Section I

Principles of Photosynthesis

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 Mechanisms of Photosynthetic Oxygen
1 Evolution and Fundamental Hypotheses
of Photosynthesis
Yuzeir Zeinalov
Institute of Biophysics, Bulgarian Academy of Sciences

CONTENTS

I. Introduction
II. The Concept of Photosynthetic Unit
A. Fundamental Results
B. Problems and Hypotheses
C. Variation in the Number of Effectively Functioning Oxygen-Evolving (Reaction) Centers
III. The Concept of Two Photosystems
A. Experimental Grounds
B. Photosynthesis with Sole Photosystem
IV. Conclusion
Acknowledgments
References

It is generally believed that these two principal

I. INTRODUCTION
Intensive investigations on the nature of photosyn-
thetic light reactions during the first half of the 20th
century led to several important discoveries and obser-
vations that were extremely complicated to explain
and resulted in the postulation of two fundamental
concepts: the concept of photosynthetic unit (PSU)
[1] and the concept of two photosystems [2]. According
to the first concept, in all photosynthesizing systems
(photosynthesizing bacteria, green unicellular algae,
and higher plants), the light-absorbing pigment
molecules are divided into two groups. Only one
highly specialized pair of chlorophyll molecules (reac-
tion center dimer) present among dozens of bacteria
and among hundreds of green photosynthesizing sys-
tems could carry out the photochemical (charge separ-
ation) reaction, while the essential part of these
molecules only absorbs light quanta and transfers the
light energy to the reaction centers [1]. According to
the second concept, the light-induced linear electron
transfer reaction of H2O to NADP is realized by the
serial operation of two different photosynthesizing
systems [2].
concepts are completely proven and verified and the
unsolved problems are connected with the elucidation
of the nature of participating components and their
mutual relationship. This chapter deals with the
basic experiments and results that have led to the
concept of the PSU and to the postulation of
the concept of photosystems in light-driven photosyn-
thetic reactions and shows that, at the time of their
postulation, the existing results and observations were
not sufficient.
II. THE CONCEPT OF PHOTOSYNTHETIC
UNIT
A. FUNDAMENTAL RESULTS
There is a limited number of experimental data that
scientists consider as crucial for the postulation of a
given concept. For the concept of the PSU, the fol-
lowing results and observations are significant:
(1) The very high (maximum) quantum efficiency
of photosynthesis under limited light intensity condi-
tions, that is, when the probability for light quanta

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absorption of a chlorophyll molecule is about one
quantum per hour.
This statement has been confirmed by investiga-
tions of the dependence of photosynthesis on light
(irradiance). It was shown in many experiments that
the photosynthetic response to very low light inten-
sities was linear (Figure 1.1, curve A). In a significant
number of experiments, the shape of light–response
curves had a logarithmic part (Figure 1.1, curve B)
with maximum slope (maximum quantum efficiency)
at the beginning of curves, that is, when the irradi-
ation was approaching zero. ‘‘S’’-shaped curves (Fig-
ure 1.1, curve C), which indicate that the quantum
efficiency under low light intensities tends toward
zero, were observed in a limited number of investiga-
tions (for review of the early investigations, see [3]).
These ‘‘S’’-shaped curves obtained in green plants
were interpreted in favor of the assumption of the
existence of a ‘‘photic threshold’’ of photosynthesis.
However, this suggestion was not accepted and the
results obtained by most researchers were in favor of
the linear shape of the light curves of photosynthesis.
Under anaerobic conditions, Diner and Mauzerall [4]
also observed nonlinear dependence. After the postu-
lation of the concept of the PSU, it was discovered
that the initial slope of the light curves below the light
compensation point was significantly higher, and
nearer to this point on the light curves an abrupt
change in the value of quantum efficiency of photo-
synthesis could be observed [5]. This observation is
called ‘‘Kok’s effect’’ and was explained by the
changes in the rate of dark respiration after irradi-
ation.
(2) The absence of induction period in the process
of oxygen evolution or carbon dioxide reduction
under very low light intensity conditions was one of
the most serious arguments of the PSU concept (Fig-
ure 1.2). Five oxygen induction curves were recorded
Oxygen-evolution rate (a.u.)
B vals between the flashes. The dependence of the oxy-
A
C
Light intensity (a.u.)
FIGURE 1.1 Different shapes of photosynthetic ‘‘light
curves’’: A, linear; B, logarithmic; C, ‘‘S’’-shaped irradiance
dependence of photosynthesis.
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A
A
Oxygen-evolution rate (a.u.)
B B
C
D C
E
ED
0 45 90
Time (sec) Irradiance (a.u.)
FIGURE 1.2 Oxygen induction curves recorded at different
irradiances after 3 min of dark adaptation (left) and the
respective ‘‘working points’’ on the ‘‘light curve’’ (right) in
Chlorella pyrenoidosa suspension with absorbance 0.05. In-
duction curves A, B, and C are recorded at 8  108 A/mm
and curves D and E at 1.2  109 A/mm sensitivity of the
polarograph (for details see text).
at different irradiances after 3 min of dark adaptation
of Scenedesmus acutus cell suspension. Curve A was
recorded at the maximum irradiance, I0 ¼ 135 W/m2,
corresponding to the oxygen-evolution rate close to
saturation (Figure 1.2, right panel point A). Other
curves were recorded at 0.76I0 (B), 0.46I0 (C), 0.19I0
(D), and 0.056I0 (E). The induction curves indicate
that the duration of the induction period decreased
simultaneously with decrease in irradiance. Under the
lowest irradiance, 0.056I0 (E), the rate of oxygen
evolution reached its steady state immediately after
the light was switched on. This observation is in
agreement with the postulate that at low irradiances
photosynthesis starts before the absorption of the
four quanta needed for the evolution of one oxygen
molecule.
(3) Oxygen flash yields depend on the dark inter-
gen flash yields on the spacing between the saturating
flashes was investigated for the first time by Emerson
and Arnold [1] with Warburg’s manometric appar-
atus. It was found that the average yields were max-
imal when intervals between the flashes were about
20 msec.
The dependence of oxygen yields produced by
separated flash groups (four saturating short flashes)
on the spacing between the flashes in groups and
recorded after reaching steady-state yields is pre-
 3500

Amplitudes of oxygen bursts (mV)

0.1msec 0.4msec 1msec 4msec 10msec 20msec 40msec 100msec 400msec 1000msec

3000

2500

2000

1500

6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15
Time (sec)

FIGURE 1.3 The steady-state oxygen yields (relative) of groups of four saturating flashes depending on the time between the
flashes in the groups in Scenedesmus obliquus suspension with absorbance 0.05 (100 mm3 sample volume). The groups of four
saturating flashes (4J, t1/2 ¼ 8 msec) are spaced 3 sec and after reaching the five steady-state several oxygen group yields
obtained at different spacing between the flashes are presented.

sented in Figure 1.3. It is clearly seen that ampli-

650
tudes of oxygen flash yields increase with increase in
3
spacing between flashes up to 10 to 20 msec, after

Oxygen flash yields (mV)

