Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 882-893 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 961-973.

882

Cationic Antiseptics Disrupt the Functioning

of the Electron-Transport Chain at the Acceptor Side

in the Photosynthetic Reaction Centres

of the Purple Bacterium Cereibacter sphaeroides

Evgenii P. Lukashev

, Mahir D. Mamedov

, Liya A. Vitukhnovskaya

Aida M. Mamedova

, Peter P. Knox

1,a

*, and Vladimir Z. Paschenko

Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University,

119234 Moscow, Russia

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119992 Moscow, Russia

e-mail: knox@biophys.msu.ru

Received March 11, 2025

Revised May 4, 2025

Accepted May 6, 2025

Abstract— Using electrometric technique, the cationic antiseptic octenidine was revealed to reduce generation

of transmembrane electrical potential difference in the chromatophores of photosynthetic bacterium Cerei-

bacter sphaeroides. This is also confirmed by measurements of electrochromic shifts of carotenoid absorption

bands in chromatophores. In reaction centers (RCs), isolated from chromatophores in the absence of external

electron donors and acceptors, the rate of recombination between photooxidized bacteriochlorophyll P

870

and

reduced secondary quinone acceptor Q

, as measured by absorption changes in the near infrared region, was

very weakly dependent on the presence of antiseptics, in contrast to the kinetics in the 400-600nm spectral

range, where absorption changes associated with the oxidation of P

870

and the formation of semiquinone

radicals Q

−

and Q

−

, as well as electrochromic shifts of the carotenoid and bacteriopheophytin RC absorption

bands, were observed. The addition of cationic antiseptics modified the flash-induced absorbance changes

in this region with the formation time of ~100-200 ms and a decay time of ~3 s. In the series: picloxydine –

chlorhexidine– octenidine– miramistin, the last one was the most effective. The maximum amplitude of such

changes was observed in the absorption region of the semiquinone radical around 460 nm. When electron

transfer from Q

−

to Q

was blocked by o-phenanthroline, the effect disappeared. Cationic antiseptics are

suggested to stimulate protonation of Q

−

with the formation of a neutral Q

−

complex.

DOI: 10.1134/S0006297925600723

Keywords: cationic antiseptics, chromatophores, reaction centers, electrogenesis, electrometric method, flash

photolysis

* To whom correspondence should be addressed.

INTRODUCTION

Currently, the widespread and uncontrolled use

of antiseptics raises a reasonable question about their

possible negative impact on the biosphere. Under-

standing the behavior of these compounds in various

environments is important for their application. Pre-

viously, we have examined the effect of cationic anti-

septics with one or two charged groups (miramistin,

octenidine, chlorhexidine, and picloxydine) on energy

transfer in the membranes of chromatophores of pho-

tosynthetic purple bacteria [1, 2], as well as on isolat-

ed nuclear complexes of photosystem II from spinach

leaves [3]. Recently, the effect of these antiseptics on

fluorescence characteristics and electron transfer was

studied in another pigment-protein complex of pho-

tosystem I from the freshwater cyanobacterium Syn-

echocystis sp. 6803 [4]. For all the abovementioned

ANTISEPTICS BLOCK Δψ GENERATION 883

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

medications, the most noticeable effect was observed

in the presence of octenidine. In bacterial chromato-

phores from Cereibacter sphaeroides (previously called

Rhodobacter sphaeroides), which represent a minimal

possible photosynthetic unit which is structurally and

functionally intact, the addition of octenidine led to a

decrease in the efficiency of electron excitation energy

migration from the light-harvesting complex LH2 to

the core complex LH1–RC (RC– reaction center) by ap-

proximately three times[1]. Miramistin had the weak-

est effect on this energy migration in C.  sphaeroides

chromatophores. It was suggested that the disruption

of effective energy transfer occurs due to the anti-

septic binding to the hydrophilic heads of negatively

charged cardiolipin molecules located in the rings of

light-harvesting pigments on the cytoplasmic surface

of the chromatophores.

The photosynthetic electron transport chain in

C. sphaeroides chromatophores catalyzes a light-driv-

en cyclic electron flow between the RC and the cy-

tochrome bc

complex involving a mobile membrane

pool of ubiquinones (UQ

) and soluble endogenous

cytochrome c

RC is a transmembrane complex comprised of

three protein subunits (designated as L, M, and H)

with a total molecular weight of about 100 kDa.

