Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1428-1437 © Pleiades Publishing, Ltd., 2023.

Published in Russian in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1731-1741.

1428

Generation of Electric Potential Difference

by Chromatophores from Photosynthetic Bacteria

in the Presence of Trehalose under Continuous Illumination

Liya A. Vitukhnovskaya

1,2

, Andrei A. Zaspa

, and Mahir D. Mamedov

1,a

Belozersky Institute of Physical-Chemical Biology, Moscow State University,

119992 Moscow, Russia

Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences,

119991 Moscow, Russia

e-mail: mahirmamedov@yandex.ru

Received June 27, 2023

Revised July 20, 2023

Accepted August 26, 2023

Abstract— Measurement of electrical potential difference(Δψ) in membrane vesicles (chromatophores) from the purple

bacterium Rhodobacter sphaeroides associated with the surface of a nitrocellulose membrane filter(MF) impregnated with

a phospholipid solution in decane or immersed into it in the presence of exogenous mediators and disaccharide trehalose

demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The mediators

were the ascorbate/N,N,N′N′-tetramethyl-p-phenylenediamine pair and ubiquinone-0 (electron donor and acceptor,

respectively). Although stabilization of photoelectric responses upon long-term continuous illumination was observed for

both variants of chromatophore immobilization, only the samples immersed into the MF retained the functional activity

of reaction centers(RCs) for a month when stored in the dark at room temperature, which might be due to the preserva-

tion of integrity of chromatophore proteins inside the MF pores. The stabilizing effect of the bioprotector trehalose could

be related to its effect on both the RC proteins and the phospholipid bilayer membrane. The results obtained will expand

current ideas on the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting

solar energy into its electrochemical form.

DOI: 10.1134/S0006297923100024

Keywords: chromatophores, reaction center, cytochromebc

complex, membrane filter, semiconductor, continuous illumi-

nation, electrical potential

Abbreviations: Δψ,transmembrane electric potential difference; Asc,ascorbate; bc

,cytochromebc

complex; cyt,cytochrome;

ITO,indium-tin oxide semiconductor; MF,membrane filter; P

870

,chlorophyll dimer; Q

and Q

,primary and secondary qui-

none acceptors, respectively; RC, reaction center; TMPD,N,N,N’N’-tetramethyl-p-phenylenediamine; UQ

,2,3-dimethoxy- 5-

methyl-1,4-benzoquinone.

* To whom correspondence should be addressed.

INTRODUCTION

Over the past decades, multiple research efforts

have been focused on the development of highly effi-

cient artificial systems based on various dyes and semi-

conductors capable of converting light energy into elec-

tricity (see reviews [1,2]). However, such systems have

some disadvantages, such as high costs and the need

for expensive components, technical maintenance, and

disposal after a certain time. In view of this, one of the

promising approaches to the development of photoelec-

trochemical energy converters is the use of natural pho-

tosynthetic systems (see reviews [3,4]).

Vesicles formed by invagination of the intracyto-

plasmic membrane (chromatophores) of nonsulfur pur-

ple bacteria contain a photosynthetic apparatus ideally

suited for such studies. Unlike the thylakoid membranes

of cyanobacteria and chloroplasts, chromatophores of

GENERATION OF ELECTRICAL RESPONSE IN CHROMATOPHORES 1429

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

photosynthetic bacteria are the minimal structural and

functional units capable of using the energy of absorbed

photons for ATP synthesis [5]. Light-dependent cy-

clic electron transfer in chromatophores, for example,

in Rhodobactersphaeroides, occurs between the reaction

center(RC) and the cytochrome bc

complex with the

involvement of mobile membrane pool of ubiquinones

(UQ

) and endogenous soluble cytochrome (cyt)c

After RC excitation by a light quantum, the photo-

activated electron is transferred from the bacteriochlo-

rophyll P

870

dimer(P) to the primary(Q

) and then to

the secondary(Q

) quinone acceptors. Reduction of the

photooxidized P

870

occurs as a result of electron trans-

fer from the peripheral cyt c

(electron donor for RC).

