ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1438-1454 © Pleiades Publishing, Ltd., 2023.
Russian Text © The Author(s), 2023, published in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1742-1760.
1438
REVIEW
Electron Transport in Chloroplasts:
Regulation and Alternative Pathways of Electron Transfer
Alexander N. Tikhonov
Faculty of Physics, Lomonosov Moscow State University, 119991 Moscow, Russia
e-mail: an_tikhonov@mail.ru
Received June 21, 2023
Revised July 9, 2023
Accepted July 9, 2023
Abstract— This work represents an overview of electron transport regulation in chloroplasts as considered in the context
of structure-function organization of photosynthetic apparatus in plants. Main focus of the article is on bifurcated oxida-
tion of plastoquinol by the cytochromeb
6
f complex, which represents the rate-limiting step of electron transfer between
photosystemsII andI. Electron transport along the chains of non-cyclic, cyclic, and pseudocyclic electron flow, their
relationships to generation of the trans-thylakoid difference in electrochemical potentials of protons in chloroplasts, and
pH-dependent mechanisms of regulation of the cytochromeb
6
f complex are considered. Redox reactions with participation
of molecular oxygen and ascorbate, alternative mediators of electron transport in chloroplasts, have also been discussed.
DOI: 10.1134/S0006297923100036
Keywords: photosynthesis, chloroplasts, electron transport, regulation
Abbreviations: Asc, MDHA, DHA, three redox forms of ascorbate (fully reduced, semiquinone, and completely oxidized);
CBC,Calvin–Benson cycle; CET,cyclic electron transport; EPR,electron paramagnetic resonance; ETC,electron transport
chain; ISP,iron-sulfur protein, part of PSI; Fd,ferredoxin; FNR,ferredoxin-NADP reductase; NDH-1,NAD(P)H-dehydro-
genase of chloroplasts type1; P
700
and P
680
,primary electron donors in PSI and PSII; Pc,plastocyanin; PGR5 and PGRL1,pro-
teins involved in cyclic electron transfer around PSI; PSI and PSII, photosystem I and photosystem II; PQ, plastoquinone;
PQH
2
,plastoquinol; PTOX,plastid (plastoquinol) terminal oxidase; ROS,reactive oxygen species.
* To whom correspondence should be addressed.
INTRODUCTION
Oxygenic photosynthesis is the most important
process of the Earth biosphere, which provides produc-
tion of molecular oxygen(O
2
) and fixation of CO
2
driv-
en by the sunlight energy absorbed by light-harvesting
pigments of plants, algae, and cyanobacteria. Photosyn-
thetic apparatus of these organisms contains two photo-
systems(PS), the pigment–protein complexes PSI and
PSII, cytochrome b
6
f complex, and ATP synthase com-
plex CF
0
–CF
1
, which catalyzes formation of ATP from
ADP and orthophosphateP
i
. Transfer of two electrons
from the water-oxidizing complex of PSII to NADP
+
(terminal electron acceptor in PSI) ensures reduction of
NADP
+
to NADPH. ATP and NADPH, as the high en-
ergy products of the “light stages” of photosynthesis, are
used in the reactions of the Calvin–Benson cycle(CBC)
for CO
2
fixation[1, 2].
Structural and functional organization of the plant
photosynthetic apparatus is well studied [3-21]. At the
same time, some questions remain unclear, related to
regulation of photosynthetic processes and acclimation
of the photosynthetic apparatus to changes in environ-
mental conditions. This review briefly examines struc-
tural organization of the photosynthetic apparatus of
oxygenic organisms and main regulation mechanisms
of the electron and proton transport, which ensure high
efficiency of light energy conversion in chloroplasts.
The first part of the article describes the processes of
non-cyclic, cyclic, and pseudocyclic electron transfer,
their role in generation of the trans-thylakoid difference
in electrochemical potentials of hydrogen ions (Δμ
~
H
+
),
and also discusses the mechanisms of pH-dependent
regulation of the cytochrome b
6
f complex functioning
in chloroplasts. The second part examines the process-
es related to participation of molecular oxygen (O
2
)
ELECTRON TRANSPORT IN CHLOROPLASTS 1439
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
and ascorbate(Asc) as mediators in electron transfer in
chloroplasts.
STRUCTURAL AND FUNCTIONAL
ORGANIZATION OF THE PHOTOSYNTHETIC
APPARATUS OF PLANTS.
ELECTRON TRANSPORT CHAIN
In plants, the processes of light-induced electron
transport and transmembrane proton transfer occur in
chloroplasts, the energy-transducing organelles of the
plant cell[18-21]. The chloroplast is separated from the
cytoplasm by the envelope, which consists of two adja-
cent membranes – the outer and the inner one. Under
the envelope, in the stroma, there are lamellae mem-
branes. Under normal physiological conditions, grana
are formed from the lamellae membranes, which are
arranged as the appressed stacks of flattened vesicles
(thylakoids) with diameter of ~350-600 nm. Individ-
ual thylakoids of the grana protrude into the stroma as
the intergranular thylakoids. Thylakoid membranes are
densely filled with photosynthetic protein complexes,
which make up to ~70-80% of the total membrane mass.
The pigment–protein electron transport complexes are
incorporated into the thylakoid membranes. The stro-
ma contains RNA, DNA molecules, ribosomes, starch
grains, as well as enzymes that ensure absorption of CO
2
in the CBC.
A diagram illustrating interaction of the electron
transport complexes is shown in Fig. 1. The energy of light
quanta, absorbed by the pigments of the light- harvesting
Fig. 1. Scheme illustrating interaction between the electron transport complexes (PhotosystemI, PhotosystemII, and the cytochromeb
6
f com-
plex) embedded in the thylakoid membrane and the mobile electron carriers (ferredoxin,Fd; plastoquinone,PQ; plastoquinol,PQH
2
; plastocy-
anin,Pc). F
X
, F
A
, and F
B
are the iron-sulfur centers of PSI; Q
A
and Q
B
are the phylloquinone molecules associated with PSI; PQ
A
and PQ
B
are
the plastoquinone molecules associated with PSII; APX, ascorbate peroxidase; FNR, ferredoxin-NADP reductase; FTR, ferredoxin thiore-
ductase; NDH-1,NAD(P)H-dehydrogenase of chloroplasts type1; PTOX,chloroplast (plastid) terminal oxidase; CBC,Calvin–Benson cycle;
SOD,superoxide dismutase; Asc, MDHA and DHA are fully reduced, semiquinone (monodehydroascorbate) and oxidized (dehydroascorbate)
forms of ascorbate, respectively; Trxf,m,isoformsf andm of thioredoxin. Explanation of other symbols and abbreviations is given in the main text
of the article. Red arrows indicate main paths of the electron transfer, blue arrows indicate transfer of hydrogen ions. The arrows marked as a and b
indicate two pathways of the Asc formation from MDHA.
