50
0
E
U)
12 5
0
5.00
2.50
DBMIB (pM)
FIG. 1. The effect of DBMIB on various electron transport reactions in
isolated spinach chloroplasts. All reaction mixtures contained chloroplasts
(0.05 mg Chl), 5 mM NH4Cl as uncoupler, and 25 mM Tris-Mes buffer at
a
specified pH,
as
shown above. In addition, the H20
--
MV
(+azide)
reaction contained 0.5 mm MV and 0.5 mm sodium azide. The H20 SM
reaction contained no DCMU at pH 7, but 5 pM DCMU was present
at pH 6 and 8; 85 ,LM SM was used at pH 6, 250 UM SM at the other
pH values. The H20 DMBQ reaction contained 10 mM DMBQ. The
DCIP reaction contained 0.5 mM DCIP. The H20
H20
FeCN
reaction at pH 6 contained 250 ,lM FeCN. The H20 FeCN reaction at
pH 8 contained 500 ,UM FeCN. The control rates for each reaction shown
in the figure from top to bottom, left column first, then the right one were
as follows: 694, 226, 90, 158, 614, 406, 403, 496 ueq/mg Chl.h. +,
Stimulation; -, inhibition of rate in relation to control.
-*
-*
--
pathway (site 1) was stimulated or inhibited <30%. It was logical
to assume that ferricyanide site 2 was on a branched pathway,
possibly forming a cyclic system around PSII, as postulated earlier
by Doring (5) in trypsin-treated chloroplasts.
The present study was undertaken with the purpose of learning
more about the thiol-sensitive, DBMIB-insensitive ferricyanide
site 2 in PSII. Could other inhibitors in the plastoquinone region,
such as BP (3) or DNP-INT according to Trebst et al. (14)
substitute for DBMIB? Stimulation or inhibition of various electron transport pathways in presence of these compounds is shown
in Figures 1 to 3. Neither DBMIB, BP, or DNP-INT totally
inhibits ferricyanide reduction, which means that the insensitive
part is potentially inhibitable by thiols.
Thiol inhibition of ferricyanide reduction with BP or DNP-INT
in place of DBMIB is shown in Figure 4. The most interesting
observation from this figure is the fact that DBMIB allows the
best thiol inhibition of ferricyanide reduction to take place. This
is followed by BP and DNP-INT, respectively, when the inhibitors
block forward electron transport to allow a possible cyclic electron
flow around PSII, a pathway on which thiol inhibition is the
criterion used for testing. It is not clear why DBMIB, BP, and
DNP-INT are not equally effective in allowing the thiol inhibition
of ferricyanide site 2, since all three compounds inhibit the
H20 -- MV (+ azide) pathway equally well. It is possible that the
linear order, in which they inhibit forward electron transport on
the main chain, has to do with their effectiveness in allowing
electron cycling around PSII, as measured by thiol inhibition of
ferricyanide site 2. We are not sure at which point the PSI cyclic
pathway joins the main electron transport chain after the plasto-
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1981 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 67, 1981
BARR AND CRANE
1192
Table I. Inhibition or Stimulation of PSII Ferricyanide Reduction by Various Thiols
Stimulation or Inhibition of Electron Transport
Concn.
Thiol
FC(DMB
FeCN(+DBMIB)
FeCN
(pH 6)
MV(+azide)(pH 7)
ratea
ratea
ratea
%b
%b
,MM
450
500
300
Control
544
+9
495
5
150
-50
1-Octane thiol
390
-22
450
7.5
150
-50
Cyclohexyl mercaptan
-14
480
20
150
-50
430
4-Nitrothiophenol
0
680
20
150
-50
500
Cysteine
-6
585
-50
470
150
25
Thiosalicylic acid
595
-50
490
-2
50
150
Butanethiol
500
0
573
150
-50
100
Glutathione (reduced)
-35
212
-15
405
100
194
2-Mercaptoethanol
a
,eq 02/mg Chl * h.
b +, stimulation; -, inhibition of rate in relation to control. Reaction conditions and components
assay as in Figures I and 2.