According to the modified concept of the Q cycle (see

review [6]), oxidation of ubiquinol molecule formed in

the RC and transferred to the ubiquinol-oxidizing center

of the bc

complex is organized in such a way that one

electron returns through the Rieske iron-sulfur protein

(Fe

) and cytochromesc

andc

(high-potential chain)

to the photooxidized P

870

and the other electron is trans-

ferred through the two-heme cytochromeb (low-poten-

tial chain) to the ubiquinone reductase center Q

for the

reduction of ubiquinone molecule from the ubiquinone

pool. Repeated initiation of the above reactions leads to

the formation of “extra” ubihydroquinone in the Q

site

and increase in the number of protons exiting into chro-

matophore lumens per absorbed photon. It should be

also noted that the light-induced vectorial transport of

charges in the RCs and cytbc

in the membrane vesicles

is associated with the generation of the transmembrane

electric potential difference (Δψ) [7-11], which is a pri-

mary form of energy storage.

A relatively simple procedure of chromatophore

preparation from bacterial cells, intact lipid environment

of energy-converting enzymes in chromatophores, abil-

ity of RCs to convert light energy with a high quantum

efficiency, and detailed knowledge of the mechanism

of Δψ generation in some parts of the electron trans-

port chain make chromatophores an attractive object for

photoelectrochemical studies. When using chromato-

phores in hybrid systems invitro, one of the important

steps is their immobilization on supports (conductors,

semiconductors, polymers, phospholipid membranes)

and measurement of the electric potentials [12-14] and/

or currents under continuous illumination [12, 15-19].

However, in the above-mentioned studies [12-14],

Δψ generation under long-term continuous illumination

has not been investigated. The current in the continu-

ously illuminated chromatophores in [15-19] was mea-

sured mainly with the three-electrode system with the

applied external electric field, so that the photocurrent

amplitude depended on the field magnitude.

In the present work, we studied generation of

electric potential difference in response to continuous

illumination in Rba. sphaeroides chromatophores im-

mobilized on the surface of a nitrocellulose membrane

filter(MF) impregnated with a solution of phospholipids

using direct electrometric method [13], which involves

Δψ registration using a pair of silver chloride (Ag/AgCl)

macroelectrodes immersed in an electrolyte solution

and connected to an operational amplifier. The ob-

tained results were compared with the electrometric data

for chromatophores immersed into the MF clamped on

both sides by semiconductor glass plates based on in-

dium-tin oxide (ITO) [20]. The obtained data clearly

demonstrated long-term retention of the stable photo-

electric activity of chromatophores in these system in

the presence of disaccharide trehalose.

MATERIALS AND METHODS

Cell cultivation and preparation of chromatophores.

Wild-type Rba.sphaeroides cells were grown under an-

aerobic conditions at 30°C in the Ormerod medium[21]

at a light intensity of 800 W·m

–2

. To obtain membrane

vesicles with a high content of endogenous soluble cyt c

we used the extraction procedure described in[22] with

minor modifications. Cells suspended in 25 mM Hepes-

NaOH (pH 7.5) containing several crystals of DNase

and protease inhibitors were disrupted with a French

press (Aminco, USA). After removing the pellet, the

supernatant was applied on a sucrose density gradient

(5-35%, wt/wt) and centrifuged in a vertical VTi-50 rotor

(acquired through the Moscow State University Develop-

ment Program) for 2 h at 27,000 rpm. The lower chro-

matophore band was collected, dialyzed twice against

25 mM Hepes-NaOH (pH 7.5) for 2 h, and concentrated.

Chromatophore suspension [~2 mg of bacteriochloro-

phyll (bChl) per ml] was frozen in liquid nitrogen and

stored at –70°C.

The concentration of bChl in chromatophores was

determined as described earlier [23]. Chromatophore ab-

sorption spectra were recorded with a Hitachi 3400 spec-

trophotometer.

