ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 873-881 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 951-960.
873
An Attempt to Increase Thermostability
of the Mutant Photosynthetic Reaction Center
of Cereibacter sphaeroides Using Disulfide Bonds
Tatiana Yu. Fufina
1
and Lyudmila G. Vasilieva
1,a
*
1
Institute of Basic Biological Problems, Russian Academy of Sciences,
Pushchino Scientific Center for Biological Research, Russian Academy of Sciences,
142290 Pushchino, Moscow Region, Russia
a
e-mail: vsyulya20@yandex.ru
Received April 2, 2025
Revised May 28, 2025
Accepted May 28, 2025
Abstract— Methods of site-directed mutagenesis are successfully used in structural and functional studies of
photosynthetic reaction centers(RCs). It has been noted that many mutations near electron transfer cofactors
reduce temperature stability of the Cereibacter sphaeroides RCs and affect amount of RCs in the membranes.
We previously reported [Selikhanovetal. (2023) Membranes, 25, 154] that introduction of inter-subunit disul-
fide bridges on the periplasmic or cytoplasmic surface of the complex promotes increase in thermal stability
of the C.sphaeroides RCs. In this work, an attempt was made to increase thermal stability of the mutant RC
with the Ile M206 – Gln substitution by introducing inter-subunit disulfide bonds. This RC is of considerable
interest for studying mechanisms of early electron transfer processes in RCs. The effect of mutations on the
amount of RCs in chromatophores was analyzed and it was found that the I(M206)Q mutation leads to twofold
decrease in the RC content in chromatophores, introduction of disulfide bonds on the cytoplasmic or peri-
plasmic sides of the complex reduces the amount of RCs in membranes by one third, the triple substitution
I(M206)Q/G(M19)C/T(L214)C reduces the amount of RCs in membranes almost 4-fold, and the substitutions
I(M206)Q/V(M84)C/G(L278)C lead to disruption of RC assembly in the membrane. It was shown that introduc-
tion of the inter-subunit S-S bond on the cytoplasmic surface of the complex did not have a significant effect
on thermal stability of the I(M206)Q RC. Our own and literature data on the factors influencing assembly
processes and ensuring stability of the structure of integral membrane complexes are discussed.
DOI: 10.1134/S0006297925600978
Keywords: photosynthetic reaction center, Cereibacter sphaeroides, protein thermal stability, absorption spectra,
disulfide bridges
* To whom correspondence should be addressed.
INTRODUCTION
Photosynthetic apparatus of the purple bacterium
Cereibacter sphaeroides includes two light-harvesting
complexes (LHC-1 and LHC-2) and a reaction center
(RC) [1]. The RC of C. sphaeroides is one of the best
studied bacterial pigment–protein complexes; it is
used to investigate mechanisms of the primary pro-
cesses of photosynthesis and is a model for studying
integral membrane proteins. This RC comprises three
protein subunits (L, M, and H) and ten cofactors ar-
ranged in two branches, active (A) and inactive (B),
for electron transfer (Fig. 1). Cofactors are represent-
ed by four molecules of bacteriochlorophyll (BChl) a,
two molecules of bacteriopheophytin (BPheo) a, two
molecules of ubiquinone (Q), a non-heme iron atom,
and a carotenoid molecule [2]. After decoding spa-
tial structure of this RC, quite a lot of works have
been devoted to studying the role of protein envi-
ronment of the cofactors in providing high quantum
efficiency of the photochemical reaction [3]. One
of the methods used in such studies is site-directed
mutagenesis. The RC of purple bacteria is consid-
ered to be a relatively stable membrane complex [4];
FUFINA, VASILIEVA874
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
however, there are cases described in the literature
where point mutations resulted in the loss of cofac-
tors, partially or completely impaired assembly of
the RC in membrane [5, 6]. Taking into account that
the photosynthetic RCs are of significant interest for
biotechnology as promising components of artificial
systems for solar energy conversion [7], it is relevant
to identify amino acid residues or polypeptide regions
in the complex structure, which are critical for its sta-
bility, as well as to search for approaches to stabilize
structure of the genetically modified RCs. The methods
used for this purpose in some works included selec-
tion of optimal detergent for RC solubilization from
the membranes as well as addition to the buffer for RC
purification and storage[8], as well as introduction of
H-bonds between the cofactors and the protein [9-11].