600
which the yields decrease. Results presented in Figure
4
1.3 confirm the turnover time of oxygen-evolving 7 8
centers (2  102 sec) estimated by Emerson and 550 12
11
Arnold [1]. 9 13 15
14
500 10
(4) When oxygen flash yields are maximal, the ratio 5
6
between oxygen molecules evolved per flash and the
number of chlorophyll molecules in the investigated 450 1 2 Dark level
suspensions is approximately constant and equal to
1O2/2500Chl. For the first time Emerson and Arnold 400
obtained this value in 1932. It was found that in Chlor-
0 5 10 15
ella pyrenoidosa suspensions with different chlorophyll Time (sec)
concentrations 4  104 M oxygen was evolved from
1 M chlorophyll after every flash.
FIGURE 1.4 Oxygen flash yields of isolated pea chloroplast
(5) Earlier studies [6–8] demonstrated that after induced by a series of 15 flashes (4 J, t1/2 ¼ 10 msec, spaced
approximately 5 min of dark adaptation of unicellu- 800 msec).
lar algae (e.g., Chlorella, Scenedesmus) or isolated
chloroplast suspensions, the oxygen yield of the first
saturating short (10 msec) flash is zero (Figure 1.4). light quanta energy and consequently at least four
(6) Oscillations in the oxygen flash yields photons are needed. For understanding and explain-
(Figure 1.4) with a period of four observed after 5 to ing the observed experimental results the following
6 min of dark incubation in algae or chloroplast principal questions arise:
suspensions [9].
At the time of the postulation of the PSU concept 1. Whether energy or photoproducts of the four
only the first four experimental observations were photons absorbed are summarized?
known. Observations 5 and 6 were obtained signifi- 2. Whether oxygen-evolving centers act independ-
cantly later and are considered as additional confirm- ently of each other or can exchange energy,
ations of the concept of the PSU. or whether the oxygen precursors (positive
charges) could migrate and cooperate in the
B. PROBLEMS AND HYPOTHESES surrounding medium?

Considering the general equation of photosynthesis, it It is well known that the average effective cross
is apparent that for the evolution of one oxygen section for light quanta absorption of a chloro-
molecule or for the reduction of one carbon dioxide phyll molecule in solution is approximately 0.2 
molecule to the level of carbohydrate, four electrons 1016 cm2. This means that under low irradiances,
should be transferred on account of the absorbed that is, 1013 to 1014hg cm2, the time needed for

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absorption of four quanta by separated chlorophyll
molecules should be approximately 1 h. Under such
conditions, if oxygen-evolving centers act independ-
ently of each other, the evolution of photosynthetic
oxygen should start after a prolonged induction time.
This is in contradiction with observation 2 and the
results presented in Figure 1.2, which show that in
reality photosynthesis starts immediately without any
induction period. The flash experiments of Emerson
and Arnold (observation 3) show that photosynthesis
decreases if the spacing between the flashes is higher
than 0.02 sec (Figure 1.3). Therefore, at low irradi-
ances, when the dark intervals between the quanta
absorption are of the order of minutes, the effective-
ness of photosynthesis should be much lower or tend-
ing toward zero. This fact is in contradiction with
observation 1, which reflects that quantum efficiency
of photosynthesis is very high under low irradiance
conditions. Observation 3 as well as additional obser-
vations 5 and 6 lead to the conclusion that oxygen-
evolving centers operate independently of each other
(noncooperative mechanism). This means that every
oxygen-evolving center should accept four light
quanta (photons) before evolving one oxygen mol-
ecule. The results observed could be explained if we
assume that oxygen-evolving reaction centers are in a
state to conserve some of the oxygen precursors (e.g.,
positive charges) for several minutes or even hours,
and upon subsequent illumination after absorption of
the first photons they could immediately start evolv-
ing oxygen. This assumption, however, is in contra-
diction with observations 3, 5, and 6. Hence, we
should conclude that the oxygen precursors are un-
stable in the dark and deactivate for about 100 sec.
If oxygen precursors are unstable in the dark, the
observed results, that is, the absence of prolonged
induction time and high quantum efficiency of photo-
synthesis under low light intensities, could be
explained by the assumption that even under limited
light conditions the oxygen-evolving centers received
photons for time intervals of about seconds or even
shorter. This assumption could be explained by an
additional speculation that hundreds of chlorophyll
molecules are functionally or even structurally assem-
bled around a given specialized chlorophyll molecule
(named reaction center), which carries out the photo-
chemical reaction and this center is supplied with the
photons absorbed by the assembled light-harvesting
(antenna) molecules. In this way, the effective cross
section of light quanta absorption of the reaction
center molecule is increased 100-fold and even under
very low light intensity conditions reaction centers
received the needed four quanta for intervals 100
times shorter than the intervals of the separated
chlorophyll molecules. This assumption explains
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both the absence of the prolonged induction period
and the high quantum efficiency under low light in-
tensities. In good agreement with this assumption is
observation 4, where after every saturating flash only
one oxygen molecule is produced from approximately
2500 chlorophyll molecules. This attractive hypoth-
esis was postulated by Emerson and Arnold in 1932
and was accepted immediately. Since then this postu-
late has been supported in many investigations and
especially with the findings of observations 5 and 6.
However, a significant number of investigations have
shown discrepancies concerning the size and structure
of the postulated PSUs [10–12]. This leads to a more
ticklish question: Are the above-considered basic ar-
guments sufficient for the postulation of the PSU
concept? Careful analysis of these arguments shows
that difficulties for a logical explanation of experi-
mental results arise from observations 3, 5, and 6,
that is, from the absence of oxygen burst or oxygen
yield at the first flash given after several minutes of
dark incubation. These observations have led us to
reject the presence of any cooperative mechanism in
the action of oxygen-evolving centers. The existence
of a noncooperative mechanism of oxygen evolution
in photosynthesis has been confirmed by observations
5 and 6 as well as by numerous flash experiments.
Especially, the model of Kok et al. [13] or the Si-states
model shows that the noncooperative mechanism
could explain both the absence of oxygen flash yield
at the first saturating flash after prolonged dark incu-
bation and the oscillations in oxygen flash yields with
a period of four.
In spite of this, a number of kinetic models have
been proposed for the explanation of various compli-
cating phenomena of the oxygen flash yield oscilla-
tions [14–16]. According to Lavorel [17,18], a special
kind of cooperative action exists in the functioning of
Si-states. In addition, there are some experimental
results that cannot be explained by Kok’s model
(e.g., the linearity of the light curves under very low
irradiance conditions). Obviously, the existence of the
noncooperative mechanism of oxygen evolution does
not exclude the participation and the existence of the
cooperative mechanism. On the other hand, the ab-
sence of oxygen flash yield after the first flash cannot
be considered as a proof for the absence of the co-
operative mechanism in oxygen evolution because of
the following reasons:
1. The first flash is applied after prolonged dark
incubation of algae or chloroplast suspensions
that leads to anaerobic conditions in cell and
chloroplast volumes.
2. Since the functioning of the cooperative mech-
anism should be realized by diffusion of oxygen
 precursors produced in different oxygen-
evolving centers, the rate constant of the reac-
tions leading to oxygen evolution through the
cooperative mechanism should be significantly
lower than the rate constant of the noncoo-
perative mechanism.
Consequently, it could be concluded that the ob-
servation of oxygen burst or oxygen production by the
first flash will be difficult and even impossible. More-
over, if we consider observation 1, that is, the linearity
of light curves under low light intensity conditions,
and observations 3 and 5, that is, the dependence of
yields on dark intervals between the flashes and the
absence of oxygen yield at the first flash, it is reason-
able to conclude that these observations are mutually
contradicting. If observations 3 and 5 reflect strictly
the photosynthetic oxygen production upon flash ir-
radiation, then even with structures like the postulated
PSUs the light curves of photosynthesis (oxygen evo-
lution) should have a nonlinear part under very low
light intensity conditions. This means that independ-
ently of the existence of photosynthetic units the light
curves of photosynthesis should be S-shaped if one
assumes that oxygen production is realized only
through the noncooperative mechanism and that the
defined deactivation reactions exist. Thus, two possi-
bilities could be considered:
1. The cooperative mechanism is functioning sim-
ultaneously with the noncooperative mechan-
ism.
2. The light curves of photosynthesis exhibit a
nonlinear part at very low light intensity con-
ditions.
The first assumption gives the explanation of the
basic arguments that have led to the postulation of
the concept of the PSU, that is, observations 1 to 3,
while observations 5 and 6 could be explained by the
functioning of the noncooperative mechanism. Obser-
vation 4 will be reconsidered in Section II.C. If we
accept the second possibility, we can explain the ‘‘red
drop’’ and ‘‘enhancement’’ effects of Emerson, which
are considered as basic observations of the concept of
two photosystems, without using this concept. Our
investigations during the last 35 years have shown
that these two possibilities exist. This means that
despite the participation of cooperative and noncoo-
perative mechanisms of photosynthetic oxygen evolu-
tion, the irradiance dependence of photosynthesis is
a nonlinear function, that is, the ‘‘light curves’’ are
‘‘S’’-shaped. Probably under low irradiance condi-
tions a significant part of the photosynthetically
evolved oxygen is consumed by dark respiration and
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under these conditions the registered light curves have
low slopes and the quantum efficiency is low.
The following observations could be considered in
favor of the cooperative mechanism:
1. In unicellular algal suspensions, prolonged (5 to
20 min) oxygen evolution is registered after
switching off the continuous irradiation.
2. Decay kinetics in oxygen flash yields are at least
biphasic, probably two different processes exist
that lead to oxygen production.
3. One cannot explain the absence of the induc-
tion period under low irradiances without the
participation of the cooperative mechanism.
4. In some photosynthesizing systems (cyanobac-
teria) one cannot register any oxygen flash
yields, despite the fact that they can produce
oxygen at a high rate under continuous irradi-
ation.
5. In our previous studies [19,20] we stressed that
the noncooperative oxygen evolving mechan-
ism operates mainly in grana regions while the
cooperative mechanism is localized predomin-
antly in stroma thylakoids.
C. VARIATION IN THE NUMBER OF EFFECTIVELY
FUNCTIONING OXYGEN-EVOLVING (REACTION)
CENTERS
If one assumes that a suspension of unicellular algae
(Chlorella, Scenedesmus, etc.) contains N0 reaction
centers, then under very general assumptions it could
be shown [21] that the following relationship exists
between the number of open reaction centers (N ) and
the rate of oxygen evolution (photosynthesis) (P):
N ¼ N0  N0 P=Pmax (1:1)
where Pmax is the saturating (maximum) rate of
photosynthesis. Obviously, the N vs. P plot is a
straight line (Figure 1.6, curve ‘‘c’’), crossing the or-
dinate at N ¼ N0 and the abscissa at P ¼ Pmax.
The experimental determination of the ratio be-
tween total and open (unoperative) centers is rela-
tively easy. According to the model of Kok et al.
[13], the oxygen-evolving centers exist in five different
oxidized states: S0, S1þ, S22þ, S33þ, and S44þ. Every
center that absorbs one photon will pass to the
next higher oxidized state. After reaching state S44þ
one oxygen molecule is produced, and the center
returns to the initial S0 state. It is easy to understand
that independently of the oxidation state, every
center after absorption of four photons separated by
dark intervals equal to or longer than the turnover
time t of the reaction centers will evolve one oxygen
molecule and attain its former state. Consequently, Figure 1.5. In contrast to our expectations, data show
the amplitudes of oxygen bursts produced by four that the amplitude of the oxygen bursts produced by
saturating flashes will reflect the number of centers the group of flashes in darkness (0) are very small and
in the unoperative (open) state. This means that if the after continuous background irradiation (1 to 4) a
flash groups are given in darkness (when all centers significant increase could be seen. On increasing the
are open) the amplitudes of bursts will reflect the total intensity of background irradiation (5 to 7) the ampli-
number of centers. tudes of oxygen bursts decrease and after reaching the
The results obtained with C. pyrenoidosa cells using saturated background irradiation (7) they are almost
excitation with groups of four saturating flashes (t1/2 invisible.
¼ 8 msec) spaced 20 msec from each other and with The relationship between the amplitudes and
7 sec dark intervals between the groups on the back- steady-state oxygen evolution is presented in Figure
ground of a gradually increasing continuous irradi- 1.6. Curve ‘‘a’’ is obtained by increasing the back-
ation with achromatic (white) light are shown in ground irradiation from zero to saturation level.
Curve ‘‘b’’ is drawn for the reverse direction, that is,
with gradually decreasing background irradiation.
7 Obviously, the difference between the two curves re-
flects an ‘‘hysteresis’’ effect and is more probably a
Oxygen yield per four flashes (a.u.)

