ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 860-872 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 937-950.
860
Protective Effect of α-Carbonic Anhydrase CAH3
Against Photoinhibition and Thermal Inactivation
of Photosystem II in Membrane Preparations
as Compared with α-Carbonic Anhydrase CA4
Vasily V. Terentyev
1,a
*, Liubov I. Trubitsina
2
, Tatyana P. Khoroshaeva
1
,
and Ivan V. Trubitsin
2
1
Institute of Basic Biological Problems, Pushchino Scientific Center for Biological Research,
Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia
2
G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms,
Pushchino Scientific Center for Biological Research, Russian Academy of Sciences,
142290 Pushchino, Moscow Region, Russia
a
e-mail: v.v.terentyev@gmail.com
Received April 13, 2025
Revised June 5, 2025
Accepted June 16, 2025
Abstract— Photosystem II (PSII) is one of the most vulnerable components of photosynthetic apparatus of
the thylakoid membrane to the action of inhibitory factors. The donor side of PSII exhibits high sensitivity
to photoinhibition and thermal inactivation, which leads to the loss of O
2
-evolving function of the water-ox-
idizing complex (WOC). The data obtained in this study demonstrated increased stability of WOC activity in
the PSII membrane preparations from the wild-type (WT) Chlamydomonas reinhardtii compared to the PSII
preparations from the cia3 mutant, which lack α-carbonic anhydrase (CA) CAH3, under conditions of moderate
photoinhibition and thermal inactivation. This effect was completely eliminated by adding a CA inhibitor to
the PSII preparations from WT. At the same time, addition of active recombinant CAH3 (rCAH3) protein to the
preparations from cia3 restored increased resistance of PSII to these factors. Under the same conditions of
photoinhibition and thermal inactivation, the PSII preparations from Arabidopsis thaliana demonstrated very
low loss of O
2
-evolving activity, regardless of the presence or absence of carbonic anhydrase α-CA4, which
is similar to CAH3. More pronounced suppression of the O
2
-evolving activity in the PSII from A. thaliana
mutants lacking CA4 was observed only when they were incubated at elevated temperature, indicating the
possibility of more significant conformational changes in the WOC proteins of PSII. Despite the clear binding
of the rCAH3 to PSII membrane preparations from A.  thaliana, the enzyme had little effect on the WOC ac-
tivity in these preparations, suggesting absence of functional interaction between the rCAH3 and PSII from
A. thaliana. The obtained results indicate different mechanisms of involvement of CAH3 and CA4, both of
which are assumed to exist in close association with PSII in live systems, in the PSII functioning.
DOI: 10.1134/S0006297925601133
Keywords: Chlamydomonas reinhardtii, Arabidopsis thaliana, carbonic anhydrase CAH3, carbonic anhydrase
α-CA4, photosystemII, water-oxidizing complex, O
2
-evolving activity, photoinhibition, thermal inactivation
* To whom correspondence should be addressed.
INTRODUCTION
Photosystem II (PSII) is a large multiprotein
pigment- containing complex of a thylakoid mem-
brane[1-3], which mediates primary charge separation
at the expense of energy of the absorbed quanta of
light. At the donor (lumenal) side of PSII this is ac-
companied by oxidation of water molecules to O
2
and
protons(H
+
), and at the acceptor (stromal) side– by re-
duction of quinone electron acceptors Q
A
and Q
B
[4-6].
PROTECTIVE EFFECT OF CAH3 861
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Based on the degree of resistance to the action of
the functional inhibiting factors, such as, in particu-
lar, excessive illumination and elevated temperature,
PSII is the most sensitive element of the photosynthet-
ic apparatus of thylakoid membrane [3, 7-9], hence,
search for approaches to maintain high photosynthetic
activity of PSII is an important and relevant task.
The mechanism of protecting PSII against exces-
sive illumination involves partial dissipation of the
absorbed light energy as heat through the energy-
dependent component of non-photochemical chloro-
phyll quenching, qE, which is mediated by the pro-
teins PsbS and Lhcsr3 (in green algae and mosses)
and activity of the xanthophyll cycle [3, 10-13]. They
also could re-target part of the absorbed light energy
to the more resistant photosystem I through re-dis-
tribution of antenna complexes between two photo-
systems (component of non-photochemical chlorophyll
quenching termed state transitions(ST))[3, 11, 14,15].
However, in the case of light intensity exceeding total
capabilities of protective mechanisms in PSII against
excessive illumination, suppression of its photosyn-
thetic activity is observed[9]. This effect is called ‘pho-
toinhibition of PSII’, and it could be both reversible
(under weak or short-term impact), and irreversible
process accompanied by the damage of proteins in
PSII, such as, in particular, D1 (PsbA), and degrada-
tion of reaction centers [3, 7, 9, 16-18]. Mechanisms
of rapid repair of damaged PSII exist in live systems,
effects of which could be eliminated by using antibi-
otics, such as chloramphenicol or lincomycin, or by
investigating photoinhibition of PSII using isolated
membrane preparations [16, 17, 19, 20]. In the pro-
cess, the rate of PSII photoinhibition depends signifi-
cantly on the intensity of the acting light [16].