TIKHONOV1440
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
antennas of PSI and PSII, migrates to the reaction cen-
ters, in which charge separation occurs, and electron
transfer along the photosynthetic electron transport
chain (ETC) is initiated [6-17]. Coordinated function-
ing of PSI and PSII accomplishes oxidation of wa-
ter in the oxygen-evolving complex of PSII (2H
2
O →
→ O
2
+ 4e
–
+ 4H
+
) and provides reduction of NADP
+
to NADPH with the help of PSI (NADP
+
+ 2e
–
+ H
+
→
→ NADPH). The cytochrome b
6
f complex and the mo-
bile electron carriers, plastoquinol (PQH
2
) and plas-
tocyanin (Pc), facilitate communication between the
low-mobility membrane-embedded protein complexes
PSII and PSI.
Electron transfer is coupled with generation of the
trans-thylakoid difference in the electrochemical poten-
tials of hydrogen ions (Δμ
~
H
+
). As a result of water decom-
position by the oxygen-evolving complex of PSII and
due to the work of the b
6
f complex, the stroma becomes
alkalized, and hydrogen ions accumulate in the lumen.
Since the thylakoid membranes have relatively low per-
meability to hydrogen ions and charged molecules,
they able to maintain Δμ
~
H
+
, which allows the mem-
brane-embedded ATP synthase complexes (CF
0
–CF
1
)
to ensure formation of ATP from ADP and ortho-
phosphate P
i
[3-5]. In chloroplasts, the pH difference,
ΔpH = pH
out
– pH
in
, where pH
out
and pH
in
are pH val-
ues of the stroma and lumen, respectively, provides
main contribution to Δμ
~
H
+
[22,23].
Photosystem II (PSII) functions as an oxidoreduc-
tase, oxidizing water and reducing plastoquinone(PQ)
to plastoquinol (PQH
2
) [6, 7, 9-11]. In photosynthet-
ic reaction centers of PSII, energy from the excited
light-harvesting pigments migrates to the primary elec-
tron donor, which consists of an ensemble of four chlo-
rophyll a molecules (Chl
D1
/P
D1
/P
D2
/Chl
D2
). The prima-
ry electron donor, known as P
680
, transfers the electron
to the pheophytin(Phe) through the chlorophyll Chl
D1
,
from which it goes to the plastoquinone PQ
A
, tight-
ly bound to PSII(P
*
680
→ Chl
D1
→ PheA → PQ
A
). PQ
A
–
re-
duces the second plastoquinone molecule PQ
B
(PQ
A
–
PQ
B
→ PQ
A
PQ
B
–
). After accepting the second elec-
tron, the PQ
B
2–
molecule becomes protonated by the hy-
drogen ions from the stroma (PQ
B
2–
+ 2H
+
out
→ PQ
B
H
2
)
and then dissociates into the lipid phase of the mem-
brane in exchange for another oxidized PQ molecule
(PQ
B
H
2
+ PQ → PQ
B
+ PQH
2
). Further step of electron
transfer along the ETC involves the PQH
2
diffusion to
the cytochromeb
6
f complex (plastoquinol–plastocyan-
in oxidoreductase). In this complex, a two-electron (bi-
furcation) oxidation of PQH
2
into PQ occurs, leading
to the reduction of Cyt f, which then reduces Pc, which
serves as an electron donor for PSI. Two hydrogen ions
absorbed from the stroma during the PQH
2
formation
(PQ + 2e
–
+ 2H
+
out
→ PQH
2
) are released into the lumen
during the oxidation of PQH
2
by the cytochrome b
6
f
complex.
Photosystem I (PSI). In plants, PSI is a mono-
meric complex that includes light-harvesting pigments
and electron carriers; PSI in cyanobacteria is, as a
rule, a trimeric supercomplex[6, 7]. Excitation of P
700
,
the primary electron donor of PSI, leads to the charge
separation in PSI and formation of the oxidized form
of P
700
(P
+
700
), which accepts an electron from the re-
duced plastocyanin (Pc
−
). From PSI, the electron is
transferred to ferredoxin (Fd) [6-8]. At the accep-
tor side of PSI, electron carriers are structured in the
form of two quasi-symmetric branches. From P
700
, the
electron proceeds (through Chl a and phylloquinone
molecules) to the iron-sulfur acceptor F
X
and further,
through the redox centers F
A
and F
B
, to the ferredoxin
located in the stroma (F
X
→ F
A
→ F
B
→ Fd). Two mol-
ecules of the reduced ferredoxin (Fd
−
) mediate reduc-
tion of NADP
+
to NADPH by the ferredoxin-NADP
reductase (FNR). Thus, due to the joint work of PSII
and PSI, the non-cyclic transfer of electrons from wa-
ter to NADP
+
occurs, ensuring formation of NADPH
molecules, which are consumed mainly in the CBC
reactions.
At the level of the Fd pool, electron flow can
branch out. In addition to the non-cyclic electron trans-
port from PSI to NADP
+
, Fd is involved in the electron
transfer around PSI (cyclic electron transport, CET),
when the electron from Fd returns to the plastoquinone
pool of chloroplasts [24-31]. Figure 2 demonstrates
possible CET pathways along which electrons from the
acceptor side of PSI could return to the pool of plas-
toquinone molecules. One of them suggests that CET
includes hypothetical protein FQR (ferredoxin-quinone
reductase), existence of which was postulated previous-
ly[24]. At present time, there are compelling reasons to
believe that the role of FQR is performed by the proteins
PGR5 and PGRL1, associated with the cytochromeb
6
f
complex [28-30]. In addition, electron transfer from
the reduced Fd to the plastoquinone pool is possible
with participation of the minor NADPH dehydroge-
nase complex type 1 (NDH-1), which forms a super-
complex with PSI[31-35].
