Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 840-859 © Pleiades Publishing, Ltd., 2025.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 7, pp. 915-936.

840

REVIEW

Photosynthetic Control and Its Role in Protection

of PhotosystemI against Photoinhibition

Daria V. Vilyanen

and Marina A. Kozuleva

1,a

Institute of Basic Biological Problems, Federal Research Center

“Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”,

142290 Pushchino, Moscow Region, Russia

e-mail: marina.kozuleva@pbcras.ru

Received April 18, 2025

Revised July 7, 2025

Accepted July 7, 2025

Abstract— This review addresses photosynthetic control as a protective mechanism that prevents photoinhi-

bition of photosystem I under conditions of imbalance between CO

assimilation during the Calvin–Benson–

Bassham cycle and light reactions in the thylakoid photosynthetic apparatus. We discuss the pathways of

photosystem I photoinhibition and describe protective mechanisms that prevent photodamage of photosys-

tem I. We propose a hypothesis regarding the influence of photosynthetic control on formation of reactive

oxygen species in photosystem I. pH-sensitivity of plastoquinol oxidation at the quinol-oxidizing (Qo) site of

the cytochrome b

f complex is analyzed, and function of two proton-conducting channels that release protons

into the thylakoid lumen from the cytochrome b

f complex is described. We examine impact of photosyn-

thetic control on the functioning of the cytochrome b

f complex itself, and propose a hypothesis regarding

the preferential activation of photosynthetic control in the thylakoid grana, which ensures operation of the

cyclic electron transport around photosystemI as a main protective mechanism.

DOI: 10.1134/S0006297925601121

Keywords: photosynthesis, photosynthetic electron transport chain, photosynthetic control, cytochrome b

complex, photosystemI, photoinhibition, PGR5, cyclic electron transport, reactive oxygen species

* To whom correspondence should be addressed.

INTRODUCTION

Variation in the intensity of environmental fac-

tors could disrupt the balance in plants between light

harvesting, charge separation within the thylakoid

photosynthetic machinery, and CO

assimilation in

the Calvin–Benson–Bassham cycle (CBB cycle), lead-

ing to decline in photosynthetic activity – the so-

called photoinhibition (PI). The primary targets of PI

are photosynthetic reaction centers, photosystem  II

(PSII), and photosystem  I (PSI). Photoinhibition of

PSII (PI(II)) has been thoroughly characterized, while

photoinhibition of PSI (PI(I)) remained undetected

in whole plants until 1994, and PI(I) was observed

only in in vitro experiments using isolated structures.

PI(I) in plants was first observed in the cold-sensi-

tive cucumber at low temperatures [1] and was later

reported for several other species (reviewed in [2]).

A breakthrough came from the experiments using

artificial fluctuating light (FL), which imitated chang-

ing natural light environment [3]. Later, other pro-

tocols for triggering PI(I) were developed, including

repetitive short pulses of saturating light (rSP) [4].

It is now evident that PI(I) poses a greater threat

to plant viability than PI(II), because repair of the

damaged PSI complexes requires a day or more, de-

pending on the damage severity [5], whereas PSII is

repaired within hours. A consensus has emerged that

PSI in plants is better protected than PSII [6]. The de-

fense mechanisms differ among the groups of photo-

synthetic organisms [7]. In angiosperms, a key protec-

tive pathway is cyclic electron transport around PSI

(CET(I)), which in C

plants operates mainly via the

Proton Gradient Regulation5 (PGR5)-dependent route.

Accordingly, the Arabidopsis thaliana mutant lacking

PGR5 protein cannot survive under FL conditions

[3] due to severe PI(I) [8, 9]. Several authors [2, 7]

have proposed existence of a universal mechanism

PHOTOSYNTHETIC CONTROL AND PHOTOSYSTEM I PHOTOINHIBITION 841

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

protecting PSI in various groups of photosynthetic

organisms, namely photosynthetic control (PhotCon).

PhotCon involves slowing down the photosyn-

thetic electron transport at the step of plastoquinol

(PQH

) oxidation within the cytochrome b

f complex

(Cyt-b

f ), triggered by acidification of the thylakoid

lumen. Imp`aired activation of PhotCon is often con-

sidered as the primary cause of the elevated PI(I) ob-

served in the pgr5 mutants [8], although this remains

a matter of debate [10]. The PI(I) phenomenon has

been reviewed in several recent publications, as well

as effects of the pgr5 mutation, and PhotCon itself [2,

6, 11, 12]. Nevertheless, the precise mechanisms by

which lumen pH modulates the rate of PQH

oxida-

tion in Cyt-b

f and the way PhotCon prevents PI(I) are

still poorly understood. Moreover, recent reviews pay

little attention to accumulating experimental evidence

that PhotCon indeed protects PSI, and they seldom

critically assess the strength of that evidence. It also

remains unclear whether PhotCon fulfils functions

beyond the PSI protection; for instance, review [12]

considers defense against PI(I) as the sole function of

PhotCon. These issues are addressed in the present

review.

STRUCTURE AND FUNCTIONING

OF THE PHOTOSYNTHETIC ELECTRON

TRANSPORT CHAIN OF HIGHER PLANTS

Light harvesting by photosynthetic apparatus initi-

ates electron flow through the photosynthetic electron

transport chain (PETC). In PSII, electrons from water

are used for reduction of lipid-soluble prenylquinone,

plastoquinone (PQ), to PQH

, which diffuses within the

lipid bilayer to Cyt-b

f, where it is oxidized, and elec-

trons are used for reduction of the lumenal copper

protein plastocyanin (Pc). Pc then donates electrons to

PSI, which reduces the stromal carrier ferredoxin (Fd).

Fd feeds electrons into various metabolic pathways of

the chloroplast [13], primarily reduction of NADP

NADPH catalyzed by ferredoxin:NADP

oxidoreductase

(FNR). Electron flow through the PETC is coupled with

generation of the proton-motive force (pmf) across the

thylakoid membrane, which consists mainly of the

trans-thylakoid pH gradient (ΔpH) [14]. The pmf drives

rotation of the thylakoid ATP-synthase, producing ATP

from ADP and Pi. The CBB cycle uses ATP and NADPH

in a ratio of 1.5. Given the number of H

-transport-

ing c-subunits of the chloroplast ATP-synthase, it was

calculated that linear electron flow yields an ATP/

NADPH ratio of only ~1.29 [15]. Therefore, additional

ATP synthesis is required for optimal functioning of

CBB cycle, which is produced in chloroplasts via al-

ternative electron transport pathways that contribute

to ΔpH formation without producing NADPH.

In angiosperms, CET(I) operates via two routes:

an antimycin-A (AA)-sensitive, PGR5-dependent path-

way that is predominant in C

plants, and an AA-in-

sensitive pathway mediated by the NADH-dehydroge-

nase-like (NDH) complex that is predominant in C

species [16, 17]. In both routes, Fd reduces the PQ

pool, which is then re-oxidized by Cyt-b

f, returning

electrons to PSI. NDH is homologous to the respiratory

Complex I but accepts electrons from Fd rather than

NAD(P)H. The Cyt-b

f itself is likely an enzyme that

oxidizes Fd and reduces PQ in the AA-sensitive CET(I)

pathway [18] (see reviews [19, 20] for details).

Molecular O

serves as an alternative electron

sink for PETC. During photorespiration, for example,

RUBISCO catalyzes an oxygenase reaction with O

in-

stead of carboxylase reaction with CO

; since regen-

eration of the formed 3-phosphoglycerate requires

ATP and reducing equivalents generated by linear

electron flow, photorespiration is considered as an

-dependent alternative electron pathway [7]. Addi-

tional routes ultimately reduce O

to H

O within the

chloroplast; collectively, these are termed water–water

cycles. One such cycle begins with direct reduction

of O

by PETC components, yielding superoxide anion

radical (O

•−

) as the primary product, and hydrogen

peroxide (H

) as the stable product. H

is then

detoxified to H

O with involvement of ascorbate and

ascorbate peroxidase, and ascorbate is regenerated

by electrons supplied by the PETC [21]. A shorter wa-

ter-water cycle is driven by the plastid terminal oxi-

dase, which reduces O

to water while oxidizing PQH

A four-electron reduction of O

to H

O by NADPH mol-

ecules catalyzed by flavodiiron proteins occurs in all

photosynthetic organisms except angiosperms [22].