Previously we have reported that introduction of in-
ter-subunit disulfide bridges on the periplasmic or cy-
toplasmic surface of the complex noticeably increases
thermostability of the C. sphaeroides RC [12]. In the
present work, an attempt has been made to similarly
stabilize the structure of the mutant RC with substi-
tution of M206 Ile for Gln, which influenced stability
of the complex to heat denaturation. RC I(M206)Q is
of significant interest for studying the mechanisms of
initial stages of photochemical charge separation, be-
cause this mutation close to bacteriochlorophylls of
the active electron transfer chain (Fig. 1) led to the
substantial decrease in the quantum yield of forma-
tion of charge-separated state P
+
Q
A
−
[13].
In the present work we attempted to increase
thermostability of the I(M206)Q RC by introduction of
inter-subunit disulfide bonds on the cytoplasmic and
periplasmic sides of the mutant membrane complex.
In addition, the effects of amino acid substitutions on
the amount of RCs in photosynthetic membranes were
investigated. Previously, in 2022, Fufina et al. [13]
showed that the properties of C. sphaeroides RCs with
the I(M206)Q substitution were significantly different
from the properties of mutant RCs of the closely relat-
ed bacterium Rhodobacter capsulatus with the simi-
lar I(M204)Q substitution. It was hypothesized that the
different consequences of the same mutation could be
due to the different interactions between these two
RCs and the membrane environment; however, at the
time of publication, spatial structure of the Rba.cap-
sulatus RC necessary for such comparison was not
available yet. The present work involves analysis of
the conserved lipid binding sites on the periplasmic
side of LHC-1–RC complexes from C. sphaeroides and
Rba.capsulatus.
MATERIALS AND METHODS
A DNA fragment with I(M206)Q mutation in
the puf-M gene of C. sphaeroides was cloned at the
NcoI/XhoI restriction sites in the puf-operon with
double mutations G(M19)C/T(L214)C resulting in for-
mation of disulfide bridges on the cytoplasmic side
of RC [12, 13]. In a similar way, this fragment was
cloned in the puf-operon with V(M84)C/G(L278)C mu-
tations resulting in formation of S–S bridges on the
periplasmic side of the wild type RC [12]. Locations
of mutation sites in the RC structure is shown in
Fig. 1. Next the puf-operons with the corresponding
Fig. 1. Structure of C.sphaeroides RC (PDB ID: 3V3Y). Inserts on the left show the sites of mutations and disulfide bridges
formed as a result of amino acid substitutions V(M84)C/G(L278)C (PDBID: 8C7C) and G(M19)C/T(L214)C (PDBID: 8C88) [12].
Insert on the right shows the site of mutation and the model of amino acid substitution I(M206)Q constructed with PyMol.
P, bacteriochlorophyll dimer; P
B
, one of bacteriochlorophylls of the dimer; B
A
and B
B
, monomeric bacteriochlorophylls;
H
A
and H
B
, bacteriopheophytins; Q
A
and Q
B
, ubiquinones.
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BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 2. Absorption spectra of chromatophores of the wild
type C. sphaeroides(1); with mutation I(M206)Q (2); with
triple mutation I(M206)Q/G(M19)C/T(L214)C (3); with a di-
sulfide bond on the periplasmic side of the membrane,
double mutation V(M84)C/G(L278)C (4); with a disulfide
bond on the cytoplasmic side of the membrane, double mu-
tation G(M19)C/T(L214)C (5); with triple mutation I(M206)Q/
V(M84)C/G(L278)C (6).
triple substitutions I(M206)Q/G(M19)C/T(L214)C and
I(M206)Q/V(M84)C/G(L278)C were cloned in a shuttle
vector pRK-415 as described by Khatypov et al. [14].