Oxidation of plastoquinol in the cytochrome b
6
f
complex. Q-cycle. The cytochrome complex b
6
f is the
link in the electron transport chain that governs inter-
actions between PSII and PSI. Oxidation of PQH
2
by
the b
6
f complex is the slowest step in the electron trans-
fer chain between PSII and PSI [36-41]. The rate-de-
termining factor in this section of the ETC is turnover
of plastoquinone (PQ → PQH
2
→ PQ), which includes
formation of PQH
2
in PSII and interaction of PQH
2
with the b
6
f complex. In a wide range of conditions
(pH, temperature), reduction of PQ to PQH
2
in PSII
and its diffusion along the membrane to the b
6
f com-
plex was experimentally proven to occur faster than ac-
tual oxidation of PQH
2
within the cytochrome complex
[37, 38, 40, 41].
ELECTRON TRANSPORT IN CHLOROPLASTS 1441
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 2. Scheme of localization of the electron transport complexes in the membranes of granal and stroma-exposed intergranal thylakoids.
The arrows labelled NET, CET-1, and CET-2 designate different paths of electron transfer at the acceptor side of PSI. NET stands for non-cyclic
electron transfer associated with NADP
+
reduction. CET-1 is the pathway for cyclic electron transfer around PSI with suspected involvement
ofthe proteins PGR5 and PGRL1, associated with the cytochrome b
6
f complex. CET-2,cyclic electron transfer involving NADP dehydrogenase
complex type1 (NDH-1).
The cytochrome b
6
f complex is organized as a di-
meric complex consisting of two identical protein frag-
ments [12-17, 42]. Oxidation of PQH
2
occurs at the
quinol-binding sites of the dimeric complex (Fig.3, Q
o
catalytic sites). Each of the Q
o
centers is located near
the lumenal side of the thylakoid membrane, between
the low-potential hemeb
6
L
and the Fe
2
S
2
cluster of the
high-potential iron-sulfur protein(ISP), often called the
Rieske protein. Catalytic functions of the monomers are
carried out by four redox centers: the Fe
2
S
2
cluster of
the ISP, two hemes of the cytochrome b
6
(b
6
L
and b
6
H
),
and cytochrome f. According to the Mitchell Q-cycle
mechanism[42-46], oxidation of PQH
2
is a bifurcated
process; two electrons donated by the PQH
2
molecule
are transferred along the different chains: one electron
goes to the oxidized Fe
2
S
2
cluster of the ISP, the sec-
ond electron is transferred to the low-potential hemeb
6
L
.
At the same time, two protons of the PQH
2
molecule
dissociate into the lumen. In the high-potential elec-
tron transfer chain, the ISP is oxidized by the heme f,
from which the electron proceeds to plastocyanin and
then to the oxidized center P
+
700
(ISP → f → Pc → P
700
).
The second electron donated by PQH
2
is transferred
along the low-potential chain of the b
6
f complex, which
includes two cytochrome b
6
hemes and heme c
n
. This
electron arrives at the PQ molecule located at the Q
i
center (b
6
L
→ b
6
H
→ PQ). According to the modified Q-cy-
cle model, the second electron arrives at the Q
i
center
from the PSI acceptor site. After the double reduc-
tion of PQ and arrival of two protons from the stroma
(PQ + 2e
–
+ 2H
+
out
→ PQH
2
), the reduced PQH
2
mol-
ecule dissociates from the Q
i
center and returns to the
catalytic center Q
o
(see [12-17, 42-45] for more details).
In the end, it turns out that two protons are transferred
into the thylakoid (H
+
/e
–
= 2) per one electron trans-
ferred to the CBC from PSI (PSI→NADP
+
)[46].
Plastocyanin diffusion in the lumen. The cytochrome
complex reduces Pc molecule. Diffusing inside the lu-
men, Pc
–
transfers an electron to PSI. The diffusion
of Pc
−
and its oxidation by PSI occurs faster (at room
TIKHONOV1442
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 3. Dimeric cytochrome b
6
f complex. The image was constructed using the Accelerys DV visualizer software package (http://www.accelrys.com)
according to the PDB data (PDB ID 1Q90).
temperatures τ
1/2
< 300 μs) than the diffusion of PQH
2
from PSII to the b
6
f complex and oxidation of PQH
2
by
the b
6
f complex(τ
1/2
≥ 4-20 ms)[36-41]. Under certain
conditions (for example, in dark-adapted chloroplasts),
steric restrictions could prevent movement of the Pc
molecules within the narrow (~4-5nm) lumen gap[47].
Width of the lumen gap can increase upon the chloro-
plast illumination, and then lateral diffusion of Pc
−
in-
side the lumen would not limit transfer of the electrons
from the b
6
f complexes to PSI[47].
Apart from the processes described above, alterna-
tive electron transfer reactions can occur in chloroplasts,
among which a special role belongs to the so-called
pseudocyclic electron transfer, when molecular oxygen
acts as the final acceptor of the electron donated by PSI
(the Mehler reaction[48-51]). Reduction of O
2
to water
can also be catalyzed by the chloroplast (plastid) termi-
nal oxidase (PTOX), which oxidizes PQH
2
[52-56]).
It should be noted that the PTOX content in mature chlo-
roplasts is very low– only one PTOX complex per 100
PSII complexes [57]. The NDH-1 content is approxi-
mately three times lower than the PTOX content[57,58].
Redox reactions involving ascorbate also play a
special role in the metabolism of plant cells, providing
detoxification of reactive oxygen species (ROS) [59].
Alternative electron transfer pathways are discussed in
more detail below.
LATERAL HETEROGENEITY OF THYLAKOID
MEMBRANES AND ALTERNATIVE PATHWAYS
OF PHOTOSYNTHETIC ELECTRON TRANSFER
Photosynthetic protein complexes are unevenly
distributed between the granal and intergranal (stro-
mal) thylakoids [18-21, 60-63]. Granal thylakoids are
enriched with PSII complexes; majority of the PSI and
ATP synthase complexes are concentrated in the in-
tergranal thylakoids, at the edges and ends of the gra-
na domains exposed to the stroma. The cytochrome
complexes are distributed approximately evenly along
the lamellar membranes, from which thylakoids of the
grana and stroma are formed [60-63]. Heterogeneous
distribution of the complexes is caused by steric restric-
tions: ATP synthases and PSI complexes have protein
fragments that significantly protrude from the mem-
brane, thereby preventing compact arrangement of
these complexes in the appressed thylakoids of grana.