Substantial evidence indicates that PSII, Cyt-b

and PSI are distributed heterogeneously within the

thylakoid membrane. Cryo-electron microscopy of

intact spinach chloroplasts, published in 2025 [23],

shows that the grana stacks contain only PSII and

Cyt-b

f, whereas stromal lamellae contain PSI, Cyt-b

and ATP-synthase. The grana-localized PSII and Cyt-b

perform linear electron transport to PSI complexes in

stromal thylakoids, with Pc shuttling electrons from

grana to stromal regions [24]. Stromal thylakoid Cyt-

f and PSI perform CET(I).

PHOTOINHIBITION OF PHOTOSYSTEM I

PSI is a multisubunit pigment–protein com-

plex embedded in the thylakoid membrane. Its elec-

tron-transfer cofactors are located in three protein

subunits – PsaA, PsaB, and PsaC. Cofactors, from the

primary donor P

700

(a chlorophyll-a special pair) to

the Fe

-S

cluster F

, are arranged in two pseudo-sym-

metrical branches (A- and B-) within the PsaA/PsaB

VILYANEN, KOZULEVA842

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig.  1. Organization of the PSI electron-transfer chain and ROS-generating pathways involving PSI cofactors: 1) formation

of

via the reaction of O

with P

700

triplet (

700

) produced during charge recombination with intermediate PSI cofactors.

2) Formation of O

•−

via reaction of O

with (a) the F

clusters and (b) the phylloquinone in the A

-sites. 3)Formation of

•

via reaction of H

with F

clusters. Thick blue arrows indicate forward electron transfer within PSI and onward

to Fd, NADP

, and the CBB cycle; dashed arrows denote charge recombination. All other arrows depict ROS-generation

routes involving PSI cofactors. The scheme is based on the structure of PSI in complex with Pc and Fd (PDB: 6YEZ) [31].

heterodimer; electrons are transferred from F

to two

terminal Fe

-S

clusters, F

and F

, on the PsaC sub-

unit (Fig. 1). In addition to P

700

, four more chloro-

phyll-a molecules participate in electron transfer: the

two most distant from P

700

serve as the A

cofactors

on the A and B branches. Between each A

and cluster

a phylloquinone molecule is located in the A- and

B-branches (the A

cofactor). The oxidized P

700

)

is reduced by Pc, while the F

clusters donate elec-

trons to Fd.

PSI can also produce reactive oxygen species

(ROS). O

•−

is produced via reduction of molecular O

by the terminal clusters F

(whose contribution sat-

urates at moderate light intensities) and the phylloqui-

nones in the A

-sites, mainly in the A branch (whose

contribution increases with increasing light intensity)

[25] (Fig. 1). Generation of O

•−

by PSI cofactors oc-

curs in parallel with Fd reduction [25]. Under NADP

limitation, the reduced Fd itself produces O

•−

, but

sufficient NADP

minimizes electron leakage from Fd

to O

[26]. In the stroma, O

•−

undergoes dismutation

to H

, which is scavenged by the chloroplast anti-

oxidant system. When H

production outpaces its

detoxification, H

accumulates. The light-reduced

clusters can then catalyze conversion of the ex-

cess H

into hydroxyl radicals (HO

•

) [27]. Several

lines of indirect evidence also suggest that PSI can

generate singlet oxygen (

) [28, 29]. Production of

is feasible via the P

700

triplet (

700

) formed during

charge recombination between P

700

and A

−

but not

between P

700

and the terminal F

clusters [30].

In the pioneering study that first demonstrated

PI(I) in a cucumber plant under chilling stress, re-

moval of O

markedly preserved PSI activity, impli-

cating ROS formation by PSI as the underlying cause

of PI(I) emergence [1]. Currently, the data have been

PHOTOSYNTHETIC CONTROL AND PHOTOSYSTEM I PHOTOINHIBITION 843

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

accumulated indicating that the ROS-dependent PI(I)

proceeds via two distinct mechanisms, depending

on the triggering stress, which leads to the primary

damage on either acceptor or donor sides of the PSI

complex.

Under chilling stress, the initial damage occurs

at the Fe

-S

clusters of PSI [32, 33], whereas destruc-

tion of P

700

itself requires more severe treatments

[32]. In vitro experiments with the illuminated spin-

ach thylakoids revealed that HO

•

scavengers provide

protection against PI(I) [34] and that exogenous H

accelerates PI(I) [35]. Importantly, photoinhibition was

strongly suppressed by adding methyl viologen (MV),

an efficient acceptor of electrons from PSI [34, 35].

Because MV itself enhances H

production during

illumination, these findings indicate that the critical

factor for PSI photodamage is not the amount of H

accumulated per  se, but rather efficiency of the elec-

tron outflow from PSI. Consistent with this view, MV

lowered HO

•

generation in thylakoids [27]. It was con-

cluded based on the data in [34, 35] that in the case

of inefficient electron outflow from PSI the reduced

cofactors reduce O

producing H

, and next, the re-

duced terminal clusters F

catalyze HO

•

formation

from that H

, acting as a Fenton-type catalyst [36].

Loss of EPR signal from the F

clusters has

been reported both in the Arabidopsis pgr5 mutant

and in the wild-type (WT) plants exposed to high light

[37, 38], suggesting that the similar PI(I) mechanism

related to the impaired electron outflow from PSI is

realized in the plants under FL and high light.

By contrast, the cucumber leaves subjected to rSP

(a long series of 300 ms saturating flashes separated

by 10 s dark intervals) show no loss of the EPR sig-

nal of the F

clusters [33]. Thus, the rSP treatment

does not promote HO

•

production on the acceptor side

of PSI, even though each flash fully reduces the PSI

cofactors and Fd [39], a condition that would favor

over-reduction. After the rSP treatment, however, the

PSI complexes show a discrepancy between the ki-

netics of charge separation and the EPR signal of F

[33], interpreted as structural damage between P

700

and A

in the A-branch, caused by the ROS generated

in close vicinity of P

700

and A

-sites. Detailed study of

the rSP-induced PI(I) in the spinach chloroplasts [28]

assumed that both O

•−

produced at the A

-sites and

contribute to the damage. The proof of

in-

volvement is based on the protective effect of an am-

phiphilic vitamin E analogue, a specific

quencher.

The similar effect of

scavengers had been observed

previously in the PSI-enriched thylakoid membranes

devoid of PSII [40]; these preparations cannot form

because electron donation to PSI is absent, yet

can be formed through charge recombination,

which is the only pathway of P

700

re-reduction un-

der those conditions. Involvement of O

•−

from the

-sites, however, is less firmly supported by the ex-

isting evidence. MV provided significant protection

during the rSP treatment [28], and MV indeed sup-

pressed O

•−

generation at the A

-sites [25]. However,

MV also diminishes charge recombination in PSI [41],

so its protective action may stem from the reduced

formation rather than from suppression of O

•−

formation at the A

-sites. Nonetheless, lack of direct

evidence does not rule out a contributory role for the

•−

generated at the A

-sites in PI(I) caused by rSP

treatment.

A series of studies employing the rSP protocol led

to formulation of the “P

700

oxidation” concept as the

principal mechanism protecting PSI against photoin-

hibition [7]. This term describes presence of the de-

tectable P

700

signal under actinic light, which is sus-

tained both by electron outflow from PSI to Fd, which

is manifested by low values of the quantum yield of

non-photochemical losses on the acceptor side of PSI,

Y(NA), and by the regulatory decrease in electron in-

flow to PSI, which is manifested by the high values of

quantum yield of non-photochemical losses on the do-

nor side of PSI, Y(ND). Accordingly, the PSI-protective

pathways fall into three categories: 1) mechanisms

regulating electron outflow from PSI, 2)  mechanisms

regulating electron inflow to PSI, 3)  CET(I), which

should be considered separately (see below).