The resultant plasmids were transferred by conjugation
into an antenna-free strain C. sphaeroides DD13 [15];
as a result, recombinant strains C. sphaeroides
DD13//I(M206)Q/V(M84)C/G(L278)C and DD13//
I(M206)Q/G(M19)C/T(L214)C were obtained. Reaction
centers used as a wild (pseudo-wild) type reaction
center were isolated from the strain C. sphaeroi-
des DD13 containing the pRK-415 derivative, which
carried unmodified copies of the puf-LMX genes [14].
Recombinant C. sphaeroides strains were grown un-
der semi-aerobic conditions in the dark on a Hutner’s
medium[16] in the presence of tetracycline (1µg/mL)
and kanamycin (5µg/mL). Inoculum density, time and
conditions of bacterial growth were the same for all
used strains; absorption of a culture suspension mea-
sured at 600 nm in a cuvette with an optical path
length of 1 cm after cultivation was 1.65-1.7. RC con-
tent in the membranes was evaluated by absorption at
800nm in chromatophores suspension prepared from
1 L of cell cultures grown under identical conditions
and dissolved in a fixed volume of 20 mM Tris-HCl
(pH 8.0). Reaction centers were purified using affini-
ty chromatography as described previously [17] with
subsequent purification by ion exchange chromatog-
raphy [18]. Membrane solubilization of the complex-
es was performed with a lauryldimethylamine ox-
ide (LDAO) detergent. Purified RCs were dissolved in
a TL buffer (20 mMTris-HCl (pH 8.0) and 0.1% LDAO).
Thermostability of isolated RCs was studied by heat-
ing them at 48°C for 60 min and recording thermod-
ependent changes in the amplitude of the Q
Y
band
of monomeric bacteriochlorophylls as described pre-
viously [8]. Thermostability of RCs within the mem-
branes was studied at 70°C by recording for 60 min
spectral changes caused by elevated temperature [9].
Electronic absorption spectra were measured at
room temperature with a Shimadzu UV-1800 spectro-
photometer (Shimadzu Corporation, Japan). Sodium
ascorbate (1 mM) was added to the samples to main-
tain the primary electron donor in a reduced state.
Fig. 3. Absorption spectra of wild type RC (1), RC with mutations I(M206)Q (2) and I(M206)Q/G(M19)C/T(L214)C(3) normal-
ized to the Q
Y
H band. The bands of low-energy exciton transition of the dimeric primary electron donor(Q
Y
P), monomeric
molecules of BChl (Q
Y
B), monomeric molecules of BPheo (Q
Y
H), as well as the band of Q
X
transitions of four molecules of
BChl (Q
X
P,B) and two molecules of BPheo (Q
X
H), are shown.
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BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Electrophoresis of RC under denaturing conditions
in 18% polyacrylamide gel was performed as de-
scribed previously [12]. Visualization of the reaction
center structure presented in Fig. 1 was carried out
using PyMol [19].
RESULTS
In the present work, as a result of combination
of amino acid substitutions in the puf-operon, a new
mutant RCs with triple substitution I(M206)Q/G(M19)C/
T(L214)C have been obtained. Absorption spectra of
chromatophores of the recombinant strain with this
triple mutation are shown in Fig. 2 (spectrum 3) to-
gether with the spectra of chromatophores from other
strains used in the work.
Combination of mutations I(M206)Q/V(M84)
C/G(L278)C aimed at introducing an inter-subunit di-
sulfide bond on the periplasmic side of the mutant RC
I(M206)Q, instead of the expected stabilization of RC
structure, seemed to result in disruption of the com-
plex assembly in the membrane, because absorption
bands typical of the BChl and BPheo RCs were absent
in the spectrum of chromatophores of this mutant
(Fig. 2; spectrum 6).
Figure 3 shows electronic absorption spectra of
the wild type C. sphaeroides RC and the mutant RCs
with single I(M206)Q and triple I(M206)Q/G(M19)C/
T(L214)C substitutions.