ELECTRON TRANSPORT IN CHLOROPLASTS 1443
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Structural and functional properties of thylakoids could
be associated with the lateral heterogeneity of the thyla-
koid membranes. The cytochrome complexes localized
in the granal and stroma-exposed thylakoids can partic-
ipate in the electron transfer along different pathways:
the “granal” b
6
f complexes are included in the chain of
non-cyclic (“linear”) electron transfer from PSII to PSI
and further to NADP
+
(PSII → PQ → b
6
f → Pc → PSI →
→ NADP
+
), the “stromal” complexes could be included
in the cyclic electron transport chain around PSI (PSI →
→ Fd → PQ→b
6
f→Pc→PSI).
Interaction between the remote low-mobility pro-
tein complexes is mediated by the diffusion of mobile
electron carriers – plastoquinone and plastocyanin.
High density of the protein complexes in the thylakoid
membrane limits mobility of plastoquinone, and poses
steric restrictions that impede movement of plastocy-
anin in the narrow gap of the lumen, thereby limiting
the rates of the diffusion-controlled stages of electron
transfer[64]. Note that, despite the steric restrictions,
diffusion of PQH
2
along the thylakoid membrane does
not limit per se the rate of electron transfer between
photosystems. As mentioned above, in a wide range of
experimental conditions (pH, temperature), formation
of PQH
2
in PSII and its diffusion occur faster than
actual oxidation of PQH
2
after its binding to the cata-
lytic center Q
o
of the cytochromeb
6
f complex [37, 38].
Despite the fact that many PSII and PSI complexes are
at a distance from each other, a significant portion of
the b
6
f complexes located in grana are positioned close
to PSII. Close arrangement of these complexes min-
imizes the average distance traveled by the plastoqui-
none pool molecules, ensuring rapid exchange between
the PQH
2
and PQ molecules formed as a result of the
PSII functioning (formation of PQH
2
) and oxidation of
PQH
2
by the b
6
f complex. Thus, due to the high mobil-
ity of plastoquinol in the membrane and rapid diffusion
of Pc inside the lumen[38, 40], effective electron trans-
fer from PSII to PSI and further to NADP
+
(non-cyclic
electron transport) is ensured.
The cytochrome b
6
f complexes located in the in-
tergranal thylakoids close to PSI complexes, could be
directly included in the cyclic electron transport (CET)
chain around PSI. Efficient functioning of CET could
be facilitated by formation of the supercomplex con-
sisting of the electron transport complexes b
6
f and
PSI[27]. Various pathways of CET are possible. PGR5
and PGRL1 proteins, which operate together with the
b
6
f complex, function as the electron transfer mediators
involved in CET[28-30].
Another CET pathway around PSI is realized with
the help of the minor NAD(P)H dehydrogenase com-
plex type1, which is an analogue of the similar complex
in mitochondria [31-33]. NDH-1 acts as an oxidore-
ductase, oxidizing ferredoxin and reducing plastoqui-
none [34, 35]. CET around PSI mediates Δμ
~
H
+
forma-
tion and, consequently, can support the ATP synthesis.
In this case, however, no reduction of NADP
+
occurs.
Coexistence of the linear and cyclic electron flows al-
lows to maintain proper stoichiometry between the ATP
and NADPH molecules, which is necessary for optimal
functioning of the CBC(ATP/NADPH=3/2)[1,2].
Distribution of the b
6
f complexes between the gran-
al and intergranal thylakoids depends on the functional
state of the photosynthetic apparatus. Changes in the
chloroplast architecture could lead to redistribution of
the electron flows between the non-cyclic and cyclic
pathways. Lateral movement of some b
6
f complexes
from the granal to the stromal regions of the thylakoid
membranes would lead to increase in the relative con-
tribution of CET to the functioning of chloroplasts.
It is assumed that such redistribution of the b
6
f com-
plexes could indeed occur, for example, due to the light-
induced changes in the distance between the neighbor-
ing granal thylakoids[65].
Two ferredoxin isoforms and alternative electron
flows. Taking into account localization of b
6
f complex-
es in different regions of chloroplasts, it is of note that
there are several isoforms of ferredoxin (at least two
fractions, minor(Fd1) and major (Fd2)), which differ
slightly in physicochemical properties (for example, in
standard redox potential values) and some functional
properties[66-70]. The Fd1 and Fd2 proteins have sim-
ilar amino acid sequences, but are present in different
quantities in the chloroplasts of C3 plants. For exam-
ple, in Arabidopsis and pea the Fd1 and Fd2 fractions
account for 10% and 90%, respectively, of the total pool
of ferredoxin molecules [66]. The ratio between the
fractions depends on the plant acclimation conditions:
practically no expression of Fd1 is observed in the plants
grown under low light; under conditions that stimulate
CET (high light intensity, drought), Fd1 expression in-
creases significantly[68, 69].
As an example illustrating the difference in the
functioning of Fd1 and Fd2, let us consider the re-
sults of experiments with the class B bean chloroplasts
(chloroplasts with damaged outer membrane, lacking
ferredoxin) presented in Fig. 4 using the kinetic data
from Gins etal. [70]. These chloroplasts retained mem-
brane-bound ferredoxin-NADP reductase, but lost
ferredoxin molecules located in the stroma. Addition of
NADP
+
, a physiological electron acceptor, did not af-
fect the kinetics of electron transfer, as assessed from the
light-induced changes in the electron paramagnetic res-
onance (EPR) signal from the oxidized P
+
700
centers[70].