The first group includes the CBB cycle and alter-

native oxygen-dependent pathways, which promote

oxidation of Fd and NADPH pools and, subsequent-

ly, of P

700

[42,  43]. The CBB cycle is the primary sink

of electrons from PSI; therefore, ensuring its optimal

operation is the key PSI-protection strategy. For in-

stance, increasing CO

concentration from 400 ppm

to 800  ppm (the CBB cycle-optimum for C

plants)

markedly reduced PI(I) in the Arabidopsis leaves un-

der FL [44]. When the CBB cycle activity is restricted,

alternative pathways provide auxiliary Fd oxidation,

mitigating PI(I). Notably, these alternative pathways

also contribute to ΔpH formation, supplying extra ATP

for the CBB cycle and other metabolic processes in

chloroplasts, as well as activating defense mechanisms

regulating electron inflow to PSI.

The second group includes (i)  PhotCon; (ii)  down-

regulation of PSII activity [45]; (iii)  diversion of elec-

trons to an alternative acceptor upstream of PSI, most

notably via oxidation of the PQ pool by O

mediat-

ed by the plastid terminal oxidase [46] and, possibly,

spontaneous PQ reactions with O

and ROS [47, 48];

(iv)dynamic modulation of grana diameter, which af-

fects the rate of Pc diffusion from the grana-embedded

Cyt-b

f to PSI complexes in the stromal lamellae [24].

A substantial body of evidence indicates im-

portance of CET(I) in protecting PSI. The Arabidop-

sis double mutants lacking both the NDH complex

and the PGR5 protein are virtually unviable [16].

VILYANEN, KOZULEVA844

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 2. Visualization of proton-releasing channels of the Qo site of Cyt-b

f. Overall arrangement of channels in spinach

Cyt-b

f (PDB 9ES9) [56](a). E-channel in the spinach Cyt-b

f (PDB 9ES9) rendered according [55](b). H-channel in the spin-

ach Cyt-b

f (PDB 9ES9) rendered according [57-59](c). H-channel with bound H

O molecules in the Cyt-b

f from Nostoc sp.

PCC 7120 (PDB 4H44) [60] (d). Channels were visualized with MOLE 2.5 (https://moleonline.cz) and ChimeraX 1.9 (https://

www.cgl.ucsf.edu/chimerax/).

The mutants that lack only the NDH complex show

enhanced PI(I) under FL [49]. Infiltrating the Arabi-

dopsis leaves with antimycin A (AA) likewise causes

a marked loss of PSI activity under FL [50]. In the

mutants with impaired binding of thylakoid FNR,

which likely acts as a regulator switch between the

linear electron transport and AA-sensitive CET(I) [51],

the increased PSI photoinhibition was observed upon

transfer of plants to high light [52]. Numerous studies

further demonstrate that the pgr5 mutations impair

700

oxidation, making PSI more vulnerable to pho-

toinhibition under both FL and high light [8, 37, 50].

Because CET(I) recycles electrons, its steady-state

operation inherently produces equal electron outflow

from and inflow to PSI; therefore, by itself it cannot

keep P

700

oxidized. Therefore, the protective role of

CET(I) is attributed to its proton-pumping activity,

which (i) generates additional ATP without concomi-

tant NADPH production, thereby optimizing CBB cycle

turnover and indirectly relieving the acceptor-side lim-

itation of PSI, and/or (ii)acidifies the thylakoid lumen,

thereby triggering PhotCon and indirectly activating

defense mechanisms regulating electron inflow to PSI.

Thus, unraveling the complete picture of molecu-

lar mechanisms protecting PSI is complicated by the

challenge of separating effects on the acceptor and

donor sides of PSI. It is possible that both factors

are at play simultaneously. Taking into consideration

existence of mechanisms of PI(I) development – one

causing primary damage on the acceptor side, and

the other on the donor side – it seems plausible that

regulation of electron outflow from PSI is crucial for

preventing the acceptor-side PI(I), whereas regula-

tion of electron inflow is crucial for preventing the

donor-side PI(I). Nevertheless, several experimental

lines of evidence (see below) indicate that PhotCon

could also protect PSI under conditions that induce

the acceptor-side PI(I).

PHOTOSYNTHETIC CONTROL

Oxidation of PQH

at the Qo site of Cyt-b

f and

proton release into the lumen. The cytochrome-b

complex (Cyt-b

f ) is a functional dimer (Fig.2a). Each

monomer comprises cytochrome (cyt) f, cyt b

, Rieske

PHOTOSYNTHETIC CONTROL AND PHOTOSYSTEM I PHOTOINHIBITION 845

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 3. Electron transfer in Cyt-b

f. Redox-potential diagram (E

m,7

) of the Cyt-b

f electron transfer cofactors(a). Red dashed

arrow marks endergonic steps of the direct electron transfer. Quinone potentials (−170 mV for PQ/PQ

•−

and +370 mV for

•−

/PQH

) are for aqueous solution because site-specific values are unknown. E

m,7

values for the low-potential branch

are from [64]; for the high-potential branch – from [65]. Scheme of ROS formation in the Qo site (b). For simplicity,

some reactants and products are omitted. 1) Oxidation of PQ

•−

(PQH

•

) and heme b

by O

. 2) Dismutation of O

•−

to H

3)Fe

-S

-catalyzed decomposition of H

to HO

•

. 4)Reduction of O

•−

to H

by PQH

bound in the Qo site. The hourglass

symbol denotes a hypothesized slowdown of the reaction when PhotCon is activated.

iron–sulfur protein (ISP), subunit IV (sub IV), and

small subunits PetG, PetL, PetM, and PetN. Its pros-

thetic groups include: Fe

-S

cluster in the ISP, two

b-type hemes in cyt b

(hemes b

and b

, oriented to-

wards the positively charged lumenal p-side and neg-

atively charged stromal n-side, respectively), c

-type

heme in cyt b

, c-type heme in cyt f, one chlorophyll a,

and β-carotene in sub IV (for detailed architecture see

[19, 20, 53]). PQH

and PQ diffuse into their respective

binding sites through the inter-monomer cavity [54]:

the quinol-oxidizing Qo site on the luminal side and

the quinone-reducing Qr site on the stromal side. A

phytyl “tail” of the chlorophyll a molecule occupies

the Qo site in either open conformation, permitting

PQH

access, or closed conformation, which restricts

PQH

access [55].

Oxidation of PQH

at the Qo site proceeds via elec-

tron bifurcation – oxidation of the two-electron donor

by the high-potential and low-potential single-elec-

tron acceptors (Fig. 3a). The first electron from PQH

is accepted by the Fe

-S

cluster of ISP, after which the

hydrophilic ISP domain rotates towards cyt f, chang-

ing its position from proximal to distal relative to the

heme b

, and the electron is then transferred to cyto-

chrome f and further to Pc (high-potential branch of

Cyt-b

f ). The second electron from PQH

is transferred

through the hemes of cyt b

to the PQ molecule in the

Qr site, thereby completing the Q-cycle (low-potential

branch of Cyt-b

f ).

Oxidation of PQH

is coupled to proton transfer

[61]. Two residues are critical for binding, stabiliz-

ing, and deprotonating PQH

and its semiquinone

form PQH

•

: H128 of the ISP [62], and E78 of sub IV

[60, 63] (spinach nomenclature, PDB 6RQF). Initially,

PQH

forms a hydrogen bond between its carbonyl

oxygen and N

atom of the H128; the first proton is

transferred to H128 (reaction 1a), and only then the

-S

cluster oxidizes PQH

−

to the neutral semiqui-

none PQH

•

(reaction 1b).

PQH

+ H128~ISP

→ PQH

−

+ H-H128~ISP

(1a)

PQH

−

+ H-H128~ISP

→ PQH

•

+ H-H128~ISP

red

(1b)

In the second step of the reaction, according to

quantum-chemical modelling, PQH

•

migrates within

the Qo site toward the heme b

[66]. PQH

•

forms a

hydrogen bond with the deprotonated carboxylate of

E78 (reaction 2a) and is next oxidized by the heme b

to PQ (reaction 2b).

PQH

•

+ E78 + heme b

→ PQ

•−

+ H-E78 + heme b

(2a)

•−

+ H-E78 + heme b

→ PQ +

+ H-E78 + heme b

red

(2b)

After oxidation, the PQ molecule leaves the Qo

site through the one-way diffusion channel [67] allow-

ing the next PQH

to bind. For the detailed thermody-

namic and kinetic aspects of PQH

oxidation, electron

and proton transfer coupling, see reviews [19, 20, 53].