Absorption spectrum of the wild type RC (Fig. 3;
curve 1) corresponds to the literature data, includ-
ing presence in the spectral region of the Q
Y
band of
low-energy exciton transition of the dimeric prima-
ry electron donor (Q
Y
P) at 865 nm, absorption band
of monomeric BChls (Q
Y
B) at 804 nm, and of BPheo
molecules (Q
Y
H) at 758 nm. In the Q
X
region of the
spectrum the band near 600 nm is associated with
Q
X
transitions of four BChl molecules. There are the
maxima of absorption bands of BPheo molecules of
the inactive (533 nm) and active (545 nm) electron
transfer chains, as well as a shoulder in the region of
500 nm associated with absorption of the carotenoid
molecule. Detailed discussion of the electronic absorp-
tion spectrum of RC I(M206)Q is given in the work of
Fufina et al. [13]; its main difference from the wild
type RC spectrum is a minor short-wave shift of the
Q
Y
P band and decrease in the amplitude of this band.
Significant differences between the absorption spectra
of the mutant RC I(M206)Q and RC I(M206)Q/G(M19)C/
T(L214)C have not been revealed (Fig. 3).
To compare the levels of RCs in the chromato-
phores of different C. sphaeroides strains, the cells
were grown under identical conditions (see Materi-
als and Methods). The results presented in Table 1
show that introduction of mutations affects the RCs
content in the photosynthetic membranes. In partic-
ular, substitution of the Ile M206 with Gln results in
2-fold reduction in the RC content in chromatophores.
Introduction of disulfide bonds on the cytoplasmic or
periplasmic sides of RCs decreases their amount in
the membranes by about one third (Table 1). Combi-
nation of amino acid substitutions I(M206)Q/G(M19)C/
T(L214)C led to almost 4-fold decrease in the RCs con-
tent in chromatophores, while mutations I(M206)Q/
V(M84)C/G(L278)C resulted in disruption of the RC as-
sembly in the photosynthetic membrane.
The results of electrophoresis under denaturing
conditions in 18% polyacrylamide gel show formation
of an inter-subunit S–S bond in the RC of the triple
mutant I(M206)Q/G(M19)C/T(L214)C, similar to the RC
with double mutation G(M19)C/T(L214)C [13], demon-
strated by disappearance of the L- and M-subunits
bands (21 and 24 kDa, respectively) and appearance of
the band between 37 and 50 kDa corresponding to the
combined mass of L- and M-subunits of about 45 kDa
(Fig. S1 in the Online Resource 1).
Changes in the RC absorption spectrum at in-
creased temperature were used as an indicator of
stability of the RC’s structure [8]. Figure 4 shows ki-
netics of thermodependent changes of the Q
Y
B band
amplitude for the wild type RC, RC I(M206)Q, and RC
I(M206)Q/G(M19)C/T(L214)C measured during 60-min
incubation at 70°C for the membrane preparations
(Fig. 4a) and at 48°C for the isolated RCs (Fig. 4b).
The results show that the I(M206)Q mutation caus-
es significant decrease in stability of both mem-
brane-bound and isolated RCs, while additional intro-
duction of a disulfide bond on the cytoplasmic side of
the complex (G(M19)C/T(L214)C mutations) to the RC
I(M206)Q has no effect on thermostability of the RC
(Fig. 4b). It should be noted that the stabilizing effect
of inter-unit disulfide bonds on thermostability of the
Table 1. Content of RC in chromatophores of recom-
binant strains of C. sphaeroides
Strain/mutation
RC content in
chromatophores,
OD
800
Wild type 19 ± 4
I(M206)Q 9± 1
V(M84)C/G(L278)C 12 ± 2
G(M19)C/T(L214)C 13 ± 2
I(M206)Q/G(M19)C/T(L214)C 5± 1
I(M206)Q/V(M84)C/G(L278)C 0
Note. OD
800
, absorption at 800 nm.
