Velthuys, B.R.

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Electron and proton transfer events in chloroplasts during a short series of flashes 

Volume 115, number 2 FEBS LE’M-ERS June 1980 
ELECTRON AND PROTON TRANSFER EVENTS IN CHLOROPLASTS DURING A SHORT 
SERIES OF FLASHES 
B. R. VELTHUYS 
Martin Marietta Laboratories, 1450 South Rolling Road, Baltimore, MD 21227, USA 
Received 21 April 1980 
1. Introduction 
The popular ‘vectorial Z’ scheme of green plant 
photosynthesis is unable to account for a number of 
observations concerning the translocation of protons 
and the role of plastoquinone and cytochromes in 
the rate limiting electron flow between the photosys- 
terns [ 11. A recently-proposed recplacement scheme 
modifies it to incorporate asite of proton transloca- 
tion located between plastoquinone and plastocyanin 
[ 1,2]. Evidence for this new scheme was obtained by 
measuring the flash-generated transmembrane el c- 
trical field, the proton uptake, and the redox reac- 
tions of cytochrome fand cytochrome b6, and by 
noting how these phenomena re affected by pre- 
reduction of the plastoquinone pool [2,3]. 
This paper examines more closely the electron and 
proton transfer events in dark-adapted chloroplasts 
exposed to a series of short saturating flashes. The 
data illustrate the field-indicating A.4s1s, the cyto- 
chrome f redox change, and the proton release inside 
the thylakoid measured by the neutral-red technique 
de&loped in [4,5]. The results confirm the involve- 
ment of the contended ‘extra’ site of proton translo- 
cation in linear electron flow and thereby support 
the earlier proposed scheme. 
2. Methods 
Broken chloroplasts were prepared by standard 
procedures [3], suspended at 5 mg chl/ml in medium 
containing 400 mM sucrose, 2mM KCl, 0.5 mM 
MgClz and 1 mM IV-2-hydroxyethyl-piperaine-N’-2- 
ethanesulfonic acid (Hepes)-NaOH (pH 7.45), and 
stored dark and cold (4’C). About 30 s before each 
measurement, a 5 ~1 sample was diluted 50-fold with 
ElsevierfNorth-Holland Biomedical Press 
reaction mixture of room temperature. The standard 
reaction mixture contained 400 mM sucrose (omitted 
in fig.l), 100 J.LM methylviologen, and 1 mM Hepes- 
NaOH (pH 7.45). Absorbance changes were induced 
by 4 consecutive flashes (at 1.2 s intervals) and mea- 
sured as in [2,3 1. (By applying the measuring beam 
only during measuring periods, a relatively high inten- 
sity could be used while still keeping its total actinic 
effect insignificant.) A small home-built computer 
(based on Heathkit ET 3401, with 4 kilobytes of 
RAM, and audio cassette mass torage), interfaced 
with the transient recorder (Biomation 802), was 
used for signal processing (storage, additions, sub- 
tractions). 
Proton release inside the chloroplast thylakoids 
was measured at 553 nm as the difference between 
alternate measurements with and without 20 JLM 
neutral red added to the standard reaction mixture. 
This method is based upon that in [5], i.e., added 
buffer of pH -7.4 easily overrides buffering (and 
color change) by neutral red in the outside medium, 
but not inside the thylakoid where neutral red is 
bound and thereby accumulated. 
Cytochrome f behavior was measured by its 
spectral changes [6] as the difference between alter- 
nate measurements with 553 nm and with 540 nm 
measuring light. Changes of the transmembrane 
electrical field were measured by its electrochromic 
effect [7] as the difference between alternate mea- 
surements with 5 15 nm and with 500 nm measuring 
light. 
3. Results and discussion 
3.1. Proton release by PS 2 
Two processes contribute to the proton release 
167 
Volume 115, number 2 FEBS LETTERS June 1980 
r//f F $L ,0-b ----- 1 
Fig.1. Proton release inside chloroplast hylakoids, recorded 
as absorbance increase of trapped neutral red, during the 
fust 4 ms after each of 4 flashes spaced by 1.2 s intervals. 
Average difference of 64 pairs of measurements with and 
without 20 pM neutral red. Dark time before the measure- 
ments, 2-6 h. Electronic time response (lo-90%), 100 US. 
Measuring beam wavelength, 553 mn (10 nm half band 
width). Optical pathlength, 2 mm. Chlorophyll (chll 100 
rg/ml. 
inside the thylakoid: water oxidation by PS 2, and 
electron transfer between PS 2 and PS 1 [4,8,9]. 