Molecular processes occurring during PSII pho-
toinhibition are associated either with the donor or
acceptor sides of PSII [7,  21,  22] and are mediated
mainly by formation and accumulation of reactive ox-
ygen species (ROS) [8, 22-25]. The donor side of PSII
is more sensitive to photoinhibition, and associated
processes occur even under exposure to light of mod-
erate intensity [9,18, 19, 26, 27].
Significant amounts of oxidized amino acids in
the preparations of PSII exposed to light has been
found close to the Mn
4
CaO
5
-cluster of the water-oxi-
dizing complex (WOC) and outlet channels for O
2
/ROS
[26,  28]. It has been suggested that the ability of Mn
ions to photoreception results in disruption of the
Mn
4
CaO
5
- cluster function under illumination and
even in removal of Mn ions from the active center of
WOC [18]. Incomplete oxidation of H
2
O molecules in
the damaged Mn
4
CaO
5
-cluster could facilitate forma-
tion of hydrogen peroxide (H
2
O
2
), which is reduced
by Mn
2+
(Fenton-like reaction) to hydroxyl radical
(OH
•
)[22, 29, 30], which, in turn, oxidizes amino acids
in its vicinity [26,28]. Insufficiently effective donation
of electrons from WOC to P
680
+
through Tyr
Z
under
illumination support their cationic forms, which oxi-
dize easily amino acids and pigments in their vicinity
[9, 18] causing functional destabilization of PSII.
The degree of PSII photoinhibition increases sig-
nificantly with increase of temperature [31], however,
decrease of photosynthetic activity of PSII at elevated
temperatures is observed also during dark incuba-
tion of the preparations (thermal inactivation)[32-37].
In the case of thylakoids, decrease of WOC activity
has been observed already at 30-40°C [35, 36, 38,39],
which is accompanied by partial removal of the WOC
proteins (PsbO, PsbP, PsbQ) from PSII, and degree of
this removal increases several folds with tempera-
ture increase to 50°C [34,  36,  40,  41]. Simultaneously
Mn and Ca ions are removed from the Mn
4
CaO
5
-clus-
ters [34, 36, 41]. Illumination of the preparations af-
ter thermal inactivation is accompanied by formation
of ROS both at the acceptor and donor sides of PSII
according the mechanisms similar to photoinhibition
mechanisms [22]. Similar degradation of the D1 and
D2 proteins is also observed [31, 35, 36].
One of the additional factors suppressing activity
of Mn
4
CaO
5
-cluster is local acidification in its close
vicinity emerging as a result of obstructed removal of
H
+
formed during photoinduced water oxidation into
a lumen [4, 42, 43]. This could be facilitated by con-
formational re-arrangements of the proteins in PSII
caused by both increased temperature and oxidative
modification of amino acids resulting in destabiliza-
tion of proton channels [4, 13, 43, 44]. Furthermore,
low pH increases significantly the probability of hy-
drogen peroxide reduction by Mn ions to OH
•
[22,30],
formation of which increases amount of oxidized ami-
no acids in close vicinity of the Mn
4
CaO
5
-cluster, thus
increasing the degree of conformational rearrange-
ments in the proteins.
It has been suggested previously, that the PSII-
associated lumenal α-carbonic anhydrase (CA) CAH3
from green algae Chlamydomonas reinhardtii plays
a role in stimulation of removal of H
+
from the
WOC active center in the case of destabilized pro-
ton channels through its dehydratase activity
(H
+
 + HCO
3
−
 → H
2
O  +  CO
2
) thus ‘neutralizing’ protons
at the channel exit to lumen [4,  42, 43,  45]. This sup-
ported photosynthetic activity of PSII of C.  reinhardtii
[4,  13,  42,  46,  47]. In the Arabidopsis thaliana plants,
the role of PSII-associated α-CA has been suggested
for the CA4 protein, however, according to the authors,
contrary to the case of CAH3, this CA catalyzes hydratase
direction of the reaction (H
2
O  +  CO
2
 → H
+
 + HCO
3
−
),
which results in additional protonation of lumenal ami-
no acids in the PsbS protein thus initiating qE [48,49].
In addition, presence of different sources of CA
activity near the WOC of PSII has been suggested in
TERENTYEV et al.862
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
a number of previous studies [49-53], including effects
of CA on WOC functioning [54-56]. However, the data
on effects of CA or CA activity on the resistance of PSII
to photoinhibition and thermal activation are practi-
cally absent at present, which emphasizes importance
and novelty of such studies.
Use of membrane preparations of PSII from
C.  reinhardtii and A. thaliana in this study includ-
ing both wild-types (WT) and mutants lacking CAH3
and CA4, respectively, allowed to examine partici-
pation of these CAs in preservation of O
2
-evolving
function of WOC in PSII during photoinhibition and
thermal inactivation. Moreover, use of the moderate-
ly high light intensities and temperatures during the
long-term exposure provided the possibility to avoid
rapid and destructive changes in the preparations,
which could mask the effects of CAs [31,  35,  36]. Ad-
dition of the highly active recombinant CAH3 protein
(rCAH3) [57] to PSII of C. reinhardtii and A. thaliana
provided the opportunity to investigate both specific-
ity of the action of this CA on photosynthetic activi-
ty of PSII from algae, and evaluate capability of the
rCAH3 to bind to PSII preparations from A.  thaliana
both containing (WT) and lacking the CA4 protein
(mutants).