Redox cofactors in the RC structure are organized

into two transmembrane branches, of which only

the L branch is functional. A special pair of bacte-

riochlorophylls (P

870

) is located near the periplasmic

surface of the photosynthetic membrane. The trans-

membrane protein L subunit contains monomeric

bacteriochlorophyll (BChl)a, bacteriopheophytin(BPh)

and the primary quinone acceptor Q

molecules,

forming the L branch, while the transmembrane sub-

unitM includes monomeric BChl, BPh, and the second-

ary quinone acceptor Q

, constituting the M branch.

A molecule of a specific carotenoid is also located in

the structure of the M subunit near BChl a. In par-

ticular, RCs of C.  sphaeroides contain spheroidene

or spheroidenone as this carotenoid. The latter is

synthesized during bacterial growth in the presence

ofO

[5]. Upon light excitation of RCs, electron trans-

fer occurs from the primary electron donor P

870

BChl with a characteristic time of ~3 ps, then to BPh

in ~0.9 ps, to Q

(~200 ps) and to Q

(~100 μs) [6-8].

RCs from C.  sphaeroides also contain three integral

lipid molecules: cardiolipin, phosphatidylcholine, and

glucosyl-galactosyl diacylglycerol[9]. This suggests the

possible existence of specific interactions between

phospholipids and electron transport components in

RCs. In particular, it was shown that the thermody-

namic and kinetic parameters of electron transfer in

the quinone acceptor unit of RCs from purple bacte-

ria depend on the presence of physiologically import-

ant lipids in their environment [9, 10]. It has been

suggested that electrostatic interactions of lipids with

quinone acceptors in RCs affect the stabilization time

of separated charges [11]. Other experiments have

shown that the packing of RCs in the lipid bilayer

of chromatophore membranes appears to increase

the pK

of the singly reduced secondary acceptor of

ubiquinone Q

, the semiquinone anion Q

−

, by al-

most three units [12] compared to its very low value

(about 4.5) in RC samples [13]. It is assumed that the

high pK

value of the secondary quinone promotes the

formation of neutral hydroquinone Q

−

and stimu-

lates the transfer of the second electron to it with the

formation of Q

2−

. This hypothesis of proton–elec-

tron events has been termed “proton-activated elec-

tron transfer” [13].

Since cationic antiseptics interact primarily with

amphiphilic molecules, it should be expected that not

only in membrane preparations but also in isolated RC

complexes containing lipid molecules and surrounded

by amphiphilic detergent molecules, electron trans-

port processes may depend on the presence of cationic

antiseptics. In this regard, the aim of this work was to

study the effect of a number of cationic antiseptics on

photosynthetic electron- and proton-transport activity

in C. sphaeroides chromatophore membranes and RC

preparations isolated from them by the electrometric

method and flash photolysis.

MATERIALS AND METHODS

Isolation and purification of chromatophores

and RC preparations from C.  sphaeroides. Chro-

matophores from the wild-type purple non-sulfur

C. sphaeroides bacteria were isolated and purified

according to the method described by Woronowicz

et al. [14], with minor modifications. In brief, cells

were collected and washed in 25 mM Hepes buf-

fer (pH 7.5) and disrupted using a French press at

12,000 psi. After centrifugation of the suspension

for 20 min at 4°C and 14,000g, the supernatant was

loaded onto a 5-35% (w/w) sucrose gradient prepared

in 25 mM Hepes buffer (pH 7.5) over a 60% sucrose

cushion and centrifuged in a VTi 50 vertical rotor for

2h at 140,000g. The bottom (third from the top) band

containing chromatophores was collected, dialyzed

twice against 25  mM  Hepes buffer (pH  7.5) for 2  h

and concentrated. The BChl concentration in the chro-

matophore suspension was determined as described

previously [15].

During RC isolation, the chromatophores were

incubated for 30 min at 4°C in 10 mM sodium phos-

phate buffer (pH 7.0) containing 0.5% lauryldimethyl-

amine oxide (LDAO) detergent. The membrane frag-

ments were then centrifuged for 90 min at 4°C and

144,000g. The RC fraction contained in the supernatant

LUKASHEV et al.884

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

was separated by chromatography on a hydroxyap-

atite column, as described by Zakharova et al. [16].

The applied method made it possible to obtain RC

preparations that had retained both quinone accep-

tors within their structure.

Electrometric method. Ag/AgCl electrodes im-

mersed in a Teflon cuvette were used to measure the

transmembrane electric potential difference (Δψ) on

chromatophores adsorbed on the surface of a collodion

film impregnated with a phospholipid (type IIS phos-

phatylcholine, Sigma-Aldrich, USA) in decane [17, 18].