Because intersystem electron transfer is slow com- 
pared to the donor side reactions of PS 2, the contri- 
butions to H’ release of PS 2 and of the intersystem 
chain can be distinguished kinetically [4]. The more 
rapid components of H+ release are shown in fig.1. 
The four different curves represent he ms-range 
H’ release induced by 4 consecutive flashes. The 
results show a large dependence on the flash number. 
As argued in [4,9,10], the probable cause of the 
flash-number dependency is that with consecutive 
photoreactions the donor side of PS 2 steps through 
a cycle of 4 states, called Se, Sr, SZ, Ss [ll]; in a 
flash series after dark-adaptation this cycling occurs 
to a large extent synchronously, with 275% of the 
centers starting in state Sr [ 111. The first flash 
induces very little H’ release; a maximum is obtained 
with the third flash (and in a longer series, not shown, 
a second minimum is obtained with the fifth and a 
second maximum with the seventh flash). Assuming 
that the individual S-state transitions cause the 
release of whole numbers of protons, and recognizing 
that their sum for a full cycle must be 4, we derive 
from the results that during the transitions of 
Se + S, + SZ + Ss + Se protons are released.accord- 
ing to a 1, 0, 1,2 pattern. This conclusion supports 
[9,10] versus [4]. Some conclusions in [4],, however, 
are upheld by fig.1, As in [4], <lo0 ~.ls half-time is 
observed for H+ release during the SZ-Ss transition 
(second flash) and -1 ms for the H+ release during 
the Ss-Se transition (third flash, the fastest com- 
ponent in the clearly biphasic release should probably 
be ascribed to a minority of centers undergoing 
Sz43). 
3.2. Total proton release inside 
Fig.2A represents an experiment similar to that of 
fig.1, but this time the measurements were extended 
over a time-span sufficiently long to observe not only 
the H’ release by PS 2 but also most of the H’ release 
associated with electron flow between PS 2 and PS 1. 
Since fig.1 showed that the PS 2 contribution to H’ 
release is negligible with the first flash, curve no. 1 
of fig2A should represent exclusively intersystem 
H’ release. To help recognize the separate contribu- 
tions of PS 2 and of the intersystem chain to the 
H’ release induced by flashes 2-4, curve 1 was sub- 
tracted from curves 2-4, resulting in curves 2’-4’ 
(fig.2B). These curves are conspicuously biphasic. 
The fast (few ms) phase should represent he H’ 
release by PS 2; and their secondary phase, the 
intersystem H’ release occurring in excess of that 
after the first flash. Fig2B clearly reveals two fea- 
tures of the measurement: 
(i) The intersystem H’ release oscillates with a 
periodicity of two, confirming [8,9]; 
B 
2’ 3’ 4’ 
crl 
3- - 
74 
l$+ 3 
--2 
--I 
-0 
2oo;;ls 
Fig.2. Proton release inside chloroplast thylakoids, recorded 
as absorbance increase of trapped neutral red, during the 
first 220 ms after each of 4 flashes spaced by 1.2 s intervals. 
Average difference of 32 pairs of measurements, with and 
without 20 PM neutral red. Dark time before the measure- 
ments, l-7 h. Electronic time response, 1 ms. Measuring 
beam wavelength, 553 mn (3 mm half band width). Curves 
2'-4' represents the difference between curves 2-4 and 
curve 1. The assumptions underlying the (w/e) scale at the 
right ordinate are discussed in the text. 
168 
Volume 115, number 2 FEBS LETTERS June 1980 
(ii) The number of protons released with intersystem 
flow (remember to add back curve 1 to each 
secondary phase in fig.2B) is appreciably larger 
than the number of protons released by PS 2. 
The oscillation indicates that the extent of inter- 
system flow is limited by the supply of electrons 
(to the plastoquinone pool) by the secondary 
acceptor R of PS 2 [12,13]. Under such conditions 
the average relative amplitudes of the H’ release by 
PS 2 and by intersystem flow should be 1: 1 according 
to the ‘classical’ Z-scheme ( .g., [4,7]) but 1:2 accord- 
ing to the ‘modified’ Z-scheme (e.g., [ 1,3]). The 
results of fig.2 clearly favor the latter scheme. Based 
on the I:2 stoichiometry, the tentative H’/e scale for 
the right ordinate of fig.2 assumes a release of 1.5 H+ 
by the first flash. The indicated average H’ release 
by PS 2 should be -1, and the given scale is reason- 
ably satisfactory in this respect. 
3.3. Electron flow into cytochrome f 
Measurements of cytochromefare shown in fig.3. 