Measurement of the electric potential difference in

chromatophores immobilized on the MF surface. Δψ was

measured using direct electrometric method developed

in our laboratory [13]. MF (Millipore, USA) with a pore

size of 0.22 μm and thickness of 150 μm was used as a

support for chromatophore immobilization. Circular

MF (diameter, 2.0 cm) impregnated with a solution of

azolectin (L-ɑ-lecithin, type II-S; Sigma) in decane

(80 mg/ml) was clamped between two compartments of

a collapsible Teflon cuvette so that the filter blocked an

opening (diameter, 0.4 cm) in the partition separating

the two compartments. Both compartments were filled

with 25 mM Hepes-NaOH (pH 7.5) containing 20 mM

MgCl

and chromatophores were added to one of the

compartments. After stirring for 1h, both compartments

were washed with a tenfold volume of the buffer without

VITUKHNOVSKAYA et al.1430

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

MgCl

using a peristaltic pump to remove magnesium

and unbound chromatophores.

Exogenous mediators were added before the mea-

surements to the cell compartment containing chro-

matophores immobilized on the MF surface.

Photoelectric responses were recorded using a pair

of silver chloride macroelectrodes (Ag/AgCl/3 M KCl)

immersed in the electrolyte solution on both sides of the

MF. The electrodes were connected to an operational

amplifier (Burr Brown 3554BM, USA); the latter was

connected to a Gage CS8012 analog-to-digital convert-

er (ADC) and a computer. An incandescent lamp with

a power of 90 W (12 V) was used as a constant light

source.

Measurement of the electric potential difference

in chromatophores immersed into the MF. The mea-

surements were performed in the ITO|chromatophores–

MF|ITO system. For this, 2.5 × 5 cm MF (pore diam-

eter, 0.22 μm; thickness, 150 μm; Millipore GSTF) was

placed on the surface of a glass slide coated with the ITO

semiconductor. Next, 30-40 μl of suspension of chro-

matophores with the bChl concentration of ~2 mg/ml

was applied dropwise to the MF surface (~1.0 cm

After adsorption for 10 min in the dark, the MF surface

was washed (2 × 250 μl) with 25 mM HEPES-NaOH

(pH 7.5) and then the second ITO electrode was placed

on the top.

It should be noted that in the ITO|chromato-

phores–MF|ITO system, the mediators had been added

to the chromatophore suspension before it was used for

the incorporation into MF.

Registration of electrical potentials in the ITO|chro-

matophores–MF|ITO system was carried out using

copper wires connected on one side to the glass slides

coated with ITO and to the operational amplifier on the

other side. The signal from the amplifier was fed to a

Gage CS8012 ADC connected to a computer. Saturating

continuous illumination was provided by an incandes-

cent lamp (12V, 90W).

Each photoelectricity measurement was repeat-

ed at least 3 times. The range of errors for the photo-

responses was ~5%. All measurements were carried out

at20±1°C.

The figures were prepared with the Origin7.5 soft-

ware package (OriginLab Corporation).

Optical spectroscopy. Photoinduced changes in the

absorbance at 603 nm, which reflect the redox proper-

ties of the primary electron donor P

870

, were recorded

with a single-beam differential spectrophotometer con-

structed in our laboratory. The measuring light from

a KGM-98 lamp passed through an HL-1 Jobin Ivon

monochromator (France), cuvette with a sample, glass

light filter, and second UM-2 monochromator, and

then hit a photomultiplier (PMT). The signal from the

PMT was fed through the operational amplifier to the

ADC (Gage CS8012) connected to the computer.

RESULTS

The aim of this work was to study Δψ generation

in intact bacterial membrane vesicles under continuous

illumination. Previously, it was shown [24] that chro-

matophores isolated under certain conditions (cell dis-

ruption with a French press at low ionic strength) from

the nonsulfur purple bacterium Rba. sphaeroides con-

tain ~70% cyt c

inside the vesicles (see “Materials and

Methods” section). In these chromatophores, reduction

of photooxidized P

870

in the RCs can occur as a result of

direct electron transfer from endogenous cytc

[24, 25].

In other words, functionally active chromatophores

should contain cyt c

, which along with a pool of ubi-

quinones, acts as a redox carrier between the RCs and

cyt bc

. The content of cyt c

was determined by record-

ing changes in the absorbance at 603 nm (which reflects

the redox properties of P

870

) in a suspension of chro-

matophores in response to single light flashes in theab-

sence and presence of 2% Triton X-100 (not shown)

(see[26]).