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BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 4. Changes in the amplitude of the Q
Y
B band in the spectra of membrane-bound(a) and isolated(b) wild type C.sphaeroi-
des RC(■ ), RC with the disulfide bond on the cytoplasmic side of the complex, G(M19)C/T(L214)C mutations(), RC with the
disulfide bond on the periplasmic side of the complex, V(M84)C/G(L278)C mutations (
▲
), RC with mutation I(M206)Q (♦),
RC with triple mutation I(M206)Q/G(M19)C/T(L214)C () measured in TL buffer at 70°C (a) or at 48°C (b).
wild type RC reported by Selikhanov et al. [12] was
more pronounced than in the present work. This is
probably associated with the method of purification
of the wild type RC: Selikhanov et al. [12] used the
longer method of ion exchange chromatography, while
in the present work all RCs were purified by more
rapid affinity chromatography, which prevents desta-
bilization of the complex.
DISCUSSION
The mutant C. sphaeroides RC with substitution
of Ile M206 with Gln is of interest for studying mech-
anisms of the initial stages of photochemical charge
separation; however, the attempts to crystallize this
complex and to decode its spatial structure have not
been successful so far[13]. One of the possible causes
could be destabilizing effect of mutation on the RC
protein structure, which manifests itself as a decrease
in resistance of the mutant RC to elevated tempera-
tures. In the literature, several possible approaches
to increase stability of the membrane proteins have
been described. In particular, replacement of LDAO
with sodium cholate in the buffer for RC dissolution
significantly slowed down RC denaturation at elevated
temperatures [8], while introduction of H-bonds be-
tween 2-acetyl group of the BChl P
B
and the protein
increased resistance of RC to temperature and elevat-
ed pressure [9, 10, 20]. However, the latter approach
increases redox potential of the primary electron do-
nor P and thereby affects the photochemical process;
therefore, it cannot always be used in the structural
and functional studies of RCs. The method for sta-
bilization of local protein structure via introduction
of S–S bridges is used mainly for globular proteins,
but there are also few known examples of using this
approach for membrane complexes [21, 22]. Recently
it has been reported that introduction of inter-sub-
unit disulfide bonds on the periplasmic or cytoplas-
mic surfaces of RC from C. sphaeroides noticeably in-
creases its thermostability [13]. In the present work
we have shown that this approach, namely, formation
of a disulfide bridge between the α-helices of L- and
M-subunits on the cytoplasmic side of the mutant RC
I(M206)Q, had no effect on resistance of the com-
plex to heat denaturation both in membranes and
in solution. The findings suggest that substitution of
IleM206 with Gln not only destabilizes local structure
of the RC close to bacteriochlorophylls of the active
electron transfer chain, but also impairs interaction
between the complex and other membrane compo-
nents. Dezi et al. [23] have shown that the RC–LHC-1
core complex from C. sphaeroides contains more than
150 molecules of lipids: cardiolipin (50%), phospha-
tidylglycerol (24%), phosphatidylethanolamine (12%),
and phosphatidylcholine(14%). Lipids are assumed to
stabilize interactions between the RC and LHC-1, as
well as between the RC–LHC-1 complex and the mem-
brane bilayer into which it is incorporated. It seems
that violation in the RC conformation and disruption
of RC’s interactions with the membrane environment
caused by the single substitution I(M206)Q cannot
be compensated by introduction of a disulfide bond.
Previously it has been shown that properties
of the C. sphaeroides RCs with I(M206)Q substitu-
tion were significantly different from properties
of the mutant RCs of the closely related bacterium
Rba.capsulatus with analogous substitution I(M204)Q,
where this mutation resulted in the loss of BChl B
A
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BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 5. Structures of LHC-1–RC complexes from C. sphaeroides (PDB ID: 7VNY) (a) and Rba. capsulatus (PDB ID: 8B64) (b),
view from the periplasmic side of the membrane. LHC-1 are gray; L-subunit of RC is pale pink; M-subunit is light blue.