Both forms of ferredoxin, Fd1 and Fd2, were active
as electron transfer mediators. In both cases, far-red
light (λ
max
= 707 nm), which predominantly excites PSI,
caused noticeable oxidation of P
700
. However, under the
white light illumination (WL), which excites both pho-
tosystems, addition of Fd1 or Fd2, which interacts with
FNR, affected kinetics of the P
700
redox transformations
TIKHONOV1444
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 4. Influence of two isoforms of pea ferredoxin, Fd1 and Fd2, on the kinetics of P
+
700
EPR signal amplitude from the isolated classB chloro-
plasts of beans (Vicia faba) , induced by the far-red light (λ
max
= 707 nm) and white light(WL) in the presence of 2mM NADP
+
. Ferredoxin input:
1)60μmFd1, 2)60μmFd2. The figure was constructed using kinetic curves from[70].
in different ways. Fd2 more efficiently catalyzed the
“linear” electron transfer from PSI to FNR and then to
NADP
+
. This is supported by the fact that white light
caused a stronger increase in the EPR signal from P
+
700
(Fig.4, parameterB) in the presence of Fd2 than in the
case of Fd1 addition. Fd1 exhibited significant activi-
ty as an electron carrier in the CET chain around PSI.
Intensity of the P
+
700
signal under the white light illumi-
nation was lower in the presence of Fd1 than in the case
of Fd2. This difference could be caused by acceleration
of the electrons outflow from PSI to NADP
+
through
Fd2, as well as by the fact that Fd1 catalyzed the cyclic
transfer of electrons around PSI, returning electrons to
PSI, thereby reducing concentration of P
+
700
. It was also
demonstrated that the addition of antimycin, the CET
inhibitor, accelerated photooxidation of P
700
. The effect
of antimycin was more noticeable in the case of Fd1
than Fd2. This indicates higher activity of Fd1 as a me-
diator of CET around PSI. It has been suggested in the
literature that variations of the expression of different
Fd isoforms allow plants to optimize the use of solar en-
ergy and avoid excessive reduction of electron transport
chain carriers under unfavorable environmental condi-
tions[68, 69].
ELECTRON TRANSPORT REGULATION
AT THE LEVEL OF b
6
f COMPLEX
PQH
2
oxidation stage limiting electron transfer be-
tween PSII and PSI. There is evidence that in a wide
range of experimental conditions (pH, temperature)
electron transfer between PSII and PSI is limited not
by the PQH
2
diffusion from PSII to b
6
f complex, but
by the electron transfer from PQH
2
to the Fe
2
S
2
cluster
of the Rieske protein that occurs after formation of the
PQH
2
–ISP complex. This conclusion, previously made
by studying the P
700
redox transformations kinetics in
chloroplasts of spinach and beans [37,38], recently re-
ceived additional confirmation in the experiments with
Arabidopsis mutants, in which the grana diameter varied
widely (from 370 to 1600 nm). In the study by Höhner
et al. [40], the characteristic time of PQH
2
formation
in PSII and its diffusion to the b
6
f complex was shown
ELECTRON TRANSPORT IN CHLOROPLASTS 1445
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
to be ~3.2÷3.6 ms in all cases. This time is significantly
less than that for the total oxidation of PQH
2
, regardless
of grana diameter. These results convincingly prove that
the limiting stage of the electron transfer from PSII to
the b
6
f complex is PQH
2
oxidation in the catalytic cen-
ter Q
o
but not the PQH
2
diffusion along the thylakoid
membrane[37, 38].
After the ISP reduction, its mobile domain con-
taining the Fe
2
S
2
cluster shifts to the cytochrome f and
donates the electron to the cytochrome f heme. From
the cytochromef the electron is transferred to Pc. After
oxidation of ISP, his labile domain returns to its original
position. The movement of the mobile domain does not
limit the total electron transfer rate: significant displace-
ments of the ISP redox center occur relatively quickly as
compared to the PQH
2
oxidation rate by Fe
2
S
2
. At room
temperatures, the electron transfer from ISP
red
to Cyt f
proceeds with the characteristic time of ~2-4 ms [71,
72], which is less than the times of PQH
2
semi-oxidation
and cytochrome f reduction (τ
1/2
> 5-20 ms [36-41]).
This means that the PQH
2
oxidation rate is ultimately
determined by the stage of electron transfer from PQH
2
to the oxidized Fe
2
S
2
cluster after formation of the sub-
strate–enzyme complex PQH
2
–ISP
ox
.
pH-Dependent regulation of electron transfer in
chloroplasts. Two main mechanisms are known for the
pH-dependent inhibition of electron transfer between
the PSII and PSI caused by the decrease in the lumen
pH (pH
in
): (i) slowdown of the PQH
2
oxidation by the
cytochromeb
6
f complex[12-15, 73-76] and (ii)attenua-
tion of the PSII photochemical activity due to phenom-
enon known as non-photochemical quenching (NPQ)
of chlorophyll excitation[77-79]. The effect of pH
in
on
the PQH
2
oxidation rate happens due to the negative
feedback mechanism: oxidation of PQH
2
is accompa-
nied by the release of protons into the lumen, acidifi-
cation of the lumen (pH
in
↓) inhibits oxidation of PQH
2
.
According to the “proton-gated” model [80, 81], the
PQH
2
deprotonation stimulates electron transfer to the
oxidized Rieske protein (ISP
ox
); slowdown of the PQH
2
deprotonation caused by pH
in
decrease should inhib-
it oxidation of PQH
2
. There is every reason to believe
that the proton is transferred to the histidine residue of
the ISP, which is a ligand for one of the Fe
2
S
2
cluster
ions, and this occurs simultaneously with the transfer of
the electron to the Fe
2
S
2
cluster of the ISP (see[12-16,
42, 44] for more details). The pK
a
value of the ISP pro-
tonated group depends on the redox state of the Fe
2
S
2
cluster[82]. Two processes– PQH
2
deprotonation (pro-
ton transfer to the histidine residue of ISP) and elec-
tron transfer to the Fe
2
S
2
cluster of the ISP– could be
considered as coupled processes that are interconnected
and occur almost simultaneously [83-87]. The depro-
tonated state of ISP is a necessary condition for the oc-
currence of the proton transfer from PQH
2
to ISP.
ISP deprotonation is associated with the proton exit into
the lumen; probability of this process depends on pH
in
.