6 consequence of the induction phenomenon in the

photosynthetic process. It should be pointed out
5
that the shapes of curves presented in Figure 1.6 are
dependent on the experimental duration and the pre-
4 ceding history of investigated alga suspensions.
3
Nevertheless, an inexplicable difference between the
straight line ‘‘c,’’ theoretically predicted on the basis
2 of the PSU concept, and curves ‘‘a’’ and ‘‘b’’ still
1 remains. The amplitudes of oxygen burst increase
under background irradiation. They reach their max-
0
imum at the level of the steady-state oxygen-evolution
Time (a.u.) rate, representing approximately one third of the
maximum value of the saturating level. Whenever
FIGURE 1.5 The amplitudes of oxygen yields (Chlorella flash groups are given under low irradiance the
pyrenoidosa) produced by four saturating flashes (4 J, t1/2 lower value of amplitudes reflects the existence of
¼ 10 msec) with 20 msec dark periods between the flashes the induction phenomenon. It is obvious that we
and 7 sec between the groups depending on the steady-state cannot estimate the exact number of reaction centers
oxygen evolution rate. The intensities of background light from amplitudes of oxygen yield under dark condi-
are: 0, 0; 1, 17.0; 2, 25.0; 3, 34.0; 4, 43.0; 5, 52; 6, 82.0; 7,
tions, that is, without background irradiation.
135 W/m2.

7 b
Number of unoperative reaction centers

FIGURE 1.6 The number of unoperative 3

(open) centers (Chlorella pyrenoidosa) de- a
pending on the oxygen evolution rate level: 2
(a) experimentally obtained results by in-
creasing the light intensity of background ir-
N 01 c
radiation from 0 to saturation level (O2 rate
from 0 to maximal [saturating-Pmax] rate);
0
(b) in the opposite direction; and (c) straight
0.00 0.25 0.50 0.75 1.00
line, predicted by the theory of the photosyn-
Oxygen-evolution rate Pm
thetic unit concept.