MATERIALS AND METHODS
Membrane preparations isolated from a green al-
gae C.  reinhardtii and A. thaliana plants and enriched
with PSII served as objects of the study.
A CC-503 strain was used as a standard WT C.  re-
inhardtii, and a cia3 mutant – as a strain without
CAH3 in a thylakoid lumen [42,  43]. Algae cultures
were cultivated under conditions described in the pre-
vious study [4] at 25°C with continuous illumination
with cold white light (LED 6500  K) of ~90  µmol pho-
tons∙m
−2
∙s
−1
intensity and aeration with air containing
5%  CO
2
. In the case of A.  thaliana plant ecotype Co-
lumbia-0(Col) was used as a WT, and plants of homo-
zygous lines 8-8 and 9-12– as mutants with knockout
of the gene encoding α-CA4 (At4g20990) [49]. Plants
were grown for 5-6 weeks in a growth chamber at
19°C and short photoperiod (8-h day/16-h night) with
illumination intensity ~120  µmol photons∙m
−2
∙s
−1
  [58].
Isolation of membrane preparations enriched with PSII
was carried out according to the protocols described
previously for C. reinhardtii [4] and A.  thaliana [49].
Maximum rates of O
2
evolution were ~280  µmol
O
2
∙mg
−1
chlorophyll∙h
−1
for PSII preparation from
C.  reinhardtii and ~310  µmol O
2
∙mg
−1
chlorophyll∙h
−1
–
for PSII preparations from A.  thaliana. These values
were accepted as 100% in the measurements. Isolated
preparations of PSII containing 10% of glycerol were
stored at −70°C.
Total chlorophyll concentration was measured
using spectrophotometry in acetone extracts (80%) of
PSII preparations[59].
Rate of photoinduced evolution of O
2
was mea-
sured in a 1-ml cell with Clark electrode (Hansatech,
United Kingdom) at 25°C, illumination with saturating
red light (~2300  µmol photons∙m
−2
∙s
−1
; λ  >  600  nm)
in the presence of electron acceptor potassium fer-
ricyanide (1 mM) and 2,6-dichloro-p-benzoquinone
(0.2 mM [60]), as has been described previously [44].
In the experiments with photoinhibition, PSII
membrane preparations diluted to concentration
15  µg of chlorophyll per ml in a buffer containing
20  mMMES (pH6.5or7.0), 35  mM NaCl, and 400  mM
sucrose were incubated in a thermostated cell at 20°C
under illumination with red light (λ  >  600 nm) of in-
tensity 1200  µmol photons∙m
−2
∙s
−1
; 1-ml aliquots of a
suspension were sampled for measuring the rate of O
2
evolution after 0-, 15-, 30-, 45-, and 60-min incubation.
Simultaneously a control sample was incubated in the
dark under the same conditions and rate of O
2
evo-
lution was measured at the same time intervals (time
shift in parallel measurements was 5 min). Thermal
inactivation of PSII was carried out according to the
similar protocol, with samples incubated in the dark
at 33°C, while the reference sample was incubated
at 20°C.
Recombinant protein rCAH3 with high CA activ-
ity (~8300  WAU∙mg
−1
) was prepared and purified as
described in the recent publication [57]. Initial used
concentration of rCAH3 was 0.71 mg∙ml
−1
. Prior to
photoinhibition or thermal inactivation rCAH3 pro-
tein [57] was added to dilutions of PSII at the ratio
0.2  µl∙ml
−1
(or 142  ng∙ml
−1
), unless stated otherwise
in the legends to figures. To perform Western blot-
ting, membrane preparations of PSII from control
and experimental samples were precipitated in mi-
crotubes by centrifugation at 12,000g for 5  min.
Samples diluted in a loading buffer (50  mM Tris-HCl
(pH  6.8); 3%  SDS; 0.005% bromophenol blue; 10% su-
crose; 5% mercaptoethanol) were loaded onto a poly-
acrylamide gel (PAAG) at the ratio 2µg of chlorophyll
per lane.
Western blotting was carried out using electro-
phoresis in a denaturing 12.5% SDS-PAAG followed
by transfer of proteins onto a PVDF-membrane (Im-
mun-Blot PVDF Membrane, 0.2 µm; Bio-Rad, USA) in
a Mini-PROTEAN 3 Cell (Bio-Rad) with a module for
wet blotting Mini Trans-Blot (Bio-Rad), as described
previously [44]. Membranes blocked with a 5% skim
milk powder solution were incubated overnight at 4°C
with primary rabbit antibodies against CAH3 protein
(Agrisera, Sweden; AS05073) at 1  :  2000 dilution, and
next for 1  h at room temperature with secondary an-
tibodies against rabbit proteins labelled with horserad-
ish peroxidase (GE Healthcare, USA) at dilution 1 :  5000.
PROTECTIVE EFFECT OF CAH3 863
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Visualization was carried out using a Pierce ECL Plus
reagent kit (Thermo Scientific, USA) and a Chemi-
Doc XRS+ system (Bio-Rad). Staining of major protein
bands on a membrane was carried out by short-term
(3-5s) immersion of a membrane into a 0.2% Ponceau
solution in 5% glacial acetic acid and membrane
washing with distilled water until clear background
was reached [44].