Figure1 shows Δψ generation under constant illu-

mination in Rba.sphaeroides chromatophores associated

with the surface of MF impregnated with a phospholip-

id solution. No photoelectric response was observed in

the absence of additives (mediators) (Fig. 1a, curve 1).

It should be noted that as in the case of a collodion film

[9, 10], association of chromatophores with MF impreg-

nated with a solution of phospholipids in decane re-

sults in the extraction of UQ both from the site of the

secondary quinone acceptor Q

in the RC protein and

the membrane pool. In addition, cytc

and the primary

electron donor bChl P

870

dimer are oxidized in the RC.

Under these conditions, light-induced electron transfer

in chromatophores is limited by the formation of the

870

–

radical ion pair inside the RC protein. However,

charge transfer outside the RC in the described system

can be reconstructed by the saturation of immobilized

chromatophores with exogenous quinone and reduction

of endogenous cytc

(see[14]).

No photoelectric response was observed in chro-

matophores associated with the MF surface in the

presence of ascorbate (Asc) and N,N,N′N′-tetra-

methyl-p- phenylenediamine (TMPD) only in the as-

say medium (Fig. 1a, curve 2). The use of 2.5 mM

Asc/150 μM TMPD pair as an electron donor was need-

ed to reduce soluble cyt c

localized inside chromato-

phores, because of poor Asc penetration through the

membranes [9].

Figure 1a, curve 3 shows Δψ generation under con-

tinuous illumination in the presence of only 150 μM

2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ

,elec-

tron acceptor). The photoresponse amplitude under

these conditions was ~1.8 mV. UQ

is an efficient ex-

ogenous electron acceptor from quinones in the RCs

[27, 28] that is widely used in photoelectrochemical

GENERATION OF ELECTRICAL RESPONSE IN CHROMATOPHORES 1431

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 1. a)Generation of electric potential difference under continuous illumination by chromatophores associated with the surface of MF (pore

size, 0.22μm; filter thickness, 150μm) impregnated with azolectin solution in decane (80 mg/ml) without additives (curve1), in the presence of

2.5mM ascorbate (Asc)/150μM N,N,N’N’-tetramethyl-p-phenylenediamine (TMPD) pair (curve2), and in the presence of 150μM UQ

only

(curve3). Incubation medium: 25mM HEPES-NaOH (pH7.5). Inset: photoresponse in the presence of 5mM Asc/150μM TMPD and 150μM

in the absence of chromatophores. Arrows(↑) and(↓) indicate light on and off, respectively. b)Generation of electric potential difference

in the medium containing 2.5mM Asc/150μM TMPD and 150μM UQ

in the absence (curve1) and presence (curve2) of 0.75M trehalose.

Fig. 2. Generation of electric potential difference under stationary illumination in the presence of 0.75M trehalose by associated with the MF sur-

face: a)chromatophores isolated by a standard method; b)chromatophores deficient in cytc

. Experimental conditions are as in Fig.1b, curve2.

systems [28]. No photoresponse was observed in the con-

trol samples containing Asc/TMPD and UQ

, but no

chromatophores (inset in Fig.1a).

Figure 1b, curve1 shows the photoelectric response

of chromatophores under continuous illumination in the

presence of both electron donor (Asc/TMPD) and ac-

ceptor (UQ

). It can be seen that only in the presence

of both mediators, a significant increase in the photo-

electric response was observed, followed by a two-com-

ponent decrease in Δψ with the fast (~3 s) and slow

(~180s) phases.

The following experiments were carried out to study

the effect of the disaccharide trehalose (bioprotector)

on the light-dependent formation of Δψ by chromato-

phores immobilized on the MF surface. In the presence

of Asc/TMPD and UQ

, addition of 0.75 M trehalose

VITUKHNOVSKAYA et al.1432

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 3. a)Generation of electric potential difference in chromatophores associated with the surface of MF impregnated with a solution of phos-

pholipids. Conditions are as in Fig.1b (curve2) in the absence (curve1) and presence (curve2) of 5μM AntA; curve3, conditions are as in Fig.3a

(curve1) in the presence of 10μM atrazine. b)Generation of electric potential difference by chromatophores after 18h (curve1) and 40h (curve2)

of storage at room temperature. Experimental conditions are as in Fig.3a, curve1.

under continuous illumination stabilized Δψ for ~190 s

(Fig.1b, curve2). Under these conditions, stabilization

of Δψ signal was also observed under much longer illu-

mination (~1800s) (Fig.2a).