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Oxygen yield per four flashes (a.u.)
Oxygen-evolution rates (a.u.)
Dark level
0 5 10
Time (min)
FIGURE 1.7 Variations in the oxygen bursts before, during,
and after the induction time of photosynthesis in Chlorella
pyrenoidosa. The suspension was kept in darkness for 5 min
and the groups of four saturating flashes (20 msec spacing
between the flashes and 7 sec between the groups) were
switched on at the time indicated by ‘‘".’’ The saturated
white light (135 W/m2) was switched on at the time indicated
by ‘‘0’’ and switched off at the time indicated by ‘‘#.’’
The results presented in Figure 1.7 show the
changes in amplitudes of oxygen burst in C. pyrenoi-
dosa produced by groups of four saturating flashes
with 20 msec spacing between the flashes and 7 sec
between each flash group before, during, and after
the induction time of photosynthesis (irradiation
with saturated achromatic [white] light). These results
demonstrate well the expressed variation in oxygen
yields from flash groups and reflect in fact the number
of open reaction centers (oxygen-evolving centers).
The results presented in Figure 1.8, where the
oxygen bursts are produced by the same flash groups
as in Figure 1.7, show that the effects of flash
groups on the background saturating ‘‘white light’’
were negligible. At time 0, the ‘‘white light’’ was
switched off and the rate of oxygen evolution de-
creased sharply to the level indicated by D, after
which the process of oxygen evolution in the dark
connected with deactivation of Si states [22] or with
the deblocking of inactivated (blocked) states began.
Immediately after switching off the continuous
saturating radiation the effect of flash groups was
very small and the amplitudes of oxygen yields in-
creased slowly in the dark up to 30 min. Conse-
quently, the increase of amplitude of oxygen group
yields in the dark (after switching off the background
radiation when all centers are in the open state)
showed that the number of effectively working oxy-
gen-evolving centers increased. This number was sig-
nificantly low immediately after switching off the
saturated background radiation and thus one might
assume that it had the same low value during the
© 2005 by Taylor & Francis Group, LLC
preceding time of irradiation with saturating ‘‘white
light.’’ This means that under saturating irradiance
conditions the essential parts of the reaction center
are in the inactivated (blocked) state. The results in
Figure 1.7 show that the initial amplitudes of four
flash-induced oxygen bursts are restored approxi-
mately 15 min after switching off the continuous sat-
urating irradiation (in the darkness).
It could be shown that the following relationship
exists between the number of operating reaction cen-
ters (Nc), the amperometric current on the polaro-
15 graph equipped with oxygen rate electrode (I ), the
turnover time of reaction centers (t), and the electric
charge of an electron (e):
Nc ¼ It=e (1:2)
If one can accept the value of Emerson and Arnold [1]
for turnover time of the centers, 2  102 sec, and
for the amperometric current of saturated oxygen-
evolution rate in Figure 1.8, 1.32  105 A, the num-
ber of oxygen-evolving centers in the investigated
sample can be calculated as
Nc ¼ It=e
¼ (1:32  105 A)(2  102 sec)=1:6
 1019 C
¼ 1:65  1012 (1:3)
1.5
Oxygen yield per four flashes (a.u.)
Oxygen-evolution rate (Ax105)
Saturated oxygen-evolution rate
1.0
D
0.5
Dark level
0
0 5 10 15 20
Time (min)
FIGURE 1.8 Oxygen bursts produced by groups of four
saturating (4 J, t1/2 ¼ 8 msec) flashes with 20 msec dark
periods between the flashes and 7 sec between the groups.
Suspension of Chlorella pyrenoidosa (4 mm3, 15 mg Chl. cm3
was irradiated with saturating white light (135 W/m2) and at
the time indicated as ‘‘0’’ the saturating light was switched
off. The groups of flashes were switched on at the time
indicated by ‘‘"’’ (for details, see text).
A comparison between the number of chlorophyll
molecules (NChl) in the sample (4 mm3 with 15 mg
Chl cm3, that is, 8.8  1014 chlorophyll molecules)
and the number of oxygen-evolving centers (Nc) leads
to
P ¼ NChl =Nc ¼ 8:8  1014=1:65  1012
¼ 533Chl=1RC
If the number of chlorophyll molecules is calculated
for one oxygen molecule evolved, the value obtained
should be increased four times, that is, about 2130
for one oxygen molecule. Consequently, the value
obtained in such a way is in accordance with the
value for the PSU of Emerson and Arnold [1].
From the results presented in Figure 1.7 and Figure
1.8, it could be concluded that the number Nc, esti-
mated above, reflects only the number of effectively
working reaction centers under saturating irradiance
conditions but not their total number. An approximate
idea about the total number of oxygen-evolving cen-
ters could be obtained if we compare the amplitudes of
oxygen yields per four flashes (Figure 1.8) during the
irradiation with saturating ‘‘white light’’ with those
obtained 20 min after switching off the light: Approxi-
mately 200 to 400 times increase was registered after
switching the light off. Keeping in mind that the ratio
between chlorophyll molecules and the operative reac-
tion center under saturating irradiance conditions is of
the order of 500 one can conclude that the total num-
ber of reaction centers is practically equal to the
number of chlorophyll molecules. This indicates that
the usual procedures used for the estimation of the
number of PSUs have to be revised. There are mainly
two reasons for this:
1. Under high light intensity or frequency of sat-
urating flashes the oxygen flash yields are low
Oxygen-evolution rate (a.u.)
FIGURE 1.9 Induction curve of photosyn-
thesis at Chlorella pyrenoidosa, recorded
after 5 min dark incubation and after ir-
radiation with 135 W/m2 ‘‘white light’’:
‘‘"’’ — light on; ‘‘#’’ — light off. For details
see text. The number of oxygen molecules
absorbed during the induction time of
photosynthesis, calculated from the dashed
area ‘‘A’’ and evolved after switching off
the irradiation in the dark (dashed area
‘‘B’’) are in order of the number of chloro-
phyll molecules in suspensions investi-
gated.
© 2005 by Taylor & Francis Group, LLC
due to inactivation of the essential part of the
reaction center.
2. Under low light intensity conditions the oxygen
flash yields are low as a consequence of the
induction phenomenon.
We found that after switching on the irradiation
(1:4) (during the induction time of photosynthesis), the
oxygen absorption reaction occurs connected with
the oxidation of oxygen-evolving centers [23]. The
amount of oxygen absorbed during the induction
time depends on the chlorophyll content and approxi-
mately the same amount of oxygen is evolved after
switching off the light (in the darkness) (Figure 1.9,
Table 1.1).
On the other hand, according to Emerson and
Lewis [24] and McAlister [25], the amount of CO2
burst during the induction period is also of the order
of the amount of chlorophyll, which was explained by
Franck and Herzfeld [26] as a result of the decompos-
ition of the ACO2 complex under light (A is the
primary acceptor of CO2 whose quantity is assumed
to be equal to the amount of chlorophyll). Thus, it
may be assumed that functioning of the oxygen-
evolving centers may be presented as follows: in dark-
ness, all oxygen-evolving centers accept CO2 mol-
ecules or HCO 3 anions. This statement is in
agreement with the results of Stemler [27,28]. At low
irradiance, every chlorophyll molecule works as a
part of the reaction center with low frequency de-
pending on the frequency of the quanta absorbed.
If the irradiance is sufficiently high, it leads to the
oxidation (blocking) of a significant part of oxygen-
evolving centers, a process connected with oxygen
consumption and leads to CO2 evolution from oxy-
gen-evolving centers during the induction time of
photosynthesis. At saturating irradiance the number
of unoxidized oxygen-evolving (working) centers can
Saturated oxygen-evolution rate
A
1 min
B
Dark level
Time
 the darkness. Reaction (b) reflects the light quanta
TABLE 1.1
absorption by the chlorophyll molecule, which forms
The Ratio Between the Number of Oxygen
a complex with the primary electron acceptor (Z). In
Molecules Absorbed During the Induction Time of
reaction (c), charge separation is accomplished and
Photosynthesis and the Number of Chlorophyll
one electron is transferred from the chlorophyll mol-
Molecules in the Investigated Suspensions of
ecule to Z. Reaction (d) shows the electron transfer to
Scenedesmus Acutus and Chlorella Pyrenoidosa
a component P on the electron transport chain. The
Samples O2/Chl electron of the bicarbonate ion fills the missing elec-
tron in the chlorophyll molecule and the bicarbonate
Scenedesmus 1.1 ion is separated as a radical (reaction [e]). The recom-
Scenedesmus 0.9
bination of four bicarbonate radicals (reaction [f])
Scenedesmus 1.0
accumulated at a given reaction center (in flash ex-
Chlorella 0.9
Chlorella 0.8
periments or under high irradiation conditions) leads
to the evolution of one oxygen molecule, two mol-
ecules of water, and four molecules of CO2 — the so-
called noncooperative or Kok’s mechanism. Under
low irradiances or after switching off the light the
cooperation of four bicarbonate radicals, produced
decrease to approximately 1 : 500; thus, the number of in different reaction centers, leads to same reaction
oxygen molecules absorbed or CO2 molecules evolved — the so-called cooperative mechanism. The restored
during the induction time would be practically equal complex of the chlorophyll molecule and the primary
to the number of chlorophyll molecules in the inves- acceptor in reaction (e) and the obtained CO2 mol-
tigated photosynthesizing system. This assertion may ecules (reaction [f]) after hydration with the partici-
explain the observed dependence of induction time on pation of carboanhydrase (CA) (reaction [g]) are
radiation intensity. According to the explanation pre- involved in reaction (a) and the cycle could start
sented above, if the quanta arrive at oxygen-evolving again.
centers after prolonged intervals (longer than several Reaction (h) takes place after irradiation and the
seconds) the centers cannot reach the higher oxidized increased oxygen concentration during the induction
states, S3 or S4, and oxygen can be evolved by the time of photosynthesis is connected with the inactiva-
cooperation of oxygen precursors obtained in differ- tion (blocking) of the oxygen-evolving centers. These
ent centers, a mechanism considered previously processes lead to the liberation of bicarbonate rad-
[29,30]. In summary, the following reaction steps icals and after their recombination (reaction [i]) the
could be presumed: process of CO2 burst [24] is accomplished. In sum-
 mary, these two reactions lead to oxygen absorption
Chl  Z þ HCO
3 ! Chl  Z  HCO3 (a) and CO2 liberation. Apparently, if the reactions pre-
 sented above reflect the molecular events in oxygen-
Chl  Z  HCO 
3 þ hv ! Chl  Z  HCO3 (b) evolving centers the isotopic experiments with labeled
 oxygen will show water as the source of photosyn-
Chl  Z  HCO þ 
3 ! Chl  Z  HCO3 (c) thetic oxygen. Water is included as the ultimate
 source of electrons in reaction (g) during the hydra-
Chlþ  Z  HCO þ
3 þ P ! Chl  Z  HCO3 þ P