VILYANEN, KOZULEVA846

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Because binding of PQH

and PQH

•

requires H128

(ISP) and E78 (sub IV) to be deprotonated, the overall

oxidation rate is governed by the efficiency of proton

dissociation from these residues, as has been shown in

studies using site-directed mutagenesis [58, 59, 63, 68].

Proton affinity of the H128 residue is redox-depen-

dent: when the Fe

-S

cluster is reduced, pK

(H128) is

~8.3-8.9, but when the cluster is oxidized, it drops to

~6-6.5 [69]. Thus, once the cluster donates its electron,

affinity of the H128 residue for H

decreases, and pro-

ton is released into the lumen. The proton from E78 is

likewise released to the lumen, but current evidence

suggests its transfer is not controlled by the redox

state of Cyt-b

f cofactors. The proton-releasing chan-

nels have been modelled in the Cyt-b

f structures [55,

57, 60]. We denote them as H-channel and E-channel,

responsible for releasing protons from H128 and E78,

respectively (Fig. 2, b-d).

The H-channel was first identified in the cyt  f

from Brassica rapa (PDB 1HCZ) [57] as an intraprotein

chain of five water molecules (Fig.2, c, d) coordinated

by highly conserved N and Q residues. The chain ex-

tends in two directions from H25 (the heme  c ligand

in cyt  f ): one water molecule is oriented toward the

ISP, while four water molecules stretch ~11  Å toward

K66, a residue involved in Pc binding on the luminal

surface. It remains uncertain how the proton leaves

H128: whether it is transferred directly to the first

water molecule or to the H25 (~3.5  Å away). Several

Chlamydomonas reinhardtii mutants carrying substi-

tutions of the residues that coordinate water mole-

cules in H-channel have been analyzed [58]. The mu-

tation N168F removed last two water molecules in the

chain, as a result the mutant lost the ability to grow

photoautotrophically. All H-channel mutants exhibited

slower cyt  f reduction and slower generation of the

slow phase of carotenoid electrochromic shift, a mark-

er for vectoral electron transfer along the low-poten-

tial branch of Cyt-b

f [58, 59]. Collectively, these data

confirm that the disruption of H-channel compromises

coordinated PQH

oxidation.

A hydrophilic region about 15.5 Å long and 4-6  Å

in diameter, beginning at E78 and extending toward

the lumen, was identified in the structure of Cyt-

f from Mastigocladus laminosus (PDB 4H13) [60];

this hydrophilic region is the primary candidate for

E-channel. In that structure, the E78 side chain faces

away from the Qo site toward the heme b

; its carbox-

yl group forms hydrogen bond with a water molecule

(at a distance of 3  Å) and with the side chain of R87 in

the cytb

(at a distance of 2.5  Å). The channel is lined

with polar residues – R87 and S91 (both in cytb

), E3

(PetG), and D58 (subunit IV). Later, a similar channel

was modelled in the spinach Cyt-b

f structure (PDB

6RQF; Fig. 2b) [55]. Here, E78, likewise, points toward

the heme b

, and the channel is lined with R87 and

S91 (cyt b

), E3 (PetG), E58 (sub IV), E5 (PetM), and

K145, E242, E34 (cyt f ).

It must be emphasized that the exact proton-re-

leasing trajectories from the Qo site to the lumen re-

mains uncertain; the existing suggestions are based

on hydrophilic intraprotein regions revealed by the

structural studies and site-directed mutagenesis data.

In the cytochrome-bc

complex (Cyt-bc

), modelling

predicts at least five distinct pathways for proton re-

lease from the Qo site, involving conserved cytb res-

idues and bound water molecules [70]. Whether the

similar multitude of proton release pathways exist

in Cyt-b

f remains unknown. No counterpart of the

H-channel has been found in cytc

[71]. Thus, proton

release from the Qo site of the Cyt-b

f could be under

tighter control than in the Cyt-bc

, which could be an

evolutionary adaptation to the lower luminal pH typ-

ical of illuminated chloroplasts.

pH-dependence of PQH

oxidation (photosyn-

thetic control). Influence of pH on PQH

oxidation

in the Qo site has been revealed from pH-dependence

of the P

700

[72-74], cyt f, and cyt b

reduction [75].

Slowdown of PQH

oxidation with the luminal pH de-

crease is attributed to accumulation in the lumen of

one reaction product, H

. However, catalytic center of

the Qo site is spatially isolated from the lumen, pre-

venting direct contact between PQH

and H

accumu-

lated in the lumen. This poses a question, how does

the Qo site mechanistically “senses” acidification of

the lumen? The prevailing view is that PhotCon aris-

es from the backpressure exerted by the increasing

content of H

in the lumen on the proton-accepting

groups in the Qo site [76]. A simple model in which

PQH

oxidation is slowed down due to the luminal

accumulation of H

, however, has been criticized [77]:

since the E

difference between PQH

and Pc is ap-

proximately 300mV (Fig. 3a), ΔpH values larger than

those normally attained under typical physiological

conditions would be required to slow down electron

transfer from PQH

to Pc (see also [20]), although

such extreme lumen acidification might occur during

stress [78, 79]. Finazzi et al. proposed an alternative

interpretation of PhotCon [77] based on existence of

a pH-dependent regulation of Cyt-b

f′s transition be-

tween two kinetic states: location of the hydrophilic

ISP domain near the Qo site or near cyt f. According

to this hypothesis, acidification of the lumen would

hinder deprotonation of H128, prolonging residence of

the ISP domain next to cyt f and thus slowing PQH

oxidation in the Qo site. Whether the ISP can revert

to the Qo proximal conformation while H128 remains

protonated is unknown; given the likelihood of sto-

chastic nature of ISP conformational shifts [80], this

possibility cannot be excluded.

Threshold of the luminal pH for PhotCon acti-

vation (~6) corresponds to the pK

of H128 when

PHOTOSYNTHETIC CONTROL AND PHOTOSYSTEM I PHOTOINHIBITION 847

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

the Fe

-S

cluster is oxidized (~6-6.5). This allowed

suggesting a hypothesis according to which PhotCon

is triggered precisely by the prolonged lifetime of

the protonated H128 state [53, 77]. By contrast, the

predicted pK

of E78 is much lower, around 3.9 [60].

Atfirst glance, such acidic pK

suggests that at luminal

pH 5-6 E78 would not retain a proton long enough

to slow down PQH

•

oxidation.

Useful insights into the protonation state of H128

and E78 come from the studies with competitive in-

hibitors of PQH

oxidation at the Qo site. One of such

inhibitors, 2,5-dibromo-3-methyl-6-isopropylbenzoqui-

none (DBMIB), initially binds to H128 in the reduced

form (DBMIBH

), which is followed by oxidation to

semiquinone by the Rieske cluster resulting in a

strong binding of the Fe

-S

cluster in Qo site [56, 81,

82]. Therefore, the protonated H128 should interfere

with binding of the reduced DBMIB. Indeed, inhibi-

tory activity of DBMIB in the isolated pea thylakoids

decreased under conditions facilitating lumen acidi-

fication [83]. Moreover, inhibitory activity of DBMIB

was 3- to 10-fold lower in the Arabidopsis pgr1 mutant

[83], in which the P194L substitution in ISP raised pK

(H128) by ~1pH unit [84]. As a consequence, at pH7.6

(the condition used in [83]) the fraction of Cyt-b

f with

protonated H128 was about 7.5-fold higher in the pgr1

mutant than in the WT, which is in agreement with

the fold difference in DBMIB binding between the

genotypes.

Changes in luminal pH had an opposite effect on

another inhibitor of PQH

oxidation at the Qo site,

2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-

INT): acidic lumen pH promoted stronger DNP-INT

inhibitory activity in the pea and spinach thylakoids

[83, 85]. Binding of DNP-INT in the pgr1 mutant was

20-50% stronger than in the WT [83]. The DNP-INT

molecule lacks proton-donating groups and can only

act as a hydrogen-bond acceptor via its nitro groups.

Therefore, improved binding under conditions that

increase the fraction of Cyt-b

f with protonated H128

implies that ISP with the protonated H128 can still

reside at the Qo site. Thus, a longer residence time of

the protonated H128 in the proximal position would

restrict PQH

oxidation in the absence of inhibitors.