CA-Activity of rCAH3 protein added to PSII prepa-
rations from C.  reinhardtii cia3 was measured ac-
cording to previously described protocol [57] using a
25 mM Tris-buffer (pH 8.5) at 0°C. Activity was pre-
sented in Wilbur-Anderson Units (WAU) per 1  mg of
rCAH3 protein or total chlorophyll. Membrane fraction
of PSII containing rCAH3 was precipitated from 5  ml
of suspension by centrifugation at 10,000g for 5 min
and resuspended in an incubation buffer (pH  6.5)
to final volume of 100 µl, which was introduced to
a reaction mixture for measuring CA activity. Incuba-
tion buffer (100 µl) without PSII was used as a neg-
ative control.
RESULTS
To investigate participation of CAH3 and CA4
in maintenance of PSII activity under conditions of
moderate photoinhibition, membrane preparations of
PSII isolated from the WT C. reinhardtii (CC-503) and
A.  thaliana (Col), as well as from the C. reinhardtii
cia3 and A.  thaliana 8-8 and 9-12 mutants not con-
taining CAH3 and CA4, respectively, were subjected to
illumination with light of moderate intensity for one
hour. This exposure caused loss of O
2
-evolving activity
Fig. 1. Change in the rate of O
2
evolution by the membrane PSII preparations isolated from C. reinhardtii (a, c) and
A. thaliana (b, d) during 1-h incubation at 20°C in the dark (1, 2) and under continuous illumination with red light (3-6).
Measurements were carried out at pH6.5(a, b) and 7.0 (c, d). 1, 3 – PSII preparations from WT (C.reinhardtiiCC-503 and
A.thalianaCol, respectively); 2, 4, 5– PSII preparations from mutants C.reinhardtiicia3 and A.thaliana 8-8, respectively;
5 – rate of O
2
evolution in the presence of 0.2µl∙ml
−1
(142ng∙ml
−1
) of rCAH3; 6– rate of O
2
evolution by the PSII prepara-
tions from C.reinhardtii CC-503 in the presence of 1µM ethoxyzolamide. Curves obtained from the PSII preparations from
A. thaliana 9-12 identical to the ones from 8-8 are omitted for clarity of presentation. Results are presented as a mean
± standard deviation of the mean (n = 3-5).
TERENTYEV et al.864
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 2. Results of Western blot analysis of the PSII preparations from C. reinhardtii (a) and A. thaliana (b) using primary
antibodies against CAH3. Recombinant protein rCAH3 was added to the PSII preparations in different concentrations shown
under the underlined lanes (4and5, 8 and 9, 12 and 13). candd)Membranes(a) and(b), respectively, with major proteins
stained using Ponceau solution.
by the PSII preparations from C. reinhardtii even at
pH6.5 optimal for WOC (Fig.1). In the case of WT the
rate of O
2
evolution decreased by ~16% after 15-min
illumination, by ~29% – after 30 min, and after one
hour – by ~44%. The decrease of activity was more
pronounced in the PSII preparations fromcia3 – ~27%
after 15-min illumination, ~39% – after 30 min, and
~59% – after one hour of photoinhibition (Fig. 1a).
Thecontrol samples containing PSII preparation from
the WT and from the cia3 mutant and incubated at
close temperature (20°C) in the dark did not exhibit
suppression of the O
2
-evolving activity. Only 10% de-
crease of the rate of O
2
evolution in these prepara-
tions was observed after 1 h of incubation.
The PSII preparations from A.  thaliana demon-
strated under the same conditions higher resistance
(Fig.1b). Rate of O
2
evolution by the PSII preparations
from Col decreased by ~5% after 15 min of incuba-
tion, by 11% – after 30min, and by ~24%– after 1 h.
This was almost 2-fold lower in comparison with the
values obtained for the PSII from WT C. reinhardtii.
The curves of suppression of the O
2
-evolving activi-
ty by the PSII in the membrane preparations isolat-
ed from the A.  thaliana mutants 8-8 and 9-12 were
identical and showed decrease by ~3% after 15 min
of incubation, by ~11%– after 30min, and by ~21%–
after 1 h, which was practically the same as for the
curve obtained for the PSII from Col (Fig. 1b). In
the process, decrease of WOC activity by 10% was
observed for the control samples incubated for 1  h
in the dark.
In order to confirm role of CAH3 in maintenance
of activity of PSII of C. reinhardtii under conditions
of moderate photoinhibition, active recombinant pro-
tein rCAH3 was added to the PSII from cia3 prior
to the start of illumination (at the ratio 0.2 µl∙ml
−1
(142  ng∙ml
−1
)), and a known inhibitor ofCA, ethoxyzol-
amide (EA) was added to the PSII preparations from
WT (at concentration 1 µM). Long-term illumination
of such PSII preparations resulted in elimination of
the inhibition of O
2
-evolving activity of WOC in the
PSII from cia3 in comparison with the PSII from WT,
while the PSII preparations from WT, on the con-
trary, demonstrated more significant suppression of
the rate of O
2
evolution, similar to one observed for
the PSII from cia3(Fig.  1a). Addition of rCAH3 to the
PSII preparations from the A.  thaliana 8-8 and 9-12
mutants did not affect significantly the degree of re-
duction of O
2
-evolving activity by the preparations
(Fig. 1b).