Protonation/deprotonation of the ISP depends on the
effective value of the pK
a
of the protonated ISP group
and is controlled by pH
in
[12, 14]. Oxidation of ISP af-
ter the electron transfer to heme f is accompanied by the
decrease in pK
a
, which should stimulate deprotonation
of ISP [82] and, accordingly, contribute to oxidation of
PQH
2
. However, with a sufficiently strong acidification
of the lumen (pH
in
≤ pK
a
), ISP deprotonation would be
hindered, and, therefore, the flow of electrons through
the cytochrome b
6
f complex would decrease. A simple
mathematical model, based on the fact that the electron
transfer through the cytochrome complex is controlled
by the processes of ISP protonation/deprotonation, ad-
equately describes pH dependence of the electron trans-
fer through the b
6
f complex [12, 14]. The alternative
hypothesis describing participation of a water molecule
as the primary proton acceptor in the ubiquinol oxida-
tion in the cytochrome bc
1
complex was suggested in the
study of Postila etal.[88].
The phenomenon of photosynthetic control. ThepH-
dependent regulation of electron transport is the basis of
a phenomenon called “photosynthetic control”[89-92].
Essence of this phenomenon is that the electron flow
between the photosystems PSII and PSI correlates with
the “phosphate potential”, P = [ATP]/([ADP] × [P
i
]),
where [ATP], [ADP], and [P
i
] denote concentrations of
ATP, ADP, and P
i
, respectively. The ratio of ATP/ADP
can vary depending on the physiological state of the
plant cell and interactions of the photosynthetic apparatus
with mitochondria and other metabolic systems[93-96].
In the state of “photosynthetic control” (“state4” ac-
cording to the terminology coined by Chance and Wil-
liams [89]), exhaustion of the pools of ADP and/or P
i
molecules leads to the decrease in the synthesis of ATP
and transmembrane proton flow through the ATP syn-
thase (CF
0
–CF
1
complex); simultaneously lumen pH
decreases significantly (pH
in
< 6), which slows down
operation of the cytochrome complex. In the “state3”,
when an intensive ATP synthesis occurs, the outflow of
protons from lumen to stroma accelerates, and, there-
fore, such strong acidification of the lumen, which
could significantly slow down the electron transfer, does
not occur (pH
in
≥ 6-6.5). This allows for an effective
ATP synthesis and, at the same time, high rate of elec-
tron transport is maintained[74-76, 90-92].
Increase of the stroma pH (pH
out
) due to the pro-
ton translocation from the stroma to lumen can also
affect the rate of the photosynthetic ETC functioning.
Increase in pH
out
up to 7.8-8.0 promotes activation of
the CBC reactions [1]. This would lead to acceleration
of the NADPH consumption and, accordingly, to the
faster outflow of electrons from PSI. Experiments on
the injections of protonophores (uncouplers) into the
leaves, which equalized pH inside (lumen, pH
in
) and
outside (stroma, pH
out
) of the thylakoids, confirmed
TIKHONOV1446
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
the above notion on the pH-dependent regulation of
electron transport in chloroplasts [97,98].
ALTERNATIVE WAYS
OF ELECTRON TRANSFER IN CHLOROPLASTS
In addition to the electron transport processes in-
volving reduction of NADP
+
and CET around PSI, de-
scribed above, chloroplasts can host other redox reac-
tion, among which molecular oxygen and ascorbate play
crucial roles in the functioning of the photosynthetic
apparatus.
Interaction of O
2
with chloroplasts. Molecular oxy-
gen(O
2
) is an active participant in metabolic processes
in the plant cell. O
2
molecules serve as electron accep-
tors interacting with the chloroplast ETC. At the same
time, active forms of oxygen may be generated: super-
oxide radicals(O
2
•−
), hydrogen peroxide (H
2
O
2
), and a
very toxic OH
•
radical[51]. In plants, formation of O
2
•−
occurs as a result of one-electron reduction of O
2
due to
its interaction with the photosynthetic ETC. The main
source of electrons for the ROS formation is the accep-
tor region of PSI, which contains low-potential elec-
tron carriers. Such electron donors can be represent-
ed by phylloquinone PhQ and low-potential iron-sul-
fur centers F
A
, F
B
, F
X
, as well as ferredoxin [99-102].
The electron from PSI is transferred to O
2
mole-
cule, which is reduced to the superoxide radical (O
2
+
+ e
–
→ O
2
•−
, the Mehler reaction[48,49]). The superox-
ide radical (O
2
•−
) is then reduced to hydrogen peroxide
and O
2
in the reaction catalyzed by superoxide dismutase
(2O
2
•−
+ 2H
+
→ H
2
O
2
+ O
2
) [51]. Quantitative estimates
of the Mehler reaction contribution to the ROS pro-
duction are ambiguous. Most authors consider that this
contribution is small, no more than ~10%; according
to other, the electron flow to O
2
measure up to 40% of
the total flow of electrons donated by PSII (for review,
see [49-51] and the literature cited therein). Neverthe-
less, even a relatively small electron flow from PSI to O
2
could be essential for the maintenance of optimal energy
balance in chloroplasts and protection of the photosyn-
thetic apparatus of plants against oxidative stress. Exam-
ples illustrating the effects of electron outflow from PSI
to O
2
include the experiments with injections of methyl
viologen– an artificial mediator of electron transport–
into a leaf, which significantly accelerates photooxida-
tion of P
+
700
, as well as experiments with varying O
2
con-
tent in the medium around the leaf, or in the suspension
of cyanobacterial cells[103-106].
Beside the electron flow to O
2
from the low-po-
tential acceptors of PSI [99-101], production of ROS
also involves the reduced ferredoxin [51, 102], the cy-
tochromeb
6
f complex, and the redox-active molecules,
plastosemiquinone and plastoquinone, located in the
thylakoid membrane [51, 107-109]. Contribution of PSII
to O
2
reduction is insignificant. When comparing the cy-
tochromeb
6
f and bc
1
complexes, it should be noted that
the b
6
f complex exhibits a noticeably higher (by an order
of magnitude) rate of O
2
reduction than the bc
1
com-
plex[107]. It is assumed that this can be explained by
the presence of chlorophyll a molecule near the PQH
2
binding site in the catalytic center Q
o
. The phytyl tail
of chlorophyll can pose steric restrictions that prevent
the shift of the plastosemiquinone molecule, which is
formed after the electron transfer reaction from PQH
2
to
the Rieske protein, towards the hemeb
6
L
. This prolongs
lifetime of the redox-active radical (plastosemiquinone)
and increases efficiency of the O
2
reduction.