Since no fold difference in the DNP-INT binding was

observed between the pgr1 mutant and the WT, as

was the case with DBMIB, these results indicate that

the protonated H128 is neither the sole nor the key

group involved in DNP-INT binding.

Inhibitory activity of DNP-INT in the spinach

thylakoids is also enhanced when gramicidin D and

valinomycin were added together [85]. This effect has

been attributed to the ability of valinomycin in the

complex with potassium ions to shield the ionized

E/D side chains of proteins [86]. Such shielding could

mimic the protonated state of E-channel and interfere

with the H

release from E78, thus prolonging its pro-

tonated state and enabling formation of a hydrogen

bond with DNP-INT. Hence, the higher Qo site affinity

for DNP-INT at low luminal pH suggests that H

may

persist on E78 and/or on other E-channel components,

thereby slowing PQH

•

oxidation.

Substitution of E78 with K or L in C. reinhardtii

significantly slowed PQH

oxidation, especially at lu-

minal pH 5-6 [63, 68]. This finding led to the idea

that at neutral pH some alternative acceptor could

take over the deprotonation of PQH

•

, whereas under

acidic conditions E78 itself is indispensable for the

efficient H

removal from the Qo site [68]. Togeth-

er with the DNP-INT data [85], these results indicate

that the E-channel contributes to PhotCon alongside

the H-channel. Given that the predicted pKa of E78 is

around 3.9 [60], at a lumen pH of 5-6, E78 should be

in an ionized state. Therefore, it can be assumed that

retention of H

on it might be related to the stron-

ger local acidification of the lumen near the Cyt-b

or it might be determined by the retention of H

the intermediate proton-accepting groups within the

E-channel, which could possess higher pKa values.

For instance, the predicted pKa for the E3 (PetG) res-

idue in the E-channel is 4.5 [60].

Altogether, the proton-releasing channels of Cyt-

f appear to serve as sensors of luminal pH for Qo

site. They are hydrated cavities lined with bound

water molecules (H-channel) and/or ionizable groups

(E-channel) through which protons are transferred

from one acceptor to the next. Acidification of lumen

slows H

transfer along these channels, extending

lifetimes of the initial proton acceptors – E78 and

H128 – in their protonated forms. Different pK

val-

ues of E78 and H128, coupled with the fact that H128

deprotonation is redox-controlled whereas E78 depro-

tonation is not, suggest two tiers of PhotCon activation

that operate at different luminal pH levels. Unlike the

H-channel, the E-channel makes direct contact with

the Qo site. Hence, the prolonged protonated state of

E78 is likely to modulate both redox chemistry of plas-

toquinone species and ROS formation within the Qo

site (see below).

Link between photosynthetic control and pro-

tection of PSI against photoinhibition. Illuminat-

ing leaves with high light results in P

700

oxidation,

whereas other components of the chain become pre-

dominantly reduced [87]. This implies existence of a

regulatory process that slows electron flow to PSI, and

PhotCon is regarded as such process [88]. Yet this does

not automatically mean that PhotCon prevents PI(I).

The view of PhotCon as a PSI-protective mechanism

under conditions of photoinhibition rests on a set of

observations that are analyzed in this section.

Studying the impact of PhotCon is challenging

due to the shortage of reliable protocols to detect

VILYANEN, KOZULEVA848

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

its activation. Researchers often use the parameter

Y(ND) (fraction of PSI centers that can be oxidized in

the light [10]) and interpret it as shortfall of electrons

at the donor side of PSI caused by PhotCon. However,

such limitation could also result from the down-regula-

tion of PSII activity, or from the increased diversion of

electrons to an alternative acceptor upstream of PSI.

Suitability of the Y(ND) parameter as a PhotCon mark-

er has been questioned recently [12]. Moreover, plants

that display severe PI(I) typically show low Y(ND) val-

ues accompanied by high Y(NA) values, which is espe-

cially important given that inefficient electron outflow

from PSI causes the acceptor-side photodamage (see

above). An alternative method to assess PhotCon is to

measure the rate of P

700

re-reduction upon switch-

ing off the light. Slowdown of P

700

re-reduction with

increasing light intensity was observed in the Silene

dioica leaves [89], but not in the pea leaves [90]; as

suggested in [87], the latter study may have omitted

the very low light intensities at which PhotCon is ac-

tivated. Restriction of electron outflow from PSI has

also been reported to activate PhotCon [89, 91]. Cor-

relation between the P

700

re-reduction rate and the

fraction of open PSII centers measured in a wide range

of light intensities has been suggested as an estimate

for PhotCon [87].

Studying protective role of PhotCon is also com-

plicated by the difficulty of separating the effects on

the donor and acceptor sides of PSI. During chilling

stress, for instance, dissociation of the coupling fac-

tor of chloroplast ATP-synthase has been proposed

to raise conductivity of the thylakoid membrane to

protons, leading to collapse of ΔpH and absence of

PhotCon activation [2]. Yet that same dissociation also

decreases ATP synthesis, thus slowing the CBB cycle

and restricting electron outflow from PSI, which is

the primary trigger of the acceptor-side PI(I). Whether

PhotCon itself protects PSI under chilling stress there-

fore remains uncertain.

Infiltration of Arabidopsis leaves with diuron, an

inhibitor of electron transport from PSII to the PQ

pool, mitigated the loss of PSI activity in high light [8].

The authors interpreted this as evidence that PhotCon

prevents PI(I). In reality, absence of electron flow to

PSI also removes the electrons that would otherwise

reduce O

and generate HO

•

by the F

clusters, the

root cause of acceptor-side PI(I). Thus, diuron more

likely suppressed ROS formation in PSI rather than

imitated PhotCon activation. Infiltration of Arabidopsis

leaves with nigericin, a proton-potassium antiporter

that dissipates ΔpH but not the electrical component

of pmf, led to the significant loss of PSI activity in

high light [88]. According to the authors, ATP synthesis

by ATP-synthase complexes was not disrupted under

these conditions. However, because neither ATP-syn-

thase activity nor CO

assimilation were measured,

we cannot completely rule out an explanation asso-

ciated with insufficient ATP supply for optimal func-

tioning of the CBB cycle.

For several Arabidopsis mutants, a correlation

has been observed between the high level of PI(I) and

elevated values of conductivity of the thylakoid mem-

branes for H

(the gH

parameter), which are accom-

panied by the decrease in pmf. In the cfq (coupling

factor quick recovery) mutant, the point substitution

E244K in the γ

-subunit of ATP-synthase disrupts the

thiol-dependent regulation of the enzyme, thereby

increasing H

leakage through the c-ring of ATP syn-

thase [92]. In the hope2 (hunger for oxygen in pho-

tosynthetic electron transport reaction 2) mutant, the

G134D substitution in the same γ

-subunit is thought

to impair metabolic regulation of ATP-synthase, like-

wise enhancing the rate of H

efflux through the

c-ring [93]. In the Arabidopsis mutant DPGRox, the

variant expressing the potassium-proton antiporter

KEA3 with G422R point substitution, the increased H

efflux from the lumen to the stroma via this antiport-

er was observed [94]. Comparison of these mutants

suggests that the cause of PI(I) is the increased dissi-

pation of ΔpH, leading the authors to conclude that the

failure of PhotCon activation underlies PI(I). However,

the elevated gH

values in the DPGRox mutant may

indicate insufficient ATP synthesis for optimal CBB cy-

cle activity. For the cfq and hope2 mutants, it has been

proposed by the authors that the enhanced H

leak-

age through the c-ring of ATP-synthase is accompanied

by the extra ATP production [92, 93]. Yet the in vitro

experiments with the cfq mutant showed that both

the synthase and hydrolase activities of ATP-synthase

were lower than in the WT [95]. Indeed, all three

mutants exhibited not only low Y(ND) values, which

could be explained by the increased ΔpH dissipation,

but also high Y(NA) values [92-94, 96] indicating re-

stricted electron outflow from PSI.

In our opinion, the most convincing evidence

comes from the Arabidopsis pgr1 mutant, in which the

P194L substitution in ISP shifts activation of PhotCon

to more alkaline luminal pH values. In this mutant,

the PSI was inhibited to a lesser extent than in the

WT upon exposure to FL, indicating protective effect

of PhotCon in the context of PI(I) [10]. Introducing

the ISP-P194L point substitution into the pgr5 mutant

likewise made PSI more resistant to FL in the mature

plants in comparison with the single pgr5 mutant.