Figure 2b shows Δψ generation in the presence of

Asc/TMPD, UQ

, and trehalose by Rba. sphaeroides

chromatophores deficient in cytc

. Illumination of these

chromatophores led to the generation of electric poten-

tial difference with an amplitude of ~1.8mV, indicating

that the reduced 150 μM TMPD penetrating through

the membrane was unable to efficiently reduce photoo-

xidized P

870

under stationary conditions [11].

Charge transfer in the RC and bc

protein complex-

es in chromatophores is associated with the generation

of Δψ that can be measured by the electrochromic shift

of carotenoid absorption bands and direct electrometric

method [10]. Figure3a (curve1) shows Δψ generation

under continuous illumination in the presence of Asc/

TMPD, UQ

, and trehalose. To determine the contri-

bution of the bc

complex to the total photoresponse, an-

timycinA (AntA, inhibitor of the ubiquinone reductase

site Q

of the bc

complex) was added to the medium,

which reduced the photoresponse amplitude by ~12%

(Fig.3a curve2). The observed effect could be due to

the fact that UQ

binds to the Q

site in the RC [28] and

receives an electron from Q

–

; its reduced form (UQ

)

acts as a “substrate” only for a small fraction of the bc

complexes.

To prove that the observed photoelectric activity of

chromatophores was due to the functioning of RCs, atra-

zine (inhibitor of electron transfer between Q

–

andQ

)

was added to the medium. Indeed, addition of 10μM

atrazine almost completely inhibited generation of the

photoelectric response (Fig.3a, curve3), indicating that

in the studied system, electron transfer in the acceptor

site proceeded from the primary quinone Q

to the ex-

ogenous acceptor Q

To study the stability of photoresponses, chromato-

phores associated with the MF surface were stored in

the dark at room temperature and illuminated at regu-

lar intervals. The amplitude of Δψ, which was initially

~14 mV (Fig. 3a, curve 1), decreased to ~1.4 mV after

18 h of storage (Fig. 3b, curve 1). No photoresponse was

observed after 40h of storage (Fig.3b, curve2).

Figure 4 shows Δψ generation in chromatophores

immersed into MF and clamped on both sides by semi-

conductor indium-tin oxide glass plates (ITO|chromato-

phores–MF|ITO) in the presence of mediators (Asc/

TMPD, UQ

), antimycin A, and trehalose. It was as-

sumed that the redox mediators moved freely inside

the MF and could interact with the RCs and ITO elec-

trodes. When the system was illuminated for ~4000 s,

a stable nondecreasing photoelectric response with an

amplitude of ~30mV was generated [20].

It should be noted that the 18-h dark adaptation of

the ITO|chromatophores–MF|ITO system at room tem-

perature resulted in a significant decrease in the Δψ sig-

nal upon illumination for 180 s (not shown). The inset

in Fig. 4 shows Δψ generation upon addition of ~40 μl

of fresh 25 mM HEPES-NaOH (pH 7.5) containing

redox mediators (Ask/TMPD and UQ

) directly to the

ITO|chromatophores–MF|ITO system after its storage

GENERATION OF ELECTRICAL RESPONSE IN CHROMATOPHORES 1433

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 4. Generation of Δψ in chromatophores immersed into MF (ITO|chromatophores–MF|ITO system) in the presence of 2.5mM Asc, 150μM

TMPD, 150μM UQ

, 0.75M trehalose, and 5μM AntA and illuminated for 4000s (control). Inset: Δψ generation after addition of fresh 25mM

HEPES-NaOH (pH7.5) containing Asc/TMPD and UQ

directly to the ITO|chromatophores–MF|ITO system on the day 30 of storage at room

temperature.