The output voltage was connected through an op-

erational amplifier (3554BM, Burr Brown, USA) to a

GaGe CS8012 analog-to-digital converter and then to

a computer. The method allows direct measurements

of undistorted electrical signals with a time resolution

of ~200 ns. Each measurement was repeated at least

3 times. The error range of the signals was ~3%. All

measurements were performed at 22  ±  1°C. Second

harmonic laser flashes of a Nd:YAG laser (532 nm;

12 ns half-width; 20 mJ pulse energy; Quantel, Les

Ulis, France) were used for light activation.

To restore the function of the loosely bound sec-

ondary quinone acceptor Q

and the pool of membrane

ubiquinones in chromatophores, CoQ

(20 mg/ml) was

added to the lipid solution for impregnating the col-

lodion film.

Flash photolysis. Light-induced absorption chang-

es in the suspension of chromatophores and RCs were

recorded using the flash photolysis setup described

earlier [4]. In the chromatophores, photoelectric re-

sponses were recorded by measuring light-induced ab-

sorption changes (electrochromic shift of carotenoid

absorption bands) at 523 nm and the redox state of

870

at 790nm. An aliquot of concentrated chromato-

phores was diluted for experiments in 25 mM Hepes

buffer (pH 7.5) to a final volume of 1 ml and a final

concentration of BChl of ~30 μM. The measurement

medium contained 25 mM Hepes buffer (pH 7.5),

2 mM potassium ferrocyanide and 50 μM N,N,N′N′-

tetramethyl-p-phenylenediamine (TMPD).

In experiments with RCs in 10 mM Hepes buffer

(pH 7.5), the BChl concentration was ~1.5 μM. In this

case, exogenous cofactors were not added. Photoin-

duced absorption changes in the RC suspension

were recorded in the visible spectrum range, 400-

650  nm, with a step of 10  nm, and in the IR range at

wavelengths of 790 and 865  nm. A 1  cm

sample in a

10×10 mm glass cuvette was placed in a thermostat-

ted qpod-2e holder (Quantum Northwest, USA) with

a set temperature of 21°C. An Nd:YAG laser (532 nm;

7ns half-width, 10mJ pulse energy, Lotis-TII, Belarus-

Japan) was used for pulsed illumination. The kinetics

of absorption changes per flash were analyzed in the

time range from 1μs to 1 s. To improve the signal-to-

noise ratio, 50 to 100 single signals were accumulated

and averaged. The time interval between flashes was

chosen to ensure complete recovery of P

870

in the

darkness; it was 1.5  s for chromatophores and 5  s

for RCs. Kinetic data analysis was performed using

Origin 9.1 (OriginalLab Corp., USA) or Mathematica 8

(Wolfram Research Inc., USA) software. Absorption

spectra were recorded on a modified Hitachi-557 spec-

trophotometer (Hitachi Ltd., Japan).

Antiseptic preparations. The following ready-

made pharmaceutical preparations were used in the

studies: chlorhexidine bidigluconate 20%, picloxydine

dihydrochloride 0.05% (Vitabact), miramistin 0.01%.

The source of octenidine was the commercial prepa-

ration “Octenisept” (Schulke & Mayr, Germany), con-

taining 0.1% octenidine dihydrochloride and 2% phe-

noxyethanol. Preliminary experiments showed that

phenoxyethanol, a component of the Octenisept prepa-

ration, did not by itself affect the spectral properties

and the studied parameters of the analyzed samples

in equimolar concentrations.

RESULTS AND DISCUSSION

The cationic antiseptics used in the study had

similar effects on the spectral characteristics and the

studied functional characteristics of photosynthetic

membranes and RCs isolated from them. The mag-

nitude of these effects decreased in the series: mi-

ramistin – octenidine – chlorhexidine – picloxydine.

At the same time, chlorhexidine and picloxydine had

a significantly less pronounced effect compared to

miramistin and octenidine. Therefore, the discovered

effects are further illustrated using octenidine and

miramistin as an example.

Light-induced vector charge transfer (electron

and proton) in bacterial membrane vesicles (chro-

matophores) results in the formation of Δψ on the

chromatophore membrane [17-19]. The electrometric

method used in this study allows to reveal both the

kinetics and dielectrically weighted distances for vec-

tor (electrogenic) charge transfer reactions within RC

protein complexes [20, 21]. Using this method, elec-

trogenic reactions such as electron transfer from P

870

to Q

, re-reduction of P

870

from cytochrome c

, and

protonation of the doubly reduced quinone acceptor

were previously shown in C. sphaeroides chro-

matophores [17, 18].