Western blot analysis using primary antibodies
against CAH3, as expected, demonstrated binding of
the rCAH3 protein with the PSII preparations from the
cia3 C. reinhardtii(Fig. 2a) with the band intensity in-
creasing with the increase of the amount of recom-
binant protein added to the PSII preparations. This
indicated the possibility of non-specific binding of a
fraction of rCAH3 with the membrane fraction, how-
ever, the effect of maintenance of the PSII activity
during photoinhibition implied functional interaction
between the WOC in the PSII preparations and rCAH3
molecules. Atthe same time, intensive binding of the
rCAH3 with the PSII preparations from A.  thaliana
was also observed, moreover, binding was observed
for both the preparations from Col, and from the mu-
tant lines 8-8 and 9-12 (Fig.2b). Intensity of the band
was also higher in the cases, when larger amounts
of recombinant proteins were added to the samples.
PROTECTIVE EFFECT OF CAH3 865
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
This indicated strong non-specific binding of the
rCAH3 protein with the membrane fraction of PSII in-
dependent on the presence of the native CA4 protein.
However, absence of the effect of the added rCAH3
on the activity of PSII from the 8-8 and 9-12 mutants
indicated either impossibility of functional interaction
of the WOC in the PSII from A.  thaliana with CAH3, or
insufficiently strong stress exposure in the case of PSII
from A. thaliana under used illumination conditions
to reveal the role of CA.
Non-optimal pH is another stress factor affecting
O
2
-evolving activity of PSII [4, 61-63]. As has been
shown previously, shift of the medium pH from 6.5 to
7.0 caused reversible suppression of WOC activity in
the PSII of C. reinhardtii, furthermore, reduction of the
O
2
-evolving activity was more pronounced (by ~20%)
in the PSII from cia3, due to the absence of CA activity
of CAH3 in close vicinity of WOC [4]. Suspension of
the membrane preparations of PSII form C.  reinhard-
tii in the medium with pH 7.0 reproduced the same
results. The rate of O
2
evolution before illumination
for the PSII from WT and cia3 were ~93% and ~74%
of the respective values observed at pH 6.5 (Fig. 1c).
The control samples incubated in the dark demon-
strated maintenance of this difference during time,
although a minor gradual decrease of the O
2
-evolving
activity of the PSII from cia3 was observed, while a
sharp drop of activity to ~83% was observed in the
case of WT PSII after 45 min of incubation. Moder-
ate photoinhibition of the preparations of PSII under
these conditions caused more pronounced loss of
O
2
-evolving activity in comparison with pH6.5. In par-
ticular, the rate of O
2
evolution in the PSII from WT
was lower by ~7% before illumination and decreased
further by ~31% after 15min, by ~46%– after 30min,
and by ~65% (versus ~44% at pH 6.5) – after 1  h of
illumination. The O
2
-evolving activity of the PSII from
cia3 was decreased by ~26% before illumination and
next decreased by ~40% after 15min, by ~50%– after
30 min, and by~64% (versus ~59% at pH 6.5) – after
1 h of illumination.
Comparison of the decrease of O
2
-evolving activ-
ity of WOC in the PSII from WT and cia3 at pH 7.0
demonstrated larger declining slope in the case of PSII
from WT during first 30 min of illumination, which
next reached the values observed for the PSII from
cia3. After 30min of photoinhibition both curves were
very similar (Fig. 1c). Higher activity of WOC in the
PSII from WT for the first 30min could be explained
by the CA activity of CAH3, which was confirmed by
the decrease of the rate of O
2
evolution in the PSII
preparations from WT to the level of PSII from cia3
on addition of EA. Moreover, addition of rCAH3 to the
PSII preparations from cia3 resulted in the curve of
decrease of the O
2
-evolving activity similar to the one
obtained for the PSII from WT (Fig. 1c).
Unlike in the case of PSII from C.  reinhardtii, the
PSII preparations from A.  thaliana at pH 7.0 demon-
strated reduced by ~20% rate of O
2
-evolution, in com-
parison with pH 6.5, for all variants (Fig. 1d). At the
same time, differences with the control samples were
minimal during first 30 min of photoinhibition. After
30 min the O
2
-evolving activity decreased by ~10%
for the PSII from Col and by ~15% for the PSII from
8-8 and 9-12 mutants. Addition of rCAH3 to the PSII
preparation from 8-8 and 9-12 mutants did not affect
the shape of the curve of suppression of the rate of
O
2
evolution during photoinhibition indicating lack of
functional interaction between the enzyme and the
WOC of A.  thaliana.
To investigate participation of CAH3 and CA4
in protection of PSII against moderate thermal in-
activation, PSII preparations from C.  reinhardtii and
A.  thaliana were incubated at 33°C in the dark at
pH 6.5 or 7.0.
At pH 6.5 activity of WOC in the PSII from WT
C. reinhardtii did not decrease significantly during the
first 15 min of incubation (by ~10%), however, after
30 min this decrease reached ~30%, and after 1 h –
~60% (Fig. 3a). The curve of the loss of O
2
-evolving
activity by the PSII from cia3 was smoother and the
values of decrease were ~22% after 15min and ~38%
after 30 min of incubation. After 45 min of incuba-
tions the curves for the PSII from WT and PSII from
cia3 practically coincided indicating loss of the pro-
tective role of CAH3. It should be mentioned that the
addition of rCAH3 to the preparation of PSII from cia3
stimulated the rate of O
2
evolution, however, during
the first 15 min of incubation, when the difference
between the curves obtained for the PSII from WT
and PSII from cia3 was the largest, this stimulation
was only ~50% (Fig. 3a).