The O
2
reduction under aerobic conditions by the
electrons donated by ETC carriers in the region be-
tween PSII and PSI, called chlororespiration, is well
studied[52-56]. In this case the PQH
2
pool is oxidized,
which can be assessed, for example, from the measure-
ments of the chlorophyll a fluorescence induction after
adaptation of the plant leaves to darkness [103, 104].
This process involves chloroplast terminal oxidase, which
oxidizes PQH
2
and provides electron transfer toO
2
mol-
ecule. Relative contribution of this pathway of O
2
re-
duction is small as compared to the contribution of the
light-induced Mehler reaction [51, 57, 58]. However,
chlororespiration emerges, for example, when we eval-
uate changes in the redox status of chloroplast ETC
during the plant adaptation to darkness. This pathway of
electron outflow to O
2
explains the relatively slow oxida-
tion (minutes to tens of minutes) of the plastoquinone
pool in the darkness. Finally, it is well known that the
oxygen uptake can occur in the intact chloroplasts at
Rubisco level due to the phenomenon called photorespi-
ration (see[1, 51] and the literature cited therein).
Another mechanism for O
2
reduction by plasto-
quinol was proposed in the works of Boris Nikolaevich
Ivanov etal. (see Ivanov et al.[51] for a review). The re-
sults of these studies suggest that the redox-active plas-
tosemiquinone molecules (mostly deprotonated form
ofPQ
•−
), which can form as a result of the compropor-
tionation reaction (PQH
2
+ PQ ↔ 2PQH
•
↔ 2PQ
•−
+
+ 2H
+
), can serve as electron donors for O
2
reduction.
Reactive radicals PQ
•−
are oxidized when interacting
with O
2
. This can also explain the fact that the content
of PQH
2
in chloroplasts gradually decreases under aer-
obic conditions in the darkness[103, 104]. In the works
of Ivanov et al.[51, 108, 109] the idea was also proposed
and substantiated that, in addition to the reduction
of O
2
molecules in the plastoquinone pool by plastose-
miquinone molecules, perhaps a more important role
is played by the reduction of superoxide anion radicals
formed both in this pool and in PSI. According to the
authors, this process is involved both in the detoxifica-
tion of O
2
•−
and, more importantly, in the production
of signal molecules of hydrogen peroxide, which, when
formed in this way, serve as messengers that transmit
ELECTRON TRANSPORT IN CHLOROPLASTS 1447
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
information to other systems of the chloroplast and the
cell about state of the ETC–sensitive sensor of environ-
mental conditions.
Permeability of thylakoid membranes to O
2
. With-
out spending much time on how the changes in O
2
concentration inside the plant cell can affect function-
ing of other metabolic systems[94-96], we should note
some properties of the chloroplast membranes that are
important for understanding the features of their in-
teraction with O
2
. It has been established that there is
a relatively rapid equalization of oxygen concentrations
inside the leaf and in the surrounding atmosphere. This
was demonstrated using EPR and solid oxygen-sensi-
tive paramagnetic particles that were injected into the
leaf with a microsyringe [110]. Such leaf samples re-
tain the ability to release O
2
in the light due to the PSII
functioning and to absorb O
2
in the darkness through
respiration. At the same time, O
2
concentration inside
the leaves, which are in contact with air, remains virtu-
ally unchanged in response to turning the light on and
switching it off[110].
It should be also noted that the rate of O
2
diffu-
sion through the thylakoid membranes is high, as de-
termined by probing the membranes with lipid-soluble
spin probes[111]. Permeability for O
2
of the lipid bilayer
of spinach chloroplast thylakoid membranes, measured
at 20°C, is 39.5 cm⋅s
–1
, which is 20% higher than the
permeability coefficient of a water layer of the same
thickness as the lipid membrane. High membrane per-
meability for O
2
can help equalize O
2
concentrations in
different compartments of the plant cell. According to
the estimates given in Ligeza et al.[110], the trans-thyla-
koid difference in O
2
concentrations is small, it does not
exceed 1 μM. Such good “ventilation” of the interior
of plant leaves indicates that significant accumulation
ofO
2
inside the plant cells, which could arise due to the
water oxidation in PSII, does not occur; this would limit
the ROS generation.
Good aeration of the plant cell should ensure that
the photosynthetic apparatus of chloroplasts would ef-
fectively interact with other metabolic systems of the
plant cell. Indicative is the fact that when O
2
is removed
by incubation of the leaves with an inert gas (N
2
orAr),
concentration of the oxidized P
+
700
centers sharply de-
creases, when the leaves are illuminated by the light
exciting both photosystems [103-105]. One of the ex-
planations for this phenomenon is restriction of the elec-
tron outflow from PSI to O
2
, which prevents oxidation
of P
700
. Excessive reduction of electron carriers on the
acceptor side of PSI, which are unable to accept elec-
trons from P
700
, would accelerate the charge recombina-
tion in PSI, preventing photo-oxidation of P
700
. Under
these conditions, singlet oxygen, a very dangerous form
of ROS, can be formed[112]. It could also be assumed
that functioning of the respiratory chain of the plant cell
mitochondria slows down and/or functioning of other
metabolic systems is disrupted during O
2
deficiency.
This would decrease consumption of NADPH, the final
electron acceptor in PSI, preventing, therefore, the P
700
oxidation.
Interaction of ascorbate with chloroplasts. A special
role is played in plant metabolism by the redox processes
involving ascorbate, which can interact with the ETC of
chloroplasts and neutralize ROS[59]. Ascorbate content
in the plant tissues can be high; in some species its con-
centration is 20-50 mM, depending on the species and
plant cultivation conditions[113-116]. Physiological role
of the reactions involving ascorbate is associated with
establishing redox homeostasis in the plant cells and
with protection of the photosynthetic apparatus from
the damage caused by ROS. Ascorbate, together with
glutathione, acts as a redox buffer when changes in ex-
ternal conditions (e.g., fluctuations in the intensity and
spectral composition of light) are capable of disrupting
redox state of the plant cell[59]. Ascorbate prevents in-
hibition of photosynthetic processes under stress condi-
tions (excess light, lack of moisture, etc.). Chloroplasts
possess a complex multi-stage system of biochemical
reactions that prevent accumulation of H
2
O
2
[51, 59].