The results further document the occurrence of an 
oscillation of intersystem electron flow under the 
conditions of dark adaptation and illumination used 
in this study. Superficially, at least, the correspon- 
dence with the &release measurements is quite good. 
Like H’ release, cytochrome f re-reduction appears 
to be twice as large after the second and the fourth 
flash than after the first and the third flash. On the 
other hand, the amplitude of the observed turnover 
23s 
y 2x10-4 
I 
Fig.3. Oxidation (downward) and re-reduction of cyto- 
chrome f in chloroplasts during the fust 220 ms after each of 
4 flashes spaced by 1.2 s intervals. Average of 32 pairs of 
measurement (553 nm minus 540 nm). The transmission 
increase of 2 X lo-’ represents a reduction of 0.1 molecule 
cytochromef/400 molecules chl (calculated as in (31). Dark 
time before the measurements, l-5 h. Addition to standard 
reaction mixture, 1 mM NaCl and 2 pM gramicidin D. 
of cytochromefis small, an av. -0.15 molecule .400 
chl-r . flash-‘. The plausible interpretation isthat 
cytochromefrapidly equilibrates with plastocyanin, 
a component of larger abundance than cytochrome 
fand of similar midpoint potential (e.g., [14]) - even 
though this interpretation isnot supported in detail 
by actual parallel measurements of plastocyanin and 
cytochromef [15,16]. 
3.4. Generation of transmembrane field 
Fig.4 shows the increase and the decay of the 
transmembrane el ctrical field under the conditions 
of fig.2. At first sight, it appears that all of the field 
increase occurs immediately with each flash and 
thus is completely due to the primary reactions of 
the photosystems [7]. According to the modified 
Z scheme, however, intersystem flow generates field 
also [ I-31. This field generation by secondary elec- 
tron flow is easily recognized when it occurs rapidly 
[2,13], but when intersystem electron flow is slow, 
as under the conditions of fig.2-4, the (supposed) 
associated field generation is hard to detect because 
of the appreciable field decay that takes place con- 
currently. 
The oscillation in intersystem flow demonstrated 
by fig.2,3 helps to make the secondary phase vident. 
Close examination of fig.4 shows that the decrease 
of the field after the primary rise is slower and more 
clearly sigmoidal after the second and the fourth 
flash than after the first and the third flash. The 
flash-number dependency isperceived better than 
A B 
------ 
v”tc,*- 
A 6 
.t, 
200 ms 
Fig.4. Transmembrane lectrical field increase (upward) and 
decrease, recorded as electrochromic absorbance changes, 
during the first 220 ms after each of 4 flashes spaced by 
1.2 s intervals. Average of 16 pairs of measurements (5 15 run 
minus 500 mn). Dark time before the measurements, 4-5.5 h. 
Chlorophyll, 60 &nl. Curves 2’-4’ represent he difference 
between curves 2-4 and curve 1. 
169 
Volume 115, number 2 FEBS LETTERS 
with the original curves after subtracting curve 
1 from curves 2-4, which produces curves 2’-4’. 
Besides a decrease of the amplitude of the imme- 
diate field increase (which suggests an incom- 
plete recovery, between flashes, of the photocenters 
of F’S 2 or of PS l), a slow phase is evident that is 
much larger in curves 2’ and 4’ than in curve 3’ 
of fig.4. This result agrees nicely with the measure- 
ments of H’ release, i.e., the manifestation of a slow 
phase in curves 2’ and 4’ that is much larger than in 
curve 3’ of fig.2. Kinetically the correspondence is 
imperfect, but this is not surprising as the curve 
manipulation that leads to curves 2’ and 4’ of fig.4 
only diminishes, but does not eliminate, the effect 
of field decay. 
Acknowledgements 
This work was supported by the US Department 
of Agriculture (Competitive Research Grants Office), 
grant 5901-0410-8-0179-0, and by the Department 
of Energy, contract DE-ACO-2-76ER03326. 
[4] Junge, W. and Ausliinder, W. (1978) in: Photosynthetic 
Oxygen Evolution (Metzner, H. ed) pp. 213-228’; 
Academic Press, London, New York. 
15 ] Junge, W., AusGnder, W., McGeer, A. J. and Runge, T. 
(1979) Biochim. Biophys. Acta 546,121-141. 
[6 ] Bendall, D. S., Davenport, H. S. and Hill, R. (1971) 
Methods Enzymol. 23A, 327-344. 
17 ] Witt, H. T. (1979) Biochim. Biophys. Acta 505, 
355-427. 
June 1980 
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170 


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Electron and proton transfer events in chloroplasts during a short series of flashes

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