(d) tion of CO2.
. The above interpretation explains the results
Chlþ  Z  HCO
3 ! Chl  Z þ HCO3 (e) presented in Figure 1.2. Induction curves showed
that the duration of the induction period decreased
4HCO.3 ! 2H2 O þ 4CO2 þ O2 (f) simultaneously with decrease in irradiation, and
 under low intensity (0.056I0) the rate of oxygen evo-
H2 O þ CO2 þ CA ! H2 CO3 ! Hþ þ HCO3 (g) lution reached its steady state very quickly after the
light is switched on — reactions (h) and (i) cannot
4Chl  Z  HCO
3 þ 4O2 þ hv ! 4Chl be accomplished as the concentration of oxygen is
(h)
Zþ  O2 .
2 þ 4HCO3 low (low irradiation). However, under these condi-
tions, the ‘‘working point’’ of the photosynthetic
4HCO.3 ! 2H2 O þ 4CO2 þ O2 (i) process enters the initial nonlinear part of the curve
depicting dependence on irradiance (Figure 1.2,
During reaction (a), oxygen-evolving centers (i.e., all right), which is characterized by a very low quantum
chlorophyll molecules) capture bicarbonate ions in efficiency. Analysis of results from flash experiments

© 2005 by Taylor & Francis Group, LLC

[31,32] showed that the linear part of the irradiance
curve corresponds to oxygen evolution connected
with successive transitions of Si states from S0 to
S44þ, while the deactivating back reactions of the
oxidized Si states take place in the region of the
initial nonlinear parts of irradiance curves. Thus, at
low irradiances when the absorption of four quanta
in the individual reaction centers needs a longer time
and the centers do not manage to pass over into the
S44þ state, the oxygen evolution is mainly a result of
the deactivation of the oxidized Si states and the
cooperation of oxygen precursors (bicarbonate rad-
icals [HCO
3 ]) produced from different reaction cen-
ters.
The concept of the PSU is now more than
70 years old. During this period, our ideas about
the size and the arrangement of these structures
have often changed. The most difficult questions
still remain: ‘‘Are the concepts of Emerson and
Arnold [1] or of Gaffron and Wohl [33] sufficiently
sound to justify the present day model?’’ Or ‘‘Are
there other possibilities for the explanation of the
existing observations?’’ I suppose that if Emerson
and Arnold [1] and Gaffron and Wohl [33] have
had in their possession the results presented in Fig-
ure 1.5–Figure 1.8, which show dramatic changes in
the number of oxygen-evolving centers during the
induction time, it could hardly be assumed that
they would have postulated their hypothesis about
the PSU. Unfortunately, all their experiments were
performed with Warburg’s manometric apparatus. It
will be useful to remember the words of Birgit Ven-
nesland [34] concerning the photosynthetic unit con-
cept:
. . . These are (having in view the hypotheses, NB)
mainly based on the assumption that a hundred or
more chlorophyll molecule operate as a unit to trans-
mit the energy of the absorbed photons to appropri-
ate, hypothetical reaction centers. The flashing light
experiments on which this view is based are of dubi-
ous significance, and the complexities and detail
in which the associated theories have been clothed
should not be confused with evidence. Freedom to
use a large number of assumptions makes it easy
to devise theories and to fit innumerable observations
to them. The most valuable experimental facts are
those which restrict such flights of the imagination.
The results presented above show the complexity
and flexibility of the oxygen-evolving system of
photosynthesis. They demonstrate that many of the
experimental data obtained cannot be understood
within the framework of the postulated PSU. Fur-
thermore, there are many observations whose explan-
ations lead to serious contradictions, which have led
© 2005 by Taylor & Francis Group, LLC
to the proposal of various models. Regarding the
basic arguments for the postulation of a PSU one
has to admit that the strongest point is the absence
of oxygen after the first saturating flash. However, it
demands a very careful reconsideration: after pro-
longed darkness the first flash hits the cells or the
chloroplasts in an anaerobic state; the rate constants
of reactions leading to oxygen evolution through the
cooperative mechanism are significantly lower than
those connected with a noncooperative mechan-
ism, since the functioning of a cooperative mechanism
requires diffusion of oxygen precursors between dif-
ferent reaction centers. Photosynthetic systems are
self-controlled and may attain a modified state after
a short saturating flash. This may be connected with
oxygen-consuming processes during the induction
period and further connected with self-regulating pro-
cesses that protect the living structure from oxidative
damage. This statement is supported by the data of
Boitchenko and Efimtcev [35], which prove that
under increased oxygen concentrations a significant
part of oxygen-evolving (PSII) centers are inactivated
(blocked).
Therefore, all three basic arguments about the
concept of the PSU could be explained by the exist-
ence of two different ways of oxygen evolution in
photosynthesis and by the different degrees of inacti-
vation (blocking) of oxygen-evolving centers. In this
respect the concept of the PSU should be accepted
as a dynamic system rather than as a structural or
statistical system.
III. THE CONCEPT OF TWO
PHOTOSYSTEMS
A. EXPERIMENTAL GROUNDS
The hypothesis of participation of two photochemical
systems in the light-driven reactions of photosynthesis
in green plants emerged after the discovery of Emer-
son’s second effect (the ‘‘enhancement’’ effect) and
was theoretically substantiated by Hill and Bendall
[2] in 1960, who assumed that both photosystems
function consecutively. In the course of the following
four decades, this hypothesis was supported by a
considerable number of experimental facts; that is,
the sites of the individual electron carriers were esti-
mated and, along general lines, were accepted by most
authors. However, as already pointed out, Emerson’s
second effect and also the ‘‘red drop’’ of quantum
efficiency, which are considered as headstones of this
concept, could be explained without resorting to the
hypothesis of two photosystems ensuing from the
nonlinearity of the light curves of photosynthesis
at low light intensities or from the principle of
nonadditiveness in the action of light [31]. On the
other hand, in the literature there is a great deal of
information that cannot be satisfactorily explained by
the concept of two photosystems. This is the reason
for the existence of several hypotheses about the se-
quence and the functioning of light reactions in
photosynthesis [2,36–39].
The existence of these hypotheses proves the dif-
ficulties that different groups of investigators have in
interpreting experimental results. Despite the fact that
significant differences exist between these hypotheses
they all contain at least two different photosystems
(PSI and PSII).
The main experimental facts supporting the con-
ception of two photosystems are the following:
1. The quantum efficiency of photosynthesis — 8
to 12 quanta are needed for the reduction of
one molecule of CO2 or for the evolution of
one molecule of O2.
2. The red drop of quantum efficiency of photo-
synthesis [24].
3. The enhancement effect (Emerson’s second
effect) [40].
4. The spectral transient effects [41].