In the case of PSII preparations from A.  thaliana
the curve of decrease of O
2
-evolving activity by the PSII
from Col differed only slightly from the control curves
up to 45min of incubation, but displayed sharp drop
by ~22% after 1h (Fig.3b). The rates of O
2
evolution
in the PSII preparations from 8-8 and 9-12 mutants
started to decrease after 30min of incubation by ~18%
and ~27%, respectively, observed after 45 min and 1h.
Addition of the rCAH3 protein to the PSII preparations
from A. thaliana 8-8 and 9-12 produced an unexpected
result. During the first 15 min of incubation presence
of rCAH3 caused slight suppression of the rate of O
2
evolution (by ~9% in comparison with the control),
which disappeared after 30 min of incubation.
In the process of moderate thermal inactiva-
tion of PSII at pH  7.0, the PSII preparation from
WT C. reinhardtii demonstrated slower reduction
of the O
2
-evolving activity in the first 15 min (by
~22% with ~9% initial inhibition at pH 7.0; Fig. 3c)
and accelerated suppression during the following
TERENTYEV et al.866
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 3. Changes of the rate of O
2
evolution by the membrane preparations of PSII isolated from C. reinhardtii (a, c) and
A. thaliana (b, d) during incubation for 1 h at 20°C (1, 2) and at 33°C (3-5) in the dark. Measurements were carried out
at pH 6.5 (a, b) and 7.0 (c, d). 1, 3 – PSII preparations from WT (C. reinhardtii CC-503 and A. thaliana Col, respectively),
2, 4, 5 – PSII preparations from mutants C.reinhardtiicia3 and A. thaliana 8-8, respectively; 5 – rate of O
2
evolution in
the presence of 0.2 µl∙ml
−1
(142 ng∙ml
−1
) of rCAH3. Curves obtained for the PSII preparation from A. thaliana 9-12 were
identical to the ones for 8-8 and are omitted for better visualization. Results are presented as a mean ±standard deviation
of the mean (n = 3-5).
30 min (by ~41%) reaching values observed for the
PSII from cia3. The decrease of the rate of O
2
evo-
lution during thermal inactivation of the PSII from
cia3 was more linear – by ~10% at each tested time
point (with initial ~26% inhibition at pH 7.0). Unlike
in the case of results obtained at pH 6.5, addition of
rCAH3 to the PSII preparation from cia3 resulted in
noticeable and complete restoration of the O
2
-evolving
activity of WOC to the levels observed for the prepa-
rations from WT even despite the more pronounced
differences between the curves obtained for the PSII
from WT and PSII from cia3, in comparison with
the curve obtained at pH 6.5 (Fig. 3c).
The PSII preparations from A.  thaliana also
demonstrated strong and rapid suppression of the
O
2
-evolving activity at pH 7.0 in the course of mod-
erate thermal inactivation in comparison with the
data obtained at pH6.5. The rate of O
2
evolution was
maintained at the level of control samples for first
15 min, by it decreased significantly after that; the
decrease was more pronounced in the PSII prepara-
tions from the 8-8 and 9-12 mutants (Fig. 3d). After
one hour of incubation the O
2
-evolving activity (with
initial ~20% inhibition at pH 7.0) decreased by ~40%
in the PSII preparation from Col and by ~50% – in
the PSII from the mutants 8-8 and 9-12. It is worth to
mention that this decrease was the highest observed
suppression of the rate of O
2
evolution in the prepara-
tions from A.  thaliana used in the study. Furthermore,
differences observed between the PSII preparations
from Col and from 8-8 and 9-12 mutants were most
significant.
Investigation of the effect of conditions of pho-
toinhibition and thermal inactivation of the PSII
PROTECTIVE EFFECT OF CAH3 867
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 4. CA activity of rCAH3. a) Activity of the enzyme per mg of protein after its addition to membrane preparations of
PSII isolated from the mutant C.reinhardtii cia3 at a ratio 0.2µl∙ml
−1
(142 ng∙ml
−1
), measured before (1, 3) and after 1-h
photoinhibition(2) or thermal inactivation (4). b)Activity of rCAH3 per mg of chlorophyll(Chl) during addition of the en-
zyme protein to PSII preparations at a ratio 0.2(1), 0.5(2), and 1 (3)µl∙ml
−1
, respectively. Results are presented as a mean
± standard deviation of the mean (n=3).
preparations on CA activity of the rCAH3 demon-
strated that in both cases the enzyme maintained its
full activity during incubation (Fig. 4a). Addition of
rCAH3 at higher ratio to the PSII preparations, which
resulted in binding of a larger amount of the enzyme
protein to the membrane fraction, as was shown pre-
viously using Western blotting (Fig.2a), also practical-
ly did not affect the rCAH3 activity (Fig. 4b). Hence,
the observed changes in the O
2
-evolving activity of
the PSII membrane preparations are not associated
with the change of CA activity of the rCAH3.