The Y(ND) values in the double mutant, though slightly

higher than in the single pgr5 mutant, still remained

significantly lower than in the WT or the single pgr1

mutant. Thus, the enhanced PSI stability in the double

mutant cannot be attributed solely to the forced acti-

vation of PhotCon at more alkaline luminal pH values.

Taken together, these results support the idea

that PhotCon might protect PSI under conditions of

PHOTOSYNTHETIC CONTROL AND PHOTOSYSTEM I PHOTOINHIBITION 849

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

photoinhibition, even those leading to the acceptor-

side damage. Yet each individual observation still al-

lows alternative explanations, such as, for example,

that the impaired ATP synthesis would slow CBB cycle

turnover at the onset of stress and thereby favor the

acceptor-side limitation of PSI.

Is the defective photosynthetic control the

cause of PSI photoinhibition in the pgr5 mutants?

The increased PI(I) in the pgr5 mutant is also often

linked to the impaired PhotCon activation. The pgr5

mutant was first isolated during the screening of Ara-

bidopsis lines for the reduced induction of non-photo-

chemical quenching due to the lower pmf values [9].

The in  vitro experiments showed that the AA-sensi-

tive CET(I) is absent in this mutant [9]. However, the

in vivo studies showed that under non-stress condi-

tions, the pgr5 mutant can perform CET(I) as effi-

ciently as WT, and the PGR5 protein is essential for

enhancing CET(I) under abrupt changes of conditions

[97]. The pgr5 mutant is unable to grow under FL, and

the reason for this is its pronounced PI(I) [8]. This ex-

ample underscores both the necessity of preventing

PI(I) for plant survival and the key role of PGR5 in

PSI protection in C

plants. As mentioned above, in-

troducing the pgr1 mutation (ISP-P194L) into the pgr5

mitigated PI(I) in mature plants [10]; nevertheless, this

does not necessarily mean that the defective PhotCon

is the primary cause of the strong PI(I) seen in the sin-

gle pgr5 mutant. Expression of the flavodiiron protein

genes of Physcomitrella patens moss, which provide

an efficient electron outflow from PSI, in the Arabi-

dopsis pgr5 mutant diminished PI(I) under FL [10,

43], confirming that PI(I) in the single pgr5 mutant is

associated with restricted electron outflow from PSI.

Screening for the secondary Arabidopsis muta-

tions that enable pgr5 seedlings to survive FL was

carried out in the study [98], where several alterations

affecting PSII function and stability, Cyt-b

f assembly,

Pc biosynthesis, chloroplast fructose-1,6-bisphospha-

tase, and other factors were identified, all of which

partially compensated negative effects of the absence

of functional PGR5 protein. However, targeted intro-

duction of the ISP-P194L substitution (the pgr1 muta-

tion) into the pgr5 did not provide seedling survival

under FL. Hence, artificially forcing PhotCon by rais-

ing the pK

of H128 cannot counteract physiological

impairment resulting from the absence of functional

PGR5 protein during the long-term exposure of the

plant to FL over the vegetative period.

Recently, it was shown that, unlike the WT, the

pgr5 mutant displays oscillatory changes in the chlo-

rophyll fluorescence, P

700

absorbance, and the signal

of electrochromic shift of carotenoid absorbance in re-

sponse to abrupt changes in light intensity or CO

con-

centration [99]. Such oscillations were not observed in

the hope2 mutant, which showed impaired induction

of the parameter Y(ND) interpreted as activation of

PhotCon. The authors concluded that the oscillations

in the pgr5 mutant are not associated with the im-

paired PhotCon activation; instead, these oscillations

arise from the ATP deficiency that develops under

abrupt environmental changes. This interpretation is

consistent with the earlier work proposing that the

PGR5-dependent CET(I) is required primarily to bal-

ance ATP production relative to NADPH [9, 100]. Itis

worth noting that the hope2 mutant also exhibited

increased activity of the PGR5-dependent CET(I) [96],

which could counteract negative effect of the elevated

and thereby dampen potential oscillations.

In summary, the recent evidence indicates that

the increased PI(I) in the pgr5 mutant is not caused

by the defective PhotCon activation but is, most like-

ly, related to insufficient ATP production and inability

to balance between ATP and NADPH.

Hypothetical mechanism by which photosyn-

thetic control might suppress ROS formation in PSI.

The proposed role of PhotCon in PSI protection is com-

monly linked to the decrease in ROS formation within

PSI, although there is no direct experimental evidence

for this claim. It remains unclear how PhotCon can

decrease ROS generation in PSI, and which specific

ROS are affected. PSI produces O

•−

, HO

•

, and

(see

above); all three have been suggested as immediate

damaging agents in PSI under different scenarios

of photoinhibition. A series of studies by K. Sonoike

showed that the key factor in the acceptor-side PI(I)

is not the amount of H

produced near the terminal

clusters but rather efficiency of the electron out-

flow from these clusters, which determines the prob-

ability of HO

•

generation (see above). We, therefore,

hypothesize that PhotCon activation could modify HO

•

generation while not affecting the rate of H

forma-

tion close to F

. There is still no clear understand-

ing of how the luminal pH influences O

reduction

in PSI. However, the work with the isolated PSI com-

plexes from the cyanobacterium Synechocystissp. PCC

6803 and the alga C.reinhardtii showed that increas-

ing concentration over a wide range of either the ar-

tificial electron donor N,N,N′,N′-tetramethyl-p-phenyl-

enediamine or the natural donor Pc did not increase

the O

•−

production (and thus production of H

) by

PSI, even though it improved efficiency of electron do-

nation to P

700

[25,  101]. High donor concentrations

in such systems mimic rapid electron flow to PSI due

to the absence of PhotCon activation. Moreover, the

rate of O

reduction in the isolated C.reinhardtii PSI

complexes changed only negligibly when the electron

outflow from PSI to Fd and NADP

was enabled [25,

26, 102]. These observations confirm that the decrease

in the P

700

reduction rate due to PhotCon activation

does not necessarily lead to the decrease in generation

of O

•−

and, consequently, of H

in PSI.

VILYANEN, KOZULEVA850

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

In the absence of MV or other efficient acceptors,

the principal route for oxidizing the F

clusters is

charge recombination with P

700

[41]. Activation of

PhotCon slows P

700

re-reduction, allowing electrons

from F

to return to P

700

and thus preventing the

reaction of F

with H

. In the case of

, which

is assumed to be responsible for PI(I) during rSP treat-

ments, enabling safe charge recombination from F

due to a slower electron flow from Cyt-b

f to PSI might

likewise diminish charge recombination with the in-

termediate PSI cofactors and thereby suppress

generation.

Photosynthetic control modulates the func-

tioning of cytochrome b

f complex. Besides directly

limiting the rate of electron donation to PSI, PhotCon

necessarily influences processes associated with Cyt-

f activity and themselves affecting energy balance

in the PETC: operation of CET(I), activation/deactiva-

tion of the STN7 kinase, and ROS formation within

Cyt-b

ROS formation. Oxidative modifications of amino

acid residues in close vicinity of the Cyt-b

f prosthetic

groups have been detected in the Cyt-b

f complexes

purified from the field-grown spinach, indicating ROS

production associated with these groups [103]. As in

PSI, Cyt-b

f can generate

(via its single chloro-

phylla molecule) [104], HO

•

(via the ISP Fe

-S

cluster),

and O

•−

(upon oxidation by O

of electron transfer

cofactors possessing sufficiently low redox poten-

tial) [47]. The semiquinone formed in the Qo site is

generally considered as the main source of O

•−

, al-

though the role of heme b

is not ruled out [103, 105].