+25
v FeCN, pH 6
* SM (+DCMU),
* SM, pH 7
Y FeCN (+DBMIB), pH 8
a MV (+azide),pH 7
* DMBQ(+DBMIB),pH7
o DCIP, pH 7
+10
0
+7
+51
+30
+32
+27
-10
for each
+25
0 SM (+DCMU), pH 6
pH 8
%/Ob
0
0
0
v
15
0
0
0
.25
C
d -25
-
hoCN, pH 8v
v FSCN, pH6
0 FOCN, pH8 + 2.5pM DBMIB
O
a
0
FCN, pH8 + 5OpM BP
FOCN, PHS +1.25pM DNP-INT
50
C
0
E
A
-75
-100
0
l
50
-100
100
150
200
250
Bathophenanthroline (pM)
FIG. 3. The effect of BP on various electron transport reactions in
isolated spinach chloroplasts. Reaction mixtures as in Figures 1 and 2.
Control rates for each reaction shown in the figure, from top to bottom,
left column first, then the right one, were as follows: 169, 113, 259, 496,
418, 203, 1150, 626 ,leq/mg Chl.h. +, Stimulation; -, inhibition of rate in
relation to control.
quinone pool, so that a branch point for the PSI cycle may be
better detected by DNP-INT than by BP or DBMIB. Another
possible explanation for the observed facts is that DNP-INT or
BP inhibition is reversed more easily by thiols than DBMIB
inhibition in this case. Reimer and Trebst (9) made the initial
observation that thiols can reverse DBMIB inhibition on the H20
NADP+ pathway. The relationships between thiol inhibition
or reversal of inhibition are tenuous at best, because the order of
addition, length of incubation period, and concentration of reagents make a difference (9). We have also found that the combination of reagents with a thiol, DBMIB and ferricyanide has to
0
25
50
75
100
Thiosalicylic Acid (pM)
FIG. 4. A comparison of thiol inhibition of ferricyanide reduction in
PSII of spinach chloroplasts, when forward electron transport is blocked
by different inhibitors. -, % inhibition of electron transport rate obtained
by blocking forward electron flow with DBMIB, DNP-INT or BP. Reaction mixtures at pH 6 and at pH 8 as in Figure 1. The control rate was 293
,ueq/mg Chl h at pH 6 and 316 at pH 8.
be handled with special caution. For example, a thiol, such as
TSA, cannot be used in concentrations higher than 100 UM,
because it reduces ferricyanide chemically, so that less oxidized
ferricyanide remains in the reaction mixture to act as an electron
acceptor. The chemical interactions between these reagents can be
minimized by incubating TSA or other thiols with chloroplast
membranes for 3 min before the addition of ferricyanide or other
electron acceptors. There are differences in the concentrations of
various thiols required to give 50%o inhibition of the H20 -* FeCN
(+DBMIB) pathway at pH 8, as shown in Table I. From this
table, it is also obvious that lipophilic thiols, such as I-octanethiol,
are inhibiting in lower concentrations than others. Water-soluble
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1981 American Society of Plant Biologists. All rights reserved.
FERRICYANIDE REDUCTION IN PHOTOSYSTEM II
Plant Physiol. Vol. 67, 1981
1193
2250
E300
E
6 50
0
%.2
~200
i75
0
1
0
C 1 2 3 4
8
6
4
Trypsin (min.)
FIG. 7. Comparison of ferricyanide reduction rates at two different
C 1 2 3 4
FIG. 5. Comparison of ferricyanide reduction rates at two different
PSII sites in various fractions isolated from discontinuous sucrose gradient
centrifugation. A, FeCN reduction at pH 6; B, at pH 8 in presence of 2.5
p.M DBMIB. Reaction mixtures for A contained chloroplasts (50 ,tg Chl),
25 mi Tris-Mes (pH 6), 5 mM NH4Cl, and 250 ,UM FeCN; for B chloroplasts
and NH4Cl as above, buffer at pH 8 instead of 6; in addition, 2.5 uM
DBMIB and 0.5 mM FeCN present.