DISCUSSION
The molecule of CA CAH3 monomer from C. re-
inhardtii without two transport peptides (1-73) has
molecular weight of ~29.5 kDa (based on the PAGE
data), has a single S-S bond (Cys90–Cys258), which
is critically important for the enzyme activity, and
structure of the catalytic center conserved for α-CAs
containing three histidine residues (His160, -162, -179)
and zinc ion (Fig.5a) [43]. At the edge of a broad ac-
tive center cavity there is a histidine residue (His134),
which plays a role of proton shuttle [43]. Presence of
a hydrophobic region at one side of the CAH3 mole-
cule has been assumed to facilitate its binding with
the membrane fraction and correct orientation rela-
tive PSII [64].
An immature CA4 protein from A.  thaliana, un-
like CAH3, contains, as suggested, only one short sig-
nal peptide (1-26; https://www.uniprot.org/uniprotkb/
F4JIK2/entry), and, therefore, despite the lower num-
ber of amino acids in the protein sequence (267 ver-
sus310), the mature CA4 protein has molecular weight
close to molecular weight of CAH3.
Spatial structure of the CA4 molecule calculat-
ed based of the amino acid sequence resembles the
structure of CAH3 protein and also has a single S-S-
bond (Cys59-Cys214), three histidine in active center
(His125, -127, -144), and His99 residue playing a role
of proton shuttle (Fig. 5b).
Hence, spatial structures of α-CAs CAH3 from
C. reinhardtii and CA4 from A.  thaliana demonstrate
close similarity both in monomer structure, and in the
presence of a single S-S-bond connecting first part of
the β-sheet forming broad cavity of the enzyme active
center and N-terminal domain of the molecule (Fig.5).
According to the previously published results,
both CAH3 [4, 13, 42] and CA4 [49] are found in the
membrane preparations enriched with PSII and iso-
lated from C. reinhardtii and A.  thaliana, respectively.
Itwas shown in the process that CAH3 maintains high
photosynthetic activity of PSII at suboptimal for WOC
pH levels due to stimulation of H
+
removal from the
active center [4, 13, 42], as well as affects structural
organization of PSII [44]. The suggested role of CA4 in-
volves inducing qE through stimulation of protonation
of the luminal amino acids in the PsbS protein [48],
as well as participation in other adaptive reactions of
A.  thaliana PSII [12].
The results of photoinhibition and thermal inac-
tivation of the membrane preparations of PSII from
C. reinhardtii and A.  thaliana demonstrated much
TERENTYEV et al.868
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig.  5. Structures of CAs CAH3 (a) and CA4 (b) with shown locations of three histidines (His) of active center that bind
Zn ion, and of H
+
-transporting His at the edge of active center cavity, as well as of two cysteines (Cys) forming a S–S-
bond. CAH3 structure is obtained based of the data of X-ray diffraction analysis of rCAH3 (PDB ID: 4xiw, https://www.
wwpdb.org/pdb?id=pdb_00004xiw). CA4 structure is calculated using AlphaFold service (https://alphafold.ebi.ac.uk/) based
on the data from UniProt (https://www.uniprot.org/) (F4JIK2ATCA4_ARATH; https://www.uniprot.org/uniprotkb/F4JIK2/entry).
Structures are visualized with the help of V.M.D.1.9.2.
lower resistance of the PSII from C. reinhardtii to
these factors even in the case of WT (Fig. 1 and 3).
Absence of CAH3 (PSII from cia3) or its inhibition
(addition of EA) resulted in the enhancement of the
negative effect of these factors, and use of highly ac-
tive rCAH3 protein, which fully maintained its activity
in the course of incubation(Fig.4a), allowed to inves-
tigate participation of the enzyme in these processes
from the other point of view, including in the PSII
preparations from A.  thaliana. It must be mentioned
that although the higher rate of O
2
evolution in the
PSII from C. reinhardtii due to CA activity of CAH3
was maintained under continuous illumination for
the entire period of the sample illumination (1  h),
in the case of thermal inactivation stimulating effect
decreased significantly after 30  min, and was com-
pletely absent after 45 min of incubation. This in-
dicated existence of additional mechanisms of WOC
inactivation at elevated temperature, which were not
compensated fully by the removal of H
+
from the ac-
tive center. These could include, for example, ther-
moinduced conformational rearrangements of proteins
in WOC or even disorganization of their structure.
On the one hand, minor removal of WOC proteins
has been demonstrated previously during incubation
of the PSII preparations at temperatures above 30°C
[36]. On the other hand, conformational changes of
the proteins in PSII caused by shift of pH to 7.0 [13]
indeed caused loss of stimulation of O
2
-evolving ac-
tivity with the help of rCAH3 after 30-min incubation
(Fig. 1c).