It should be noted that PQH

•

can reside transiently

inthe spin-coupled state with the reduced Fe

-S

clus-

ter, a state that does not react with O

[106]. O

•−

un-

dergoes dismutation with another O

•−

to yield H

but it is unclear how efficient is this reaction directly

within the Qo site, because proton availability is a lim-

iting factor for this reaction. In any case, O

•−

could

produce H

upon exiting into the lumen, which

could occur via diffusion through the E-channel. It is

also plausible that the PQH

molecule within the Qo

site or en route to it inside the complex, could re-

duce O

•−

to H

, as has been demonstrated for PQH

molecules in the membrane [107]. In this regard, it

is worth mentioning that quantitative assessments

of the O

•−

generation rate by Cyt-b

f [105] were de-

rived from the experiments that monitored fluores-

cence of resorufin, produced in the reaction of Am-

plex Red dye with H

, but not O

•−

Discussing effects of PhotCon on generation of

in Cyt-b

f is complicated by the absence of informa-

tion on how conformational changes in the chloro-

phyll a molecule position might be linked to lumen

pH, and how these changes could affect probability

of the molecule transitioning to triplet state, which

is required for generation of

. By contrast, there

is a stronger evidence to suggest that PhotCon sup-

presses electron leakage to O

. When PhotCon is ac-

tivated, the PQ pool becomes more reduced, limiting

availability of PQ molecules for the Qr site and for

Q-cycle turnover. This would increase the probability

of reverse electron transfer from heme b

to heme

, which increases the probability of heme b

oxi-

dation by O

; however, the reaction of heme b

with

should be suppressed by the subsequent electron

transfer from heme b

to quinone species in the Qo

site. Reduction of PQ or PQ

•−

by heme b

is thermo-

dynamically unfavorable, whereas reduction of PQH

•

by heme b

is thermodynamically more favorable.

As stated above, lifetime of PQH

•

is governed by the

protonated state of E78, manifestation of PhotCon ac-

tivation. Prolonging the lifetime of PQH

•

itself in the

Qo site decreases the probability of O

•−

formation,

because PQH

•

reduces O

less efficiently than PQ

•−

due

to its more positive redox potential [108]. Another

manifestation of PhotCon activation is longer lifetime

of the protonated H128. On the one hand, this could

retain the Fe

-S

cluster in its distal position, away

from the low-potential cofactors capable of produc-

ing O

•−

and H

with the Qo site, thereby minimiz-

ing the risk of forming the most dangerous ROS, HO

•

On the other hand, it would hinder catalytic oxidation

of PQH

in the Qo site, increasing likelihood of PQH

reacting with O

•−

and converting it into the less re-

active H

. Thus, PhotCon activation should decrease

formation of both O

•−

and HO

•

, lowering the chance

of oxidative protein modifications that could impair

the entire complex. However, the amount of H

pro-

duced would depend on the combined reactions, so

PhotCon could in principle increase the H

levels

in Cyt-b

f by both preventing H

decomposition into

•

within the Qo site and stimulating the reaction

of O

•−

with PQH

instead of O

•−

dismutation.

Activation of the STN7/STT7 kinase. The STN7/

STT7 kinase is structurally and functionally associ-

ated with Cyt-b

f [19, 20] and phosphorylates sever-

al proteins [109, 110], primarily the light-harvesting

complex II (LHCII) proteins. Redistribution of phos-

phorylated LHCII from PSII to PSI could also reduce

PI(I) under FL [111]. STN7 has also been shown to

phosphorylate FNR [110], which is likely involved

in switching between the linear electron transport

and CET(I) [51], and Thylakoid Soluble Protein 9

[109], a protein that binds to the stromal side of Cyt-

f and, likely, contributes to CET(I) regulation [67].

Within the STN7 context, two distinct but related is-

sues remain unresolved: (i) the mechanism of kinase

activation, which is tied to PQH

oxidation in Cyt-

f, and (ii) the mechanism of kinase inactivation in

high light. In particular, one activation model posits

that the lumen-facing N-terminus of STN7 contacts

PHOTOSYNTHETIC CONTROL AND PHOTOSYSTEM I PHOTOINHIBITION 851

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

the hydrophilic domain of ISP; during the conforma-

tional changes accompanying electron transfer from

PQH

to cyt f; this contact induces the kinase switch

to its active dimeric state via formation of an intermo-

lecular disulfide bridge between the N-termini of two

STN7 proteins [112]. Suppression of the STN7 activity

in high light is believed to involve reduction of this

intermolecular disulfide by the trans-membrane pro-

tein system that transfers electrons from stromal thi-

oredoxins to the luminal thiol-regulated targets [113]

followed by formation of an intramolecular disulfide

bridge that renders STN7 inactive. H

produced by

the PETC components in high light may participate in

this inactivation; indeed, adding catalase, which de-

composes H

, to the Arabidopsis thylakoids led to

the increased accumulation of phosphorylated LHCII

proteins in high light [114].

In vitro experiments showed that lumen acidifi-

cation lowered accumulation of the phosphorylated

LHCII proteins in the maize chloroplasts [115] indi-

cating correlation between the conditions that trigger

PhotCon and STN7 kinase activity. One possible expla-

nation is that lowering lumen pH raises the midpoint

redox potentials (E

) of the thiol groups in STN7 and

in other proteins involved in regulating its activi-

ty [113] with simultaneous increase the E

of H

making it a stronger oxidant. Another explanation is

direct effect of PhotCon on the STN7 activity [20]. For

instance, when the luminal pH drops below the pK

of H128, the hydrophilic domain in ISP may reside

longer near the cyt f, which could restrict the domain

interaction with the N-terminus of STN7 and thereby

suppress kinase activation. As noted above, PhotCon

could hypothetically increase the H

production in

Cyt-b

f; the produced H

might inactivate STN7 by

oxidizing its thiols with formation of an intramolecu-

lar disulfide bond.

The Q-cycle and the AA-sensitive CET(I). Electron

transfer along the low-potential branch of Cyt-b

f re-

duces the PQ molecule to PQH

in the Qr site via the

Q-cycle, which can operate in two modes: (i) when

both electrons needed to reduce PQ in the Qr site orig-

inate from the Qo site via sequential oxidation of two

PQH

molecules (an electron from the first PQH

mol-

ecule is transferred to heme c

, an electron from the

second PQH

molecule is transferred to the PQ mole-

cule from heme b

simultaneously with the transfer

of an electron from hemec

to PQ) and (ii)when one

electron for PQ reduction comes from the Qo site (via

heme b

), while the second is supplied by the reduced

Fd through heme c

[19]. This latter mode is realized

during the AA-sensitive CET(I), in which Cyt-b

f, either

alone or in complex with FNR, oxidizes Fd and re-

duces PQ, provided that PGR5 is present. In the C.  re-

inhardtii lacking PGR5, switching between these two

Q-cycle modes is impaired [18]. Several hypotheses

have been proposed to explain how competition be-

tween the two modes is regulated when Fd can donate

electrons to Cyt-b

f [20, 64]. Recent measurements of

the redox potentials of cytb

hemes have shown that

the E

of heme b

is about 30-50  mV more negative

than that of heme b

[64]. In the process, endergonic

electron transfer from heme b

to heme b

becomes

feasible because it is coupled to the subsequent exer-

gonic electron transfer to the PQ molecule in the Qr

site or to heme c

, resulting in the net decrease in free

energy. Consequently, thermodynamic efficiency of the

electron flow through the low-potential branch under

steady-state conditions would depend on the propor-

tion of oxidized PQ molecules in the membrane.

As noted above, activation of PhotCon could re-

strict availability of PQ for operation of the Q-cycle

(although it is not ruled out that a PQ molecule from

the Qo site may reach the Qr site without ever leav-

ing Cyt-b

f [67]). Prolonging the lifetime of proton-

ated H128 could keep the ISP in its distal position,

slowing turnover of the complex. Extending lifetime

of the protonated E78 should slow oxidation of PQH

•

by heme b

. However, pK

of E78 is lower than pK

H128 (see above), and with a moderate drop in lumi-

nal pH, PhotCon would be mediated mainly through

the longer lifetime of the protonated H128, whereas

E78 would remain predominantly ionized. Under these

conditions the overall turnover of the complex would

be slower, but the efficiency of electron transfer along

the low-potential branch would be unaffected, main-

taining rapid reduction of PQ at the Qr site.