5. Myers’ and French’s effect [42,43].
6. Cytochrome f oxidation by light with 700 nm
wavelength and its reduction by light with at
680 nm (or shorter wavelength).
7. The existence of alga mutants [44], one of
which (mutant no. 8) does not accomplish pho-
tolysis of water and does not evolve oxygen
(does not show Hill activity) but has the ability
to reduce NADPþ and CO2, while the other
(mutant no. 11) evolves O2 and posseses Hill
activity but is not able to reduce NADPþ and
CO2.
8. The existence of chloroplast fragments pos-
sessing different activities, that is, some ac-
complish the Hill activity while the others
reduce NADPþ.
9. The results of experiments with specific inhibi-
tors of electron transport such as CMU,
DCMU, hydroxylamine, and others.
10. Some results obtained by studying photopho-
sphorylation coupled with electron transport
in the light reactions of photosynthesis.
Besides the above-cited experimental facts, there
are many other results that are interpreted with the
aid of the hypothesis of two photosystems, but pre-
sumably they could also be explained with the same
level of acceptance by leaving out this concept.
The most important experimental result that sug-
gested the idea for two photosystems was Emerson’s
© 2005 by Taylor & Francis Group, LLC
second effect or the so-called ‘‘enhancement effect.’’
As is well known, in 1956 Emerson [40] looked for
an explanation of the red drop of quantum efficiency
that was observed at wavelengths above 700 nm.
During the experiments he observed that if short-
wavelength light was added to the less efficient long-
wavelength light the efficiency of this light increased.
In other words, the effect of simultaneous action of
two light beams with different wavelengths is greater
than the sum of the effects of their independent
action. The principal reason for including the two
photosystems in the light induced reactions of photo-
synthesis is just to explain this nonadditive light ac-
tion. This raises the question: Is it possible to explain
this effect with the operation of a single photosystem?
As discussed in Section I, the answer to this question
would be positive if one assumes that the light curves
of photosynthesis are nonlinear at low light inten-
sities, that is, they are S-shaped.
A suspension of C. pyrenoidosa was irradiated
with two light beams (Figure 1.10), one of which is
700 nm modulated (1 sec light/1 sec dark) and the sec-
ond is background light with different wavelengths
between 600 and 700 nm. The amplitude of the modu-
lated oxygen rate induced by the 700 nm beam
changed after applying background radiation of dif-
ferent wavelengths whose intensities were chosen in
such a way as to give an equal oxygen-evolution rate
in the linear part of the ‘‘light curve.’’ The intensity of
the 700 nm modulated beam was kept constant. The
amplitude of the modulated oxygen-evolution rate
10 sec
b
Oxygen-evolution rate (a.u.)
600
700
10
50
80
90
20
30
40
60
70
Background wavelengths
Dark level
a
Time
FIGURE 1.10 Amplitudes of the modulated (0.5 Hz) oxy-
gen-evolution rate in Chlorella pyrenoidosa induced by a
700 nm beam without background radiation (a) and after
compensation of the initial nonlinear part of the ‘‘light
curve’’ with background radiation of different wavelengths
between 600 and 700 nm (b).
remained constant (in the limit of experimental errors) 1 min
in all investigated spectral regions (600 to 700 nm). If
Emerson’s second effect exists as a separate appear- I 650(1)

Oxygen-evolution rate (a.u.)

V650(1+2)
ance we should not obtain any enhancement in the
case of addition of 700 nm background radiation to
the 700 nm modulated beam. But this was not ob- I650(2)
served: the enhancement did not depend on the wave- V650(2)
length of the background radiation but on its intensity
and on the obtained oxygen-evolution rate. The equal
degree of enhancement with 700 nm and other wave-
V650(1)
lengths showed that Emerson’s second effect is only a
particular case of the principle of nonadditive action I650(2) Dark level
of radiation in photosynthesis [31] and that it does not I650(1)
exist even as a second-order effect. Obviously, this Time
suggestion is in sharp contradiction with the accepted
concepts and literature data. Mann and Myers [45] FIGURE 1.11 ‘‘Enhancement effect’’ in Chlorella pyrenoi-
even obtained a negative enhancement effect in the dosa obtained by means of two monochromatic light beams
case of superposition of two beams of the same wave- of the same wavelength (650 nm): ", turning on; #, switching
length. Such a ‘‘negative enhancement’’ (attenuation) off the light beams.
exists in different regions of Emerson’s second effect
action spectra. According to Heath [46], there is no
reasonable explanation for this negative effect. Our
efforts to find such attenuation after having observed 3 sec
the conditions ensuing from the nonlinearity of the
‘‘light curves’’ were unsuccessful. Probably both ab-
Oxygen-evolution rate (a.u.)

sence of enhancement in the case of superposition of

two beams of same wavelength and observation of
attenuation in different regions of Emerson’s second
effect action spectra are consequences of reaching sat-
uration with radiant energy. A correct compensation
of the initial nonlinear part of the ‘‘light curves’’ is
impossible not only in suspensions with high absorb-
ance (>0.5) but also in suspensions with very low
absorbance because of the nonhomogeneous distribu-
tion of pigments in them (in the cell and the chloro-
plast volumes). When one tries to compensate the
lowest sublayer in suspensions or in chloroplasts of
higher absorbance, the oxygen-evolving centers situ-
ated in the surface sublayers always reach the region of
saturation with radiant energy. Due to the difference
in the wavelengths of exciting radiation the distribu-
tion of absorbed light quanta in various sublayers of
suspension or of chloroplast volumes is also different.
This means that the action of light with different ab-
sorption coefficients will be different even after equal-
ization of their summary effects.
The graph in Figure 1.11 clearly shows the ap-
pearance of the effect of enhancement after excitation
of photosynthesis by two continuous monochromatic
rays with the same wavelength (650 nm).
Figure 1.12 represents an original protocol from
the experiment in which two monochromatic 650 nm
light beams are focused on the suspension layer of
C. pyrenoidosa. One of the beams, I1, is modulated
and the other, I2, is continuous. In the left part only
I1 I1 I2 I1 I2
A B C
Dark level
Time
FIGURE 1.12 The effect of two monochromatic 650 nm
light beams depending on their positions on the suspension
layer of Chlorella pyrenoidosa (for details see the text).
the modulated beam is used and the obtained modu-
lated oxygen-evolution rate (designated by ‘‘A’’) is
seen on the ‘‘zero’’ dashed line. In the middle part
of the figure the continuous light beam I2 is switched
on but is focused on different regions with respect to
the modulated beam (I1). It is seen that the continu-
ous oxygen-evolution rate increases; however, the
amplitudes ‘‘B’’ of the modulated oxygen-evolution
rate remain unchanged. In the right part of the
figure both beams are directed on one and the same
surface of the suspension and a significant increase
in the amplitudes of the oxygen evolution rate is
observed.