+100
MV (+azide), pH 7
v FeCN, pH 6
FeCN, (+DBMIB), pH 8
0
8
+500
0
'4-
-1000
0
5
10
15
Chloroplast Fragmentation (min.)
FIG. 6. Comparison of ferrcyanide reduction rates at two different
PSII sites in fragmented chloroplasts. The pH 6 and pH 8 FeCN sites were
assayed as in Figure 5. The H20 --l MV reaction mixture contained
chloroplasts, 25 mm Tris-Mes (pH 7), 0.5 mm Na azide and 0.5 mm MV.
The control rate for H20--+ FeCN (pH 6) was 581 ,uEq/mg Chl-h; for
H20-3- FeCN (+DBMIB), pH 8-474, and for H20 --) MV- 1032. +,
Stimulation; -, inhibition in relation to control rates.
2
PSII sites in trypsin-treated chloroplasts. The reaction mixtures for the
various reactions as in Figure 6. The control rate for H20 -. FeCN (pH
6) was 519 Leq/mg Chl *h, for H20 -* FeCN (+DBMIB), pH 8-305, and
for H20 -* MV-802. +, Stimulation; -, inhibition of rate in relation to
control.
compounds, such as 2-mercaptoethanol, presumably do not reach
the active site of DBMIB-insensitive ferricyanide reduction in
PSII, because they don't give 50%o inhibition of this pathway,
regardless of concentration.
Another approach in investigating the two ferricyanide reduction sites in PSII was to attempt their physical separation by
mechanical means. Here we employed the discontinuous sucrose
density gradient centrifugation technique, fragmentation, and
fragmentation combined with sucrose density gradient centrifugation. The original aim was to see if fragmentation of chloroplasts
selectively destroyed one or the other of the two ferricyanide
reduction sites. As can be seen in A in Figure 5, ferricyanide
reduction at pH 6, which measures the activity of the site on the
main electron transport pathway, decreased from 30 to 50%o in the
various sucrose gradient fractions. Site 2 ferricyanide reduction,
the DBMIB-insensitive activity measured at pH 8, increased in all
fractions, except in fraction 1, as shown in B of Figure 5. The
stimulation of activity in these fractions indicates that ferricyanide
site 2 became more accessible, possibly due to stripping of chloroplast outer membranes in the gradient. Fragmentation as the
basis for stimulation of activity at this site can be excluded,
because lighter material was expected to collect itself in fraction
1 of the gradient.
Ferricyanide is a nonpenetrating reagent. It is generally assumed
that PSII is found on the inside of the thylakoid membranes (1 1).
However, a PSII redox loop in the vicinity of Q, the primary
electron acceptor of PSII, is more easily accessible from the outside
than the water oxidation end of PSII (6). Ferricyanide reduction
site 2 must be located in this particular redox loop, since it is
DCMU-sensitive, but DBMIB-insensitive, indicating a location
between Q and plastoquinone A in the electron transport chain of
chloroplasts. To get stimulation of ferricyanide site 2, as in fractions 2 to 4 in Figure 5, more PSII redox loops would have to be
exposed to the outside of the thylakoid membrane.
The sucrose-NaCl chloroplasts (SN) used in this study contain
about 60%o whole chloroplasts, 35% envelope-free chloroplasts and
3% chloroplast fragments (unpublished data). To test if fragmen-
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1981 American Society of Plant Biologists. All rights reserved.
BARR AND CRANE
1194
tation of chloroplast membranes could lead to increased ferricyanide site 2 activity, chloroplasts were expelled from a syringe for
specified time periods (Fig. 6). Mild fragmentation of chloroplasts
for 5 min stimulates the DBMIB-insensitive ferricyanide reduction
at pH 8 (Fig. 6). At this point, about two-thirds of the fragmented
chloroplast membranes remain in the top fraction on sucrose
density gradient centrifugation, indicating that they have been
fragmented efficiently. No further separation of the fragments was
made, but it is clear that fragmentation can be used as a means to
stimulate ferricyanide site 2 activity.