When electrons are donated from Fd, the PhotCon-

induced slowdown in the Cyt-b

f turnover would also

slow CET(I), because the PQH

generated in the Qr

site must still be oxidized on the luminal side of Cyt-

f, and PhotCon, due to protonated H128, would lim-

it the steady-state CET(I). Contradictions arise in this

case, since it is often assumed that under the abrupt

environmental changes CET(I) accelerates to acidify

the lumen and activate PhotCon [2]. These contradic-

tions could be resolved by assuming that Cyt-b

f in

the grana and unstacked lamellae perform different

functions: electron transfer from PSII to Pc and oper-

ation of CET(I) respectively [23, 24]. The key factor is

heterogeneity of the luminal pH [116]: in the grana,

where PSII releases H

into the lumen and efficient

efflux pathways are scarce, the lumen pH drops

more sharply than in the stromal thylakoid regions

enriched with ATP-synthase complexes that dissipate

ΔpH. Consequently, abrupt environmental changes

would activate PhotCon in the granal Cyt-b

f rather

than in the stromal Cyt-b

f, thereby slowing PQH

ox-

idation specifically during linear electron flow from

PSII to PSI, but not during CET(I) (Fig. 4). Moreover,

activation of PhotCon in the grana would favor CET(I)

over the linear flow, thereby maintaining proper

VILYANEN, KOZULEVA852

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig.  4. Schematic representation of PhotCon activation under moderate lumen acidification, taking into account lateral

heterogeneity of both the thylakoid membrane and lumen pH. The color gradient from red to blue depicts luminal pH

gradient between grana and stromal lamellae (pH ≪ 7 in grana; pH < 7 in stromal regions). The red arrow indicates H

diffusion; the blue arrow shows Pc diffusion (dashed: linear electron flow; solid: CET(I)). The black arrow denotes electron

transfer from PSI to the Cyt-b

f via Fd during CET(I).

ATP/NADPH balance. It is this effect, rather than di-

rect suppression of ROS formation in PSI, that could

explain how PhotCon protects PSI under conditions

that cause the acceptor-side PI(I), particularly under FL.

Under more severe stress, when ATP-synthase

activity declines, the luminal pH of both the granal

and stromal thylakoid regions is expected to drop, so

PhotCon could also be triggered in the stromal Cyt-

f, thereby influencing CET(I). In this case, there is

obviously no physiological need to supply additional

to the lumen. Nevertheless, the electrons from Fd

could still enter heme c

, and because the PQ pool

is virtually fully reduced under such conditions, ef-

ficiency of the electron transfer into the PQ pool is

decreased. A hypothetical electron transfer from Fd

to quinone species in the Qo site – a speculative (i.e.,

experimentally unverified) model of CET(I) that by-

passes the bulk PQ pool – has been discussed in the

review [19]. The possibility of this pathway depends

on which quinone species occupies the Qo site. Re-

verse electron transfer from heme c

to PQ in the

Qo site comprises two endergonic steps (Fig. 3a) not

coupled to a sufficiently exergonic reaction, making

the process thermodynamically unfavorable. Electron

transfer to the PQ

•−

radical (forming the dianion PQ

2−

)

is also thermodynamically unfavorable in the absence

of free protons. By contrast, transferring an electron

to PQH

•

(forming PQH

−

) is favorable and energetically

profitable, compensating for the endergonic step of

electron transfer from hemec

to heme b

. As noted

above, likelihood of PQH

•

deprotonation to PQ

•−

in the

Qo site should be governed by the lifetime of proton-

ated E78. Therefore, activation of PhotCon at the level

of E78 would facilitate an alternative CET(I) pathway

where electrons do not exit the Cyt-b

f into the PQ

pool. This alternative CET(I) would not pump extra

to the lumen (which is unnecessary under the de-

scribed conditions), yet it could still play a physiolog-

ical role by ensuring steady-state PSI operation with

zero net redox balance.

CONCLUSION

In this review we have thoroughly examined the

causes of PSI photoinhibition and proposed that it pro-

ceeds by two distinct mechanisms leading to primary

damage on either the acceptor or the donor side of

PSI. We have raised the question of how experimental-

ly substantiated is the notion that PhotCon functions

PHOTOSYNTHETIC CONTROL AND PHOTOSYSTEM I PHOTOINHIBITION 853

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

as a protective mechanism preventing PI(I). Our analy-

sis of the literature on the pgr5 mutant indicates that

the key role of the PGR5 protein in preventing PI(I)

lies in regulating CET(I) for supplying additional ATP,

rather than for PhotCon activation. We suggest that

activation of PhotCon under conditions of photoin-

hibition could suppress generation of the more re-

active species (HO

•

and

) in the PSI rather than

•−

or H

We have summarized current knowledge on

the proton-releasing channels of the Qo site of the

Cyt-b

f and their role in PQH

oxidation, providing

a more comprehensive understanding of the mech-

anisms underlying the Cyt-b

f sensitivity to luminal

pH and activation of PhotCon. We conclude that both

amino acid residues accepting H

during PQH

oxi-

dation, H128 of the ISP and E78 of the subunit IV,

are involved in PhotCon activation. However, due to

the differences in pK

values, PhotCon activation at

the level of these residues would occur at different

lumen pH values. We have considered how PhotCon

activation, including at the level of protonated E78,

could hypothetically influence Cyt-b

f functioning: its

ROS production, STN7 kinase activation and deactiva-

tion, and operation of the AA-sensitive CET(I) pathway.

Based on the notion of lateral heterogeneity of both

thylakoid structure and luminal pH, we propose that

PhotCon slows linear electron transport in the Cyt-b

located in the grana. Meanwhile, in the stromal lamel-

lae, where the lumen pH drops to a lesser extent, the

Cyt-b

f activity under these same conditions would

not slow down, thus facilitating CET(I). This could ex-

plain the apparent paradox in the scientific commu-

nity: abrupt environmental changes simultaneously

accelerate CET(I) and activate PhotCon. The enhanced

CET(I) in the stromal thylakoids is crucial for addition-

al ATP synthesis and maintenance of an optimal ATP/

NADPH ratio for the CBB cycle and other chloroplast

metabolic pathways. This, in turn, promotes efficient

electron outflow from PSI, preventing PI(I). It is this

competitive advantage provided to CET(I) in the stro-

mal lamellae, achieved by suppressing linear transport

at the grana level, that may underlie the protective

action of PhotCon, preventing PI(I) on the acceptor

side of PSI.

Ultimately, this review encourages readers to

re-evaluate the mechanisms of PhotCon activation and

its physiological role in regulating photosynthetic ap-

paratus function.

Abbreviations. ΔpH, trans-thylakoid pH gradi-

ent; AA, antimycin A; CET(I), cyclic electron transport

around PSI; Cyt, cytochrome; Cyt-bc

and Cyt-b

f – cy-

tochrome bc

and b

f complexes, respectively; DBMIB,

2,5-dibromo-3-methyl-6-isopropylbenzoquinone; DNP-

INT, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol;

E- and H-channels: proton-releasing channels that

remove protons from E78 and H128, respectively; Fd,

ferredoxin; FL, fluctuating light; FNR, ferredoxin-NA-

DP-reductase; ISP, iron-sulfur protein; LHCII, light-har-

vesting complex II; MV, methyl viologen; Pc, plastocy-

anin; PETC, photosynthetic electron transport chain;

PGR5, proton gradient regulation 5; PhotCon, photo-

synthetic control; PI(I) and PI(II), photoinhibition of

photosystemI and photosystemII, respectively; pmf,

proton motive force; PQ, PQH

, PQH

•

, plastoquinone,

plastohydroquinone, and plastosemiquinone, respec-

tively; PSI and PSII, photosystemI and photosystemII,

respectively; ROS, reactive oxygen species; rSP, repet-

itive short pulses; WT, wild type; Y(NA) and Y(ND),

quantum yields of non-photochemical losses on the

acceptor and donor sides of PSI, respectively.

Acknowledgments. The authors are grateful to

Dr. I. A. Naidov for assistance in preparing illustra-

tions.

Contributions. M.A.K. developed the concept;

M.A.K. and D.V.V. analyzed the available literature and

wrote the manuscript; M.A.K. edited the manuscript.

Funding. The work was financially supported

by the Russian Science Foundation [project no. 25-

24-00597 (https://rscf.ru/project/25-24-00597/) (in Rus-

sian)].

Ethics approval and consent to participate.

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest. The authors of this work de-

clare that they have no conflicts of interest.

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