BChl dimer P and monomeric CHhl B
A
and -B
B
are dark gray. Isoleucine M206 (C. sphaeroides) and M204 (Rba. capsula-
tus) are bright pink. Cardiolipin is blue; 1,2-diacyl-sn-glycero-3-phosphocholine is green; 1,2-distearoyl-sn-glycerophospho-
ethanolamine is violet.
from the RC structure [5, 13]. Two structures of
RC–LHC-1 from Rba. capsulatus obtained by the
method of cryoelectron microscopy were published
in 2023 [24, 25], making it possible to compare mem-
brane environments of these complexes in the two
bacteria. Figure 5 shows the RC–LHC-1 structures from
C. sphaeroides and Rba. capsulatus, view from the
periplasmic side of the membrane. It can be noticed
that the regions of tight binding of the conserved lip-
ids in the structures of both bacteria are localized
mainly on the side of the active chain of cofactors
(Fig. 5). Qualitative and quantitative lipid compo-
sitions in the two bacteria are noticeably different.
Close to the site of mutation, M206, in C. sphaeroi-
des there is 1,2-diacyl-sn-glycero-3-phosphocholine;
in the structure of Rba.capsulatus, there is 1,2-distea-
royl-sn-glycerophosphoethanoamine (Fig. 5). These
structural data are in agreement with the assumption
that interactions between the two RCs and the mem-
brane environment are significantly different, which
could be a potential cause of different consequences
of the same substitution Ile → Gln [5, 13].
In the present work we have demonstrated that
introduction of mutations could have effect not only
on thermostability of RC but also on the amount of
these complexes in photosynthetic membranes. It has
been shown that the double mutations resulting in
formation of inter-subunit disulfide bonds on the peri-
plasmic or cytoplasmic surface of RC lead, simulta-
neously with the increase in thermostability of RC,
to the decrease in the membrane level of RC by one
third. The substitution of Ile M206 with Gln leads to
2-fold reduction of the RC level in chromatophores;
addition of the disulfide bridge on the cytoplasmic
side of RC I(M206)Q leads to 4-fold decrease in the
level of RC in the membranes, while introduction of
the disulfide bond on the periplasmic side complete-
ly disrupts assembly of the RC in the photosynthetic
membrane. The findings indicate that point substitu-
tions of amino acid could affect the processes of in-
corporation of the complex into the membrane.
In view of the above, theoretical studies of the
factors that determine stability of α-helices in the pro-
tein are of interest. Calculations show that deforma-
tion energy, which counteracts protein folding, arises
when position of the side-chain amino acid group
introduced as a result of site-directed mutation does
not coincide with the most stable conformation of this
group in a free α-helix [26, 27]. This factor seems to
cause disruption of the assembly of the mutant RCs
and, as a consequence, decrease in their quantity in
the membranes.
At present, the general concept of protein trans-
port and assembly in the cytoplasmic membrane of
bacteria has been formed in the literature (https://
simbac.gatech.edu/translocon/). During the synthesis
on ribosome, the proteins that must pass through or
be embedded into the membrane enter the translocon,
a heterotrimeric molecular complex with the shape of
a channel. The translocon “analyzes” hydrophobicity
THERMOSTABILITY OF THE C. sphaeroides PHOTOREACTION CENTER 879
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
ofthe polypeptide passing through it and, upon reach-
ing a certain hydrophobicity threshold, directs the
polypeptide not through the channel to the extracel-
lular space but through the regulated cavity in the
channel wall to the membrane lipid bilayer. There is a
large amount of literature data on formation, sizes, and
functional models of the membrane vesicles of pur-
ple bacteria; spatial structures and interrelationships
between the pigment–protein complexes involved in
light energy absorption and conversion are also well-
known [28]. In C. sphaeroides, membranes are formed
both in the presence of light and in the dark under
low aeration conditions [29]. The number of mature
chromatophores per cell may vary depending on cul-
tivation conditions and light intensity, but composition
of a single vesicle is relatively constant. The mature
chromatophore of C. sphaeroides has a photosynthetic
apparatus containing, on the average, 63-67 peripheral
light-harvesting complexes LHC-2, 11 dimers of core
complexes RC–LHC-1–PufX, 2 monomeric complexes
RC–LHC-1–PufX, 4dimers of cytochromebc1 complex,
and 2 complexes of ATP synthase [30, 31].