A photoelectric signal consists of an unresolved

fast phase of Δψ generation (resolution time of the

measuring system is ~ 200 ns), which is caused by

charge separation between P

870

and Q

, followed by

Δψ dissipation. Since the primary and the secondary

quinone electron acceptors are located parallel to the

membrane plane, the stage of electron transfer from

−

to Q

is not coupled with the formation of Δψ,

ANTISEPTICS BLOCK Δψ GENERATION 885

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 1. Photoelectric responses induced by the first (curves1) and second (curves2) light flashes in the presence of 100μM

octenidine (lower curves) and 100 μM miramistin (upper curves), respectively. Arrows indicate the moment of the laser

flash. The incubation medium contains 25 mM Hepes buffer (pH 7.5), 2 mM potassium ferrocyanide, and 50 μM TMPD.

20 mg of ubiquinone Q

was added to 1 ml of the phospholipid solution in decane to impregnate the collodion film.

The inset shows the electrical responses of C. sphaeroides chromatophores adsorbed on the surface of a collodion film

impregnated with a phospholipid solution in the absence of antiseptics induced by the first (curve1) and second (curve 2)

laser flashes. The subtraction of curve 2 from curve 1 is marked as 2-1. The time interval between the first and second

flashes is 1 s here and below.

i.e., it is non-electrogenic. In early studies [17,  18],

it was shown that the association of chromatophores

with a collodion film impregnated with phospholipids

in decane leads to the extraction of loosely bound sec-

ondary quinone Q

and a pool of ubiquinones (UQ).

Indeed, in the absence of exogenous electron donors

and acceptors, the kinetics of Δψ decay caused by the

reverse electron transfer reaction between Q

−

and

870

is almost fully completed within 100 ms. In this

case, the Δψ signal decay is approximated fairly well

by a single exponential with τ ~  66  ms (not shown).

It should be noted that the redox conditions in the

medium in the presence of 50 μM TMPD and 2 mM

potassium ferrocyanide (the redox potential of the me-

dium is about +320 mV) allow the detection of slow

electrogenic stages caused by vector charge transfer

involving both RC and the cytochromebc

complex in

chromatophores [17,  18]. As can be seen on the inset

to Fig. 1, in response to the second light flash, an ad-

ditional electrogenic phase appears in the kinetics of

the photoelectric response with a rise time of ~190  μs

(~14% of the amplitude of the fast phase, see the dif-

ference between the 2nd and 1st flashes). This submil-

lisecond rise phase is inhibited by o-phenanthroline,

which displaces the loosely bound secondary quinone

acceptor Q

from the binding site in the RC protein

(not shown). The dissipation time of the photopoten-

tial in the darkness under these conditions is ~500-

600 ms and reflects passive membrane discharging.

The main part of Fig. 1 shows the kinetic curves

of photopotential generation in chromatophores in the

presence of 100 μM cationic antiseptics. At a concen-

tration of 50 μM, both antiseptics had a weak effect

on the additional submillisecond electrogenic phase

in response to the second flash of light (not shown),

but octenidine completely removed it already at a

concentration of 100μM, while miramistin was much

less effective. It should also be noted that while the

photoresponse decay time was greater than 0.5 s in

the control sample (insert to Fig. 1), in the presence

of antiseptics, especially octenidine, the decay kinet-

ics became faster (<12 ms) (Fig. 1). This is probably

due to the disordering effect of the antiseptic on both

the RC protein and the chromatophore membrane, re-

sulting in an increase in passive ionic conductivity of

the chromatophore membrane.

In parallel experiments, we investigated pho-

toinduced absorption changes in a chromatophore

suspension using flash photolysis, choosing a wave-

length of 523  nm to monitor the carotenoid shift and

a wavelength of 790  nm to monitor P

870

redox trans-

formations. It is important to note that in bacterial

photosynthetic chromatophores of C.  sphaeroides,

measurements of the electrochromic shift at 523 nm

in response to single light flashes allow real-time de-

tection of generation and dissipation of the electri-

cal component of both the local and transmembrane

fields (Δψ and ΔμH

) [22-24]. Its formation is due to

LUKASHEV et al.886

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 2. Kinetic curves of the absorption changes at 523 nm (curves 1 and 2) and 790 nm (curves 3 and 4) recorded by

flash photolysis in C.  sphaeroides chromatophore preparations in the absence (control, 1 and 3) and in the presence of

100μM octenidine (2 and4). The curves were normalized to the maximum amplitude of absorption changes in the control.