© 2005 by Taylor & Francis Group, LLC

 The results presented lead to the conclusion that
the ‘‘enhancement effect’’ depends on the ‘‘working
point’’ of the oxygen-evolving system on the light
curve or on some feature belonging to cell or chloro-
plast structure, but not on the concentration of oxy-
gen in the surrounding volume. The changes of
oxygen-evolving amplitudes obtained after irradi-
ation with modulated light beams before switching
on the background irradiation, during the induction
time (after switching on the background irradiation
[arrow ‘‘a’’]), and in darkness (after switching off the
continuous irradiation [arrow ‘‘d’’]) are presented in
Figure 1.13. The wavelength of the two light beams
is 650 nm. Arrow ‘‘b’’ indicates switching off and
arrow ‘‘c’’ switching on the modulated irradiation. It
is seen that the amplitudes of the modulated oxygen-
evolution rate do not reach their maximum immedi-
ately after the induction of the photosynthetic pro-
cess. The amplitudes increase simultaneously with
increase in the continuous oxygen-evolution rate.
After switching off the continuous irradiation the
amplitudes do not reach their initial value and during
a certain dark period they decrease continuously.
A comparison of the enhancement values (ap-
proximately 5 to 10) obtained in our experiments
with those in Emerson’s second effect investigations
(approximately 1.2 to 2.2) shows that the effect pro-
voked by nonlinearity of the irradiance curves is
much stronger that that observed for Emerson’s en-
2500
Oxygen-evolution rate (mV)
2000
1500
1000
500
a
0 FIGURE 1.14 A tentative model of photosynthetic electron
0 10 20 30
Time (sec)
FIGURE 1.13 Dependence of the amplitude of the modu-
lated (0.5 sec light/0.5 sec dark) oxygen evolution in Scene-
desmus obliquus during the induction time of photosynthesis.
The two light beams have the same wavelength (650 nm) and
allow 10 and 6 mmol/m2/s irradiances for modulated and
continuous beams, respectively. The continuous light is
switch on (arrow ‘‘a’’) and switch off (arrow ‘‘d.’’) Arrows
‘‘b’’ and ‘‘c’’ show switching off and switching on of the
modulated light beam, respectively.
© 2005 by Taylor & Francis Group, LLC
hancement effect. Obviously, the effect of irradiance
on photosynthesis is nonadditive not only for the
beams with different wavelengths (Emerson’s en-
hancement effect) but also for the beams with the
same wavelength. This statement was confirmed by
Warner and Berry [47] and Milin and Sivash [48]. As
pointed out earlier this effect is considered as a ‘‘cru-
cial experiment’’ for the assumption that the electrons
from water to NADP are transferred through two
consecutive photoacts.
B. PHOTOSYNTHESIS WITH SOLE PHOTOSYSTEM
Figure 1.14 presents a tentative diagram of electron
transport light reactions of photosynthesis in
green plants by a single photosystem on the basis
of the existing diagrams of Hill and Bendall and
Arnon’s group (cf. Hall and Evans [49]). The best
known electron carriers according to their corre-
sponding redox potentials are arranged in three
groups. The group of electron carriers at the reduc-
tion side of the photosystem, consisting of the pri-
mary acceptor of that photosystem Z (FRS; Fe-S),
feredoxine (FD), and flavoprotein (fp), is determined
P* Phe Artificial e-acceptors
NADP
FRS; Fe-S; FD
P~
Cytb563
b DCMU PQ
d HOQNO Cytb559[L.P.]
NH2OH
DCIPH2 P~
DPC DCIP
FeCy
(Mn)
c hn P
Cytb559[H.P.]
Z Cl− HCO−3
Cytf
PC O2
DCMU
Tris
40 50 60 transport with only one photosystem. P, oxygen-evolving
(reaction) center; P*, excited state of P; Phe, pheophytin;
FRS, ferredoxin reducing substance; Fe-S, bound iron sulfur
protein; FD, ferredoxin; NADP, nicotinamide adenine di-
nucleotide phosphate; DCMU, 3-(3,4-dichlorophenyl)-1,
1-dimethylurea; HOQNO, 2-heptyl-4-hydroxyquinoline-N-
oxide; PQ, plastoquinone pool; Cyth, cytochromes; DCIP,
2,6-dichlorophenolindophenol; DCIPH2, reduced form of
DCIP; PC, plastocyanin; FeCy, potassium ferricyanide;
NH2OH, hydroxylamine; DPC, 1,5-diphenylcarbazide;
DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquin-
one (for detail see the text).
with the highest degree of significance. This group of
electron carriers has the highest negative redox poten-
tial and is closely connected with the reduction of
NADPþ. Besides this, it is known that FD partici-
pates also in the process of cyclic photophosphoryla-
tion, and on this account it is assumed that at this
point the electron transport chain branches off to-
ward the cyclic electron transport or the group of
electron carriers consisting of cytochrome b563, cyto-
chrome b559(L.P.), and probably plastoquinone (PQ).
The group of electron carriers consisting of plasto-
cyanine (PC), cytochrome b559(H.P.), and cytochrome
f shows a tendency toward oxidation upon illumin-
ation and is probably situated at the donor part of the
electron-transport chain. It is possible that some of
the carriers of this group take part in the cyclic elec-
tron transport.
The figure also shows the possible sites of photo-
phosphorylation at the cyclic electron transport, the
expected sites of action of best known inhibitors of
the individual reactions, and the artificial electron
donors and acceptors. With the exception of the nat-
ural electron acceptor in the reducing part of the
photosystem the electron carriers and the reactions
taking place in this part are relatively well known. All
electron carriers shown in the figure are at their re-
spective places in the cyclic transport according to
Hill and Bendall [2] and Knaff and Arnon [36]. Of
course, many details in both structural and functional
aspects should be clarified after a profound analysis
of the existing literature data.
Figure 1.14, indicating the functioning of the elec-
tron transport reactions of photosynthesis in green
plants, could explain the following experimental facts:
1. Emerson’s effects, the red drop and the en-
hancement, are explained by the principle of
the nonadditiveness in the action of light dur-
ing photosynthesis.
2. The existence of mutant forms algae (no. 8 of
Bishop) and also of different fragments from
chloroplasts (light fragments), which cannot
evolve oxygen, could be explained with dam-
ages of electron-transport chain in the oxida-
tive part (Z, Cythf, Cyth559[H.P.], PC, different
kinds of polypeptides), and for mutant no. 11
and for heavy chloroplast fragments with de-
structions in the reduction side (Phe, Fe-S, Fd)
[44].
3. The qualitatively different behaviors of the
photosynthesizing system toward light of wave-
lengths over and below 700 nm is probably due
to the unequal number of absorbed quanta;
hence, depending on the degree of reduction
of NADPþ, a change occurs in the relative
© 2005 by Taylor & Francis Group, LLC
number of the electrons participating in the
cyclic and noncyclic pathways.
4. Depending on the sites of action of the various
inhibitors, they will lead to different effects. It
is possible that some of these substances may
have nonspecific action as well. Certainly, the
final effect of the action of individual inhibitors
will depend also on the corresponding sequence
of the electron carriers in the various groups.
5. As shown in our earlier work [30] the spectral-
transient effect of Blinks [41] and Myers and
French [42] could be considered as a result of
the superposition of the induction-type transi-
ent phenomenon observed during oxygen
evolution. As a consequence of different
permeabilities of the pigmented sections in
chloroplasts for light beams with different
wavelengths a change occurs in the frequency
of turning of the functioning reaction centers