Although photosynthetic membranes of purple
bacteria are well characterized, little is known about
the details of the assembly of photosynthetic com-
plexes. This is a complex multistage process, which
includes polypeptide translocation and incorporation
into the membrane, posttranslational modifications
and protein folding, and cofactor attachment [28].
According to the recent data, the LhaA protein plays
an important role in the RC–LHC-1 assembly; the gene
of this protein, lhaA, is localized in the photosynthet-
ic gene cluster along with most of the genes related
to biosynthesis, assembly, regulation, and function of
RC–LHC-1 complexes in phototrophic bacteria [32].
It has been shown that the LhaA protein forms oligo-
mers at the sites of initiation of membrane invagi-
nation during chromatophore formation and that it
interacts with RC, BChl synthase (BchG), protein YajC
translocase subunit, and membrane protein YidC in-
sertase [33]. It has been suggested that LhaA is part
of the membrane nanodomain, where close proximi-
ty of the components of biosynthesis and membrane
translocation contributes to coordinated delivery of
the pigments (cofactors), co-translational insertion of
polypeptides, their folding and assembly for formation
of photosynthetic complexes [28].
Swainsburyetal. [28] drew some general conclu-
sions about the sequence of events that form RC and
then surround it with LHC-1 subunits. It is believed
that RC–LHC-1 complexes are built from the inside out:
first L- and M-subunits are attached to the H-polypep-
tide, which is permanently present in the membrane,
thereby forming RC. Next, the first subunit of LHC-1
binds to the specific region of RC and, beginning from
this point of initiation, a ring of LHC-1 subunits begins
to form [34]. Taking into account that our work was
carried out with the genetically modified strains of
C. sphaeroides with chromatophores containing RCs
without light-harvesting antenna complexes, it could
be suggested that the mutations mentioned in the
work could influence the processes such as translo-
cation and insertion of L- and M-subunits into the
membrane, protein folding, as well as interaction be-
tween the RC proteins and membrane lipids. Further
research is needed to draw more detailed conclusions.
CONCLUSIONS
It was shown in the present work that introduc-
tion of the mutations resulting in formation of an in-
ter-subunit disulfide bridge on the periplasmic surface
of mutant RC with substitution of Ile M206 with Gln
leads to disruption of RC assembly in the membrane.
Formation of the disulfide bond on the cytoplasmic
side of RC I(M206)Q does not promote increase in
thermostability of the mutant complex both in mem-
branes and in solution. In addition, introduction of
the I(M206)Q, V(M84)C/G(L278)C, G(M19)C/T(L214)C,
I(M206)Q/G(M19)C/T(L214)C, and I(M206)Q/V(M84)C/
G(L278)C mutations significantly reduces the amount
of RCs in chromatophores to varying degrees, appar-
ently affecting assembly of an integral membrane
complex. Thus, in spite of the fact that introduction of
disulfide bonds increases thermostability of the wild
type RC, this method is not suitable for stabilization
of the structure of the mutant RC with I(M206)Q ami-
no acid substitution close to bacteriochlorophylls of
the active chain cofactors.
Abbreviations. BPheo, bacteriopheophytin; BChl,
bacteriochlorophyll; RC, reaction center; LHC, light-
harvesting complex; B
A
and B
B
, monomeric bacte-
riochlorophylls; P, dimer of bacteriochlorophylls P
A
andP
B
; Q,ubiquinone.
Supplementary information. The online version
contains supplementary material available at https://
doi.org/10.1134/S0006297925600978.
Acknowledgments. The equipment used in the
work was provided by the Center for Collective Use,
Pushchino Scientific Center for Biological Research,
Russian Academy of Sciences (no.670266; https://www.
ckp-rf.ru/ckp/670266/).
Contributions. T. Yu. Fufina and L. G. Vasilye-
va – experimental part, discussion and editing of the
manuscript; T. Yu. Fufina – preparation of figures;
L. G. Vasilyeva– writing the manuscript.
Funding. The work was financially supported by
the State Assignment no. 122041100204-3 of the Min-
istry of Science and Higher Education of the Russian
Federation.
FUFINA, VASILIEVA880
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
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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