Fig. 5. Electron transfer pathways induced by stationary illumination in Rba.sphaeroides chromatophores associated with the MF surface(a)

andimmersed into MF(b).

for 30 days in the dark at room temperature. Under

these conditions, the photoresponse amplitude was

~70% of the control.

Figure 5 shows presumable electron transfer path-

ways in chromatophores immobilized by two different

methods.

DISCUSSION

The studies of the mechanism of Δψ generation in

chromatophores associated with the surface of a collo-

dion film impregnated with a lipid solution in response

to single light flashes using direct electrometric method

VITUKHNOVSKAYA et al.1434

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

demonstrated the presence of the following electrogen-

ic reactions: 1) charge separation between P

870

and Q

;

2) re-reduction of photooxidized P

870

by endogenous

cyt c

; 3) protonation of the doubly reduced secondary

quinone acceptor Q

(see review [11]); 4)electron and

proton transfer in the cytbc

complex [10].

The purpose of this work was to demonstrate Δψ

generation under stationary illumination of chromato-

phores immobilized on the surface of MF and immersed

into it and to study its mechanism.

It should be noted that adsorption of biomaterials

on a solid surface (Au, TiO

, ITO electrodes) can lead to

structural changes and emergence of electrostatic repul-

sion between the studied sample and the support surface

([29] and references therein). Taking into account the

biocompatibility, biodegradability, nontoxicity, and low

cost of porous nitrocellulose membrane filter [30, 31],

we used it as a matrix for chromatophore immobiliza-

tion. Interaction of chromatophores with the MF sur-

face was achieved by neutralization of negative charges of

the polar heads of chromatophore membrane phospho-

lipids and MF by addition of Mg

or Ca

cations [13]

(see “Materials and Methods” section).

To generate stable electric signals, we used func-

tionally active chromatophores with a high content of

endogenous cyt c

. Since the redox cofactors in the RC

and bc

transmembrane complexes are immersed deeply

in the protein matrix, the recording of Δψ under contin-

uous illumination invitro (in a semi-synthetic system)

was performed in the presence of exogenous mediators.

The Asc/TMPD pair was used because of the chromato-

phore membrane permeability for the reduced TMPD

and capability of this compound to reduce endogenous

cyt c

[9]. Soluble UQ

maintained the functional ac-

tivity of RCs by accepting electrons from the primary

quinone Q

–

in chromatophores associated with the MF

surface and from the secondary quinone Q

–

in the case

of chromatophores immersed into the MF [20,28]. UQ

was an efficient mediator between the quinone acceptor

site of the RC and electrodes [28,32].

In Rba. sphaeroides chromatophores associated with

the MF surface, loosely bound Q

and UQ molecules

was washed out with n-decane used as a hydrophobic

solvent for phospholipids during MF impregnation,

which resulted in the blockade of cyclic charge trans-

fer [13]. Under these conditions, ubihydroquinone

(UQ

) generated under stationary illumination could

not act as a substrate for the bc

complex, probably, be-

cause of its high hydrophilicity (Fig.5a).

On the other hand, when chromatophores were im-

mersed into MF, the functioning of the cyclic electron

transport chain with the participation of the RC and bc

complexes, UQ pool, and cytc

, was preserved. This was

confirmed by the fact that addition of AntA (bc

com-

plex inhibitor) led to a significant (almost twofold) in-

crease in the amplitude of the steady-state Δψ [20].

The increase in the amplitude of the photoelectric re-

sponse upon inhibition of the bc

complex was due to the

redirection of cyclic electron flow (which does not con-

tribute to the membrane potential in the studied system)

toward linear electron transfer between the ITO elec-

trodes and RCs (Fig.5b). This type of chromatophore

immobilization allowed to record stable non-decreasing

Δψ signal during prolonged illumination (~4000s) [20].

Therefore, the recorded maximal photoelectric re-

sponse was probably due to the light-induced direct

electron transfer along the cyt c

→ RC → Q

→ Q

→ O

path in the case of chromatophores associated with the

MF surface (Fig.5a) or ITO → cyt c

→ RC → Q

→ Q

→

→ UQ

→ ITO path for chromatophores immersed into

MF pores (Fig.5b). In other words, in both cases, gen-

eration of stationary Δψ was due to the operation of RCs

themselves. This was evidenced by the suppression of

electrical response generation in the presence of atra-

zine, an inhibitor of electron transfer between quinone

acceptors in RCs (Fig.3a, curve3).