Additional proof, that DBMIB-insensitive ferricyanide reduction is located on a redox loop close to the thylakoid membrane
surface, comes from trypsin digestion studies. Earlier studies by
Renger (10) showed that ferricyanide reduction lost its sensitivity
to DCMU after trypsin digestion. After an 8-min incubation
period with trypsin only about 50%o of the DCMU-sensitive rate
has been lost, as measured in the ferricyanide reduction site I
pathway at pH 6. However, this correlates with a total loss of
DBMIB-insensitive ferricyanide reduction at pH 8 (Fig. 7). Even
during a 2-min incubation period, more than 50%o site 2 activity
has been destroyed. This again emphasizes the fact that PSII
ferricyanide site 2 protrudes through PSI, which is located on the
outside of thylakoid membranes. The data presented here also
support a zig-zag arrangement of electron transport components
across the thylakoid membrane.
Acknowledgments-We thank Dr. R. A. DiLley for providing fresh spinach every
week and Dr. A. Trebst for supplying DNP-INT.
LITERATURE CITED
1. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in
Beta vulgaris. Plant Physiol 24: 1-15
Plant Physiol. Vol. 67, 1981
2. BANASZAK J, R BARR, FL CRANE 1976 Evidence for multiple sites of ferricyanide
reduction in chloroplasts. J Bioenerget 8: 83-92
3. BARR R, FL CRANE 1976 Organization of electron transport in photosystem II of
spinach chloroplasts according to chelator inhibition sites. Plant Physiol 57:
450-453
4. BARR R, KS TROXEL, FL CRANE 1980 EGTA, a calcium chelator, inhibits
electron transport in photosystem II of spinach chloroplasts at two different
sites. Biochem Biophys Res Commun 92: 206-212
5. DORING G 1976 The chlorophyll al, reaction in trypsin-treated spinach chloroplasts in the presence of potassium ferricyanide. Z Naturforsch 3 lc: 78-81
6. GIAQUINTA RT, RA DILLEY, BR SELMAN, BJ ANDERSON 1974 Chemical modification studies of chloroplast membranes. Water oxidation inhibition by
diazoniumbenzene sulfonic acid. Arch Biochem Biophys 162: 200-209
7. HAUSKA G 1977 Artificial acceptors and donors. In A Trebst, M Avron, eds,
Encyclopedia of Plant Physiology, Vol 5, Photosynthesis I. Springer-Verlag,
Berlin, pp 253-265
8. MALKIN R, PJ APPARICIO 1975 Identification of a g = 1.90 high-potential ironsulfur protein in chloroplasts. Biochem Biophys Res Commun 63: 1157-1160
9. REIMER S, A TREBST 1976 Reversal of dibromothymoquinone inhibition of
photosynthetic electron flow by thiol compounds. Z Naturforsch 3 lc: 103
10. RENGER G 1976 Studies on the structural and functional organization of system
II of photosynthesis. The use of trypsin as a structurally selective inhibitor at
the outer surface of the thylakoid membrane. Biochim Biophys Acta 440: 287300
1 1. SANE PV 1977 The topography of the thylakbid membrane of the chloroplast. In
A Trebst, M Avron, eds, Encyclopedia of Plant Physiology, Vol 5, Photosynthesis I. Springer-Verlag, Berlin, pp 522-542
12. SIRECI JE, A PLOTNER, R BARR, FL CRANE 1978 Selective thiol inhibition of
ferricyanide reduction in photosystem II of spinach chloroplasts. Biochem
Biophys Res Commun 85: 976-982
13. TREBST A, E HARTH, W DRABER 1970 On a new inhibitor of photosynthetic
electron-transport in isolated chloroplasts. Z Naturforsch 25b: 1157-1159
14. TREBST A, H WIETOSKA, W DRABER, HJ KNOPS 1978 The inhibition of photosynthetic electron flow in chloroplasts by the dinitrophenylether of bromo- or
iodo-nitrothymol. Z Naturforsch 33c: 919-927
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 1981 American Society of Plant Biologists. All rights reserved.