The dots represent the experimental values; the solid line is an approximation as the sum of exponentials (details given in

the text). The medium was 25 mM Hepes buffer, pH 7.5, 50 μM TMPD, and 2mM potassium ferrocyanide.

the generation of Δψ as a result of the functioning

of both the RC and the cytochromebc

complex, and

the decline in the photoelectric response reflects ion

fluxes across the chromatophore membrane, includ-

ing the proton current through the F

-ATP synthase

channel [25] and the passive diffusion of ions across

the phospholipid bilayer[24].

It should be noted that just as in the experiments

on recording the Δψ generation using the electromet-

ric method, 50  μM TMPD and 2  mM potassium ferro-

cyanide were also added to the medium for absorp-

tion measurements.

The parameters of the approximation of the ki-

netic curves of the carotenoid shift (Fig. 2, curves 1

and2) are given in Table1. The unresolved fast phase

(shorter than 0.1μs), reflecting the formation of a local

electric field during charge separation between P

870

and Q

in the control comprises ~60% of the total am-

plitude. The remaining 40% can be represented by two

phases with half-times of ~20 μs and ~2.4 ms, which,

in our opinion, are associated with the formation of

the transmembrane field. The decline in carotenoid

absorption changes occurs over tens to hundreds of

milliseconds with characteristic times of about 50 and

300 ms and an amplitude ratio of 3 :  7. When octeni-

dine is added at a concentration of 100 μM, the total

signal amplitude drops by about 2.5times, and signif-

icant changes occur in the kinetics of both its forma-

tion and decay. The fast phase of formation (<0.1 μs)

dominates, accounting for more than 80%, and the

microsecond phase (~20 μs) completely disappears.

A small-amplitude phase with the length of the order

of several milliseconds remains, the nature of which

is not entirely clear. And while the dark decay kinetics

at 523nm was much slower than the recovery of P

870

in the control sample, in the presence of octenidine

the dissipation of the carotenoid shift accelerated, and

these events occurred in the same time range.

Changes in P

870

absorption depend weakly on

the presence of octenidine in the medium. Both the

amplitude of P

870

photooxidation and the kinetics of

dark reduction of P

870

remain practically unchanged

(Fig. 2, curves 3 and 4). Since the primary photooxi-

dation of P

870

occurs on a subpicosecond time scale,

the instrumental capabilities allowed us to record only

the slower processes of dark reduction of P

870

on a

micro-millisecond time scale.

In Table 2 we present the results of fitting the

kinetics at 790 nm by a sum of 4exponentials.

The following assumptions can be made about

the nature of individual phases of P

870

reduction af-

ter the activating flash. Since the medium contains

reduced TMPD and some preserved amount of cyto-

chromec

, it is possible that phasesτ

and τ

, lasting

about 50-500  μs, reflect electron transfer from cyto-

chromec

, and the next two millisecond-range phases,

and τ

, are associated with the reduction of P

870

by the redox mediator TMPD. It is important that the

amplitude of changes in P

870

absorption after the ad-

dition of octenidine remains constant and the kinetics

ANTISEPTICS BLOCK Δψ GENERATION 887

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Table 1. The parameters (τ and A,%) of the multiexponential approximation of the kinetics of the photoinduced

shift of carotenoid absorption band at 523 nm

Parameters

Formation Dissipation

, %) τ

, %)

Control < 0.1 μs (~60) 20 μs (~16) 2.4 ms (~24) 52 ms (~30) 310 ms (~70)

+ 100 μM octenidine < 0.1 μs (~83) – 3.1 ms (~17) 18 ms (~50) 117 ms (~50)

Table 2. The parameters (τ and A,%) of the multiexponential approximation of the kinetics of dark reduction

of P

870

at 790 nm

Parameters τ

, %) τ

, %)

Control 50 μs (~15) 0.5 ms (~15) 14 ms (~46) 79 ms (~24)

+ 100 μM octenidine 50 μs (~20) 0.5 ms (~17) 12 ms (~36) 74 ms (~27)

of dark reduction of P

870

changes insignificantly,

while the carotenoid shift decreases in amplitude, the

microsecond phase of its generation disappears from

the kinetics, and the dark decline accelerates.

Flash photolysis data obtained on chromato-

phores showed that octenidine does not affect the

primary charge separation between P