and this leads to the difference in oxygen in-
duction curves. The same interpretation will be
valid also for the so-called ‘‘State 1–State 2’’
phenomenon. There is no doubt that these ef-
fects as well as data obtained upon investiga-
tion of photophosphorylation cannot be
considered as irrefutable arguments for the ser-
ial operation of the two photosystems in the
light reactions of photosynthesis.
IV. CONCLUSION
In every field of science the relevant and correct
choice of the basic principles or postulates has de-
cisive action on its future progress and development.
In photosynthesis, there are still many principal ques-
tions concerning the light reactions of photosynthesis
that remain unanswered. If the ‘‘enhancement effect’’
is a consequence of the nonlinearity of the irradiance
curves under low irradiances, then the idea about the
two consecutive photoacts in bringing the electron
from the primary electron donor to NADP loses its
crucial evidence. However, if the electrons are trans-
ferred in only one photoact then a problem from the
energetic point of view arises. According to Bolton
[50,51], if the photosynthetic process is affected by
one photosystem only (using only four photons),
then the fraction of photon energy («) at lmax (the
maximum wavelength at which photosynthesis could
be affected) should reach 0.73. This value is approxi-
mately equal or even higher than the theoretically
calculated thermodynamic limit. As a consequence,
it is postulated that the quantum requirement of
photosynthesis cannot be less than 8 to 12 quanta
per oxygen molecule evolved. However, as pointed
out by Brown and Frenkel [52], the experimental
determination of the minimum quantum requirement
of Chlorella photosynthesis has become one of the
most strenuously contested problems in all of biology
and thus before the acceptance of the idea about the
two photosystems there was no real agreement on the
value of the quantum efficiency. According to Bell
[53], an analysis of the available literature data
allowed the drawing of histograms in which from
nine studies, four reported quantum requirement
less then eight or even seven quanta. I believe that it
is possible that this contradiction can be overcome if
one accepts the idea of Warburg [54], Metzner [55],
and Stemler [28,56] that HCO 3 is almost certainly the
immediate source of photosynthetically evolved oxy-
gen. In this case, the energy of one quantum with
wavelength of even 700 to 730 nm will be sufficient.
Obviously, if the bicarbonate ions and CO2 partici-
pate only as catalysts (reaction steps [a] to [i] in Sec-
tion II.C), the experiments with labeled oxygen
cannot be considered as evidence in support of the
statement that PSII receives its lost electrons directly
from water. The only conclusion that could be drawn
from these experiments is that the photosynthetic
oxygen comes from water, but this does not mean
that water is the immediate electron source to the
reaction centers of photosynthesis [55]. It seems that
we have no decisive experiments to prove the nature
of the electron donor of the reaction centers of photo-
synthesis. It is, therefore, necessary to undertake
a thorough study of the arguments considered in
favor of the participation of H2O and against the
participation of HCO 3 ions as an immediate electron
source in the process of photosynthesis. Consider-
ing this statement the estimated values of the quan-
tum requirement, 5–6–9 quanta per oxygen [57–61],
which are lower than the estimated theoretical min-
imum quantum requirements (maximum efficiency)
of photosynthesis (10 quanta per oxygen), predicted
by the Z-scheme [62,63] seem entirely correct. Keep-
ing in mind that the entire photosynthetic process
contains a significant number of very complicated
biochemical steps, it is not possible to understand
how every photon is used with almost 100% effective-
ness without any losses even while believing that Na-
ture is built absolutely perfectly. The other strange
fact is that in many experiments (including Emer-
son’s) on action spectra of photosynthesis it is
shown that oxygen evolution could be observed even
at wavelengths around 720 to 730 nm where only
photosystem I should be active. Obviously, these re-
sults are in sharp disagreement with the concept of
two photosystems and consequently with the assump-
tion that the oxygen-evolving reaction centers receive
their lost electrons immediately from water. Thus, if
© 2005 by Taylor & Francis Group, LLC
the energy for electron removal from bicarbonate ions
is twice lower [55] than from water molecules and the
electrons could be transferred with a single photosys-
tem (with one photon energy) then Nature will use
electrons from bicarbonate ions and will not create a
second photosystem.
Interpreting the sense of Warburg’s statement that
‘‘in a perfect nature photosynthesis is perfect too,’’ we
can state that Nature is built with maximum simpli-
city and at minimum expense.
There is no need to point out that the postulate of
two photosystems originates from the initial results
obtained during the investigation of mechanisms of
photosynthetic light reactions and in particular from
the results of oxygen evolution. All the other results
concerning the structural aspects of the photosyn-
thetic machinery, especially the polypeptide compos-
ition of thylakoid membranes and the ‘‘water-
oxidizing’’ system, the existence of heavy and light
fragments cannot be considered as evidence here. In
our previous works [20,64], we hypothesized that a
close relationship exists between the grana and
stroma localized PSII (PSIIa and PSIIb centers) and
the participation of two different mechanisms for
oxygen evolution. Obviously, during the process of
the development of the photosynthetic apparatus the
entire electron transport system cannot be con-
structed simultaneously. Consequently, in every
given time we could find different sorts of particles
similar to the observed heavy or light particles pos-
sessing different functional properties [65]. Moreover,
the different kinds of photosystems (PSIIa, PSIIb,
PSIg, and PSIs centers) should not be on any account
considered as artifacts and nonexisting. The main
problem is what is the real function of these structures
and whether the electron transfer from water (the
electrons after all are coming from water) to NADP
is accomplished with the participation of two con-
secutive photoacts or with a single one.
In conclusion, it should be stressed that the rejec-
tion of the ‘‘generally accepted’’ hypotheses with
more than 40 years of history is a very complicated,
difficult, and painful process and needs the cooper-
ation and efforts of many investigators in this field.
The aim of this work is only to show that there are
serious difficulties concerning the explanation of
existing experimental data supporting the concepts
of the PSU and the generally accepted ‘‘Z’’ scheme
of photosynthesis based on the assumption of two
photosystems operating in series [2] but also to em-
phasize the alternative pathways and mechanisms
explaining the basic principles of photosynthetic pro-
cesses. I hope that the young scientists in the 21th
century will reconsider more carefully the basic argu-
ments of these two hypotheses and speed up the
understanding of photosynthesis, the unique and im-
portant process for life on Earth.
ACKNOWLEDGMENTS
This paper is dedicated to Otto Warburg, Birgit Ven-
nesland, and Helmut Metzner.
This work was supported in part by the National
Council for Scientific Investigations (Contract K-
808).
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