Ascorbate is an effective antioxidant; it serves as a ROS
scavenger by participating in the enzymatic reduction of
H
2
O
2
and superoxide radicals; enzymatic oxidation of
Asc to monodehydroascorbate (MDHA) in the ascor-
bate peroxidase reaction catalyzing conversion of H
2
O
2
to H
2
O could be an example. Ascorbate is an electron
donor for the violaxanthin–deepoxidase reaction (vio-
laxanthin → zeaxanthin), which results in the increase of
thermal energy dissipation in the PSII light-harvesting
antenna (non-photochemical quenching of chlorophyll
excitation)[117-119].
Ascorbate and its oxidized forms (monodehydro-
ascorbate, MDHA, and dehydroascorbate, DHA) in-
teract with chloroplasts at various sites in the ETC.
Theaverage redox potential (E
m
′) of the Asc/DHA pair
is ~60-90 mV [120]. This means that ascorbate (in its
fully reduced form) can interact as an electron donor
with the chloroplast ETC in the region between PSII
and PSI, while its oxidized forms serve as electron ac-
ceptors for the electrons donated by PSI. Due to this,
ascorbate helps to maintain optimal redox status of the
plant cell. Figure 5 shows a diagram demonstrating how
the reduced and oxidized forms of ascorbate interact
with the chloroplast ETC. The fully reduced form of
ascorbate(Asc) can serve as an electron donor on the
donor side of PSII and at the plastocyanin section of
ETC between PSII and PSI [121,122]. Ascorbate pen-
etrating into thylakoids is capable of reducing Pc, which
in turn, serves as an effective electron donor for PSI.
The flow of electrons from Asc to PSI can reach ~50-70%
of the non-cyclic electron transport[121]. In one-electron
oxidation of Asc, it is converted into MDHA radical,
which produces the characteristic doublet EPR signal.
TIKHONOV1448
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 5. Interactions of various forms of ascorbate (Asc, MDHA, DHA) with chloroplasts. Designations: APX,ascorbate peroxidase; GR,glutathi-
one reductase; GSH and GS-SG,reduced and oxidized forms of glutathione; Cat,catalase; MV,methyl viologen; MDHAR,MDHA reductase;
SOD,superoxide dismutase.
It should be noted that Asc can directly reduce oxidized
P
+
700
centers, which has been clearly demonstrated by
the experiments with the isolated PSI complexes[122].
Inthis case, however, the reduction rate of P
+
700
is low;
the characteristic reduction time of P
+
700
is ~20-30 s,
which is significantly slower as compared to the reduc-
tion of P
+
700
by Pc
–
.
Asc oxidation is a reversible process. Reduction of
the semiquinone form of MDHA to the fully reduced
Asc form occurs via different ways. MDHA can receive
an electron directly from PSI[51,123]. The characteristic
rate of electron transfer from PSI to MDHA, leading to
formation of the fully reduced form of Asc, is compa-
rable to the rate of electron transfer between photosys-
tems. MDHA molecules can also accept electrons from
the reduced ferredoxin (Fd
–
) and NADPH [51, 124].
The rate of MDHA reduction depends on Fd
–
con-
centration and, under certain conditions, can be many
times higher than the rate of the light-induced reduction
of NADP
+
by FNR.
Another pathway of the reduced Asc form forma-
tion is disproportionation reaction of MDHA radicals
(2MDHA ↔ Asc + DHA)[122]. The resulting fully ox-
idized DHA form can then be reduced to Asc. In this
process, an important role is played by the glutathione
oxidase reaction, in which reduced glutathione serves as
an electron donor[59].
Thus, in the same way as the pseudocyclic electron
transport (the “water–water” cycle) and cyclic electron
transfer around PSI protect the photosynthetic appara-
tus from the excessive light energy, reactions involving
ascorbate protect the photosynthetic apparatus from
the damage under excessive light, promoting energy
dissipation into heat and antioxidant protection of the
plant cell. Ascorbate can serve as an alternative medi-
ator of electron transfer in chloroplasts: by stimulating
the outflow of electrons from PSI and Fd
–
, MDHA
prevents excessive reduction of carriers at the accep-
tor site of PSI. On the other hand, reduced ascorbate
molecules could compensate for the reduction of pho-
tochemical activity of PSII caused by adverse envi-
ronmental factors and could serve as electron donors
in the electron transport chain section between PSII
and PSI[125-127].
ELECTRON TRANSPORT IN CHLOROPLASTS 1449
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
CONCLUSION
Optimal functioning of the photosynthetic electron
transport in chloroplasts is achieved primarily through
regulation of the electron transfer in two sections of
the electron transport chain: between PSII and cyto-
chromeb
6
f complex and at the stage of electron outflow
from PSI to the CBC. An important role in these pro-
cesses is associated with the structural and functional
rearrangements of the photosynthetic apparatus, which
determine sustainability of chloroplasts and their capa-
bility to respond quickly to the changes in external con-
ditions, as well as alternative redox mediators (O
2
and
ascorbate), which ensure redox homeostasis of the plant
cell and the ETC stability.
Funding. This work was carried out within the
framework of the research topic of the Faculty of Phys-
ics of Lomonosov Moscow State University “Phys-
ical Foundations of the Structure, Functioning and
Regulation of Biological Systems” (State registration
no. 012004 085 35) and with partial financial support
from the Russian Foundation for Basic Research (grant
no.21-04-20047).
Acknowledgments. This article is dedicated to the
memory of L. A. Drachev– one of the greatest Russian
experimental biophysicists, whose scientific and techni-
cal developments and pioneering research in the field of
photosynthesis contributed to a deeper understanding of
the processes of charge separation and transfer in the re-
action centers of photosynthetic systems.
The author is grateful to E. K. Ruuge, G. B. Khomu-
tov, and L. Yu. Ustynyuk, together with whom the main
results of the experimental and theoretical study of the
regulation of electron transport in chloroplasts were
previously obtained, to which the author refers in this
review. The author thanks the anonymous reviewers for
their helpful comments and recommendations.
Ethics declarations. The author declares no conflict
of interest in financial or any other sphere. This article
does not contain any studies with human participants
oranimals performed by the author.
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