In studies on the conversion of light energy, it is

important not only to obtain a significant amplitude of

electric current under continuous illumination, but also

to identify conditions for maintaining the functional ac-

tivity of samples during their long-term storage at room

temperature. In chromatophores associated with the MF

surface, an almost complete drop in the Δψ amplitude

was observed after 40 h of storage (Fig. 3b, curve 2).

The decrease in the photoresponse amplitude could be

associated either with a decrease in the resistance of

MF impregnated with a lipid solution [13] or with the

‘degradation’ of added mediators, in particular, Asc.

However, the recovery of the MF resistance (2 × 10

Om)

and addition of a fresh portion of the buffer contain-

ing mediators (Asc/TMPD, UQ

) to the assay medium

does not lead to recovery of Δψ (not shown). Therefore,

the decrease in the Δψ amplitude with time in the case

of chromatophores associated with the MF surface was

most likely associated with the conformational and struc-

tural changes in the RCs.

As for the stability of photoelectric responses of

chromatophores immersed into the MF, addition of a

fresh buffer with the electron donor and acceptor to the

ITO|chromatophores–MF|ITO system stored at room

temperature under aerobic conditions for 30 days, caused

a recovery of the Δψ amplitude to ~70% (inset in Fig.4)

relative to the control sample (Fig. 4), indicating that

the majority of chromatophores immobilized on the MF

remained functionally active for at least 30 days.

Hence, in both systems, stable maximal photore-

sponses were observed only in the presence of trehalose,

a disaccharide with unique physicochemical properties.

Previously, similar stable photoelectric responses were

detected in the presence of this osmolyte in isolated pig-

ment–protein complexes of PSI and PSII immersed into

MF [33, 34]. Note that trehalose also affects the nature

GENERATION OF ELECTRICAL RESPONSE IN CHROMATOPHORES 1435

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

of the electric response (Fig. 1b, curve 2), namely, sup-

presses the rapid burst and decay of Δψ at the start of

illumination (Fig. 1b, curve 1), which can be due to a

decrease in the ionic permeability of the chromatophore

membrane [35,36]. A similar effect of trehalose on the

photoresponse rapid burst and decay was observed in

chromatophores immersed into MF [20]. Stabilization

of photoresponses could be associated with an improve-

ment in the efficiency of interactions between the me-

diator(s) and proteins of the photosynthetic electron

transport chain. Preservation of a thin hydration shell

of transmembrane proteins in the presence of this disac-

charide can change their conformation into a conforma-

tion more optimal for their efficient functioning [37-39].

Trehalose might also stabilize lipid bilayers by replacing

water during formation of hydrogen bonds between its

hydroxyl groups and polar lipid heads [40]. In this case,

interactions of trehalose with the transmembrane pro-

teins and phospholipids in chromatophores can occur

only near the outer side of the membrane because of the

impermeability of chromatophore membrane for treha-

lose.

Therefore, trehalose stabilized photoelectric sig-

nals generated by both types of immobilized chromato-

phores; however, the most stable photoresponses were

observed when chromatophores were immersed into the

MF. This extremely simple method of sample immo-

bilization might help to preserve intact photosynthetic

proteins of chromatophore inside the MF pores at room

temperature for a long period of time.

The obtained results will expand modern ideas on

the use of semisynthetic structures based on various in-

tact photosynthetic systems (cyanobacteria and purple

bacteria cells [41], plant thylakoid membranes) capable

of converting solar energy into its electrochemical form.

Contributions. M.D.M. developed the concept and

supervised the study, analyzed electrical measurements,

and wrote the text of the article; L.A.V. and A.A.Z. iso-

lated bacterial membrane vesicles (chromatophores) and

performed electrical measurements.

Funding. The work was supported by the Russian

Science Foundation (project no.23-74-00025).

Ethics declarations. The authors declare no conflict

of interest in financial or any other sphere. This article

does not contain any studies with human participants

oranimals performed by any of the authors.

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