High functional stability of A.  thaliana PSII both
from Col, and from 8-8 and 9-12 mutants under
used conditions of photoinhibition indicated lack of
need of CA4 participation in maintenance of WOC
activity. However, taking into account the possibility
of conformational changes in the WOC proteins at
pH 7.0 and elevated temperature mentioned above,
it could be suggested that the PSII from mutants 8-8
and 9-12 were subjected to WOC disorganization,
which was manifested by greater loss of O
2
-evolv-
ing activity after 30 min of incubation in compari-
son with the PSII from Col (Fig. 1d and 3b). Addition
of rCAH3 did not affect the curve of decrease of the
rate of O
2
evolution by the PSII from 8-8 and 9-12
mutants during photoinhibition (Fig. 1, b and d) in-
dicating either non-specificity of its interaction with
PSII from A.  thaliana, or lack of need in participa-
tion of CA in maintenance of WOC activity of the
A.  thaliana PSII.
Interestingly enough, transfer of the PSII prepa-
rations from A.  thaliana to the buffer with pH 7.0
decreased the rate of O
2
evolution by ~20% in these
preparations in comparison with the one at pH 6.5
(Fig. 1d and 3d). In the case of C. reinhardtii similar
decrease was observed only for the PSII from cia3 not
containing CAH3 that was completely eliminated by
addition of rCAH3 to the preparations (Fig.1a), which
binds strongly to the membrane preparations (Fig.2).
Another unusual fact is that suppression of O
2
-evolv-
ing activity observed in the preparations from the
mutants without CAs was different in the PSII from
PROTECTIVE EFFECT OF CAH3 869
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
C. reinhardtii and A.  thaliana. While in the first case
inhibition appeared immediately and was maximal
after 15 min of photoinhibition or thermal inactiva-
tion as a result of absence of protective effect medi-
ated through CA activity of the enzyme, in the second
case inhibition manifested itself only after 30 min of
incubation, when conformational changes accumu-
lated in the proteins (Figs.  1 and 3). All these facts
could be considered as an additional confirmation of
absence of participation of CA in functioning of the
PSII from A.  thaliana.
Surprisingly, addition of rCAH3 during thermal
inactivation of the PSII preparations from the A.  thali-
ana mutants 8-8 and 9-12 at pH 6.5, resulted in a
slight suppression of the O
2
-evolving activity of PSII
after 15 min of incubation, which disappeared after
30 min of incubation (Fig. 3b). Comparison with the
data on photoinhibition revealed that this effect was
present there too, although was insignificant (Fig.1b).
Considering the Western blotting data on strong bind-
ing of rCAH3 with the membrane preparations of
PSII from A.  thaliana (Fig. 2), it could be suggested
that this inhibiting effect indicated existence of albeit
small, but functional interaction between rCAH3 and
PSII from A.  thaliana, i.e., between the highly active
CA (Fig. 4) and WOC in PSII from A. thaliana. How-
ever, unlike in the case of PSII from C.  reinhardtii,
similar interaction is, likely, not natural for the PSII
from A.  thaliana thus providing an opposite effect.
Addition of rCAH3 to the PSII preparations A.  thali-
ana mutants 8-8 and 9-12 at pH 7.0, did not reveal
the similar effect, which indicated loss of function-
al interaction between rCAH3 and PSII at this pH. At
the same time, at pH 7.0 protective effect of CAH3
on the O
2
-evolving activity of PSII from C. reinhard-
tii was most pronounced especially during the first
15 min of thermal inactivation both in the case of
native protein (PSII from WT), and of recombinant
protein (Fig. 3c).
Hence, based on the obtained data, the follow-
ing conclusions could be made on the properties of
α-CAs CAH3 and CA4 (in the membrane PSII prepa-
rations): 1) CAH3 exerts protective effect on the ac-
tivity of PSII from C. reinhardtii under conditions of
moderate photoinhibition and thermal inactivation;
2)  CAH3 protein binds strongly with the membrane
preparations of PSII, including those isolated from
A.  thaliana, while preserving full activity; 3)  CAH3
does not provide noticeable protective effect under
conditions of moderate photoinhibition and thermal
inactivation of the PSII preparations from A.  thaliana
or even results in suppression of O
2
-evolving activity;
4)  CA4 does not participate in supporting activity of
WOC in PSII similar to CAH3, however, absence of the
CA4 protein results in larger disorganization of WOC
under the action of stress factors.
Abbreviations. 8-8 and 9-12, mutant lines of
A. thaliana with knockout of the gene encoding α-CA4;
CA, carbonic anhydrase; cia3, Chlamydomonas rein-
hardtii mutant lacking CAH3 in thylakoid lumen; Col,
Arabidopsis thaliana ecotype Columbia-0; PSII, pho-
tosystem II; rCAH3, recombinant CAH3 protein; ROS,
reactive oxygen species; qE, energy-dependent com-
ponent of non-photochemical chlorophyll quenching;
WOC, water-oxidizing complex; WT, wild-type.
Acknowledgments. Equipment of the Center for
Collective Use of the Pushchino Scientific Center for
Biological Research, Russian Academy of Sciences, was
used in the study (no. 670266; https://www.ckp-rf.ru/
ckp/670266/) [in Russian].
Contributions. V. V. Terentyev – concept and
supervision of the study, writing and editing text of
the paper; V. V. Terentyev, L. I. Trubitsina, T. P. Khoro-
shaeva, and I. V. Trubitsin– conducting experiments;
V. V. Terentyev, L. I. Trubitsina, and T. P. Khoroshaeva
– discussing results of the study.
Funding. This study was financially supported by
the Russian Science Foundation, grant no. 23-24-00550,
https://rscf.ru/project/23-24-00550/ [in Russian].
Ethics approval and consent to participate.
This article does not contain any studies with human
participants or animals performed by any of the au-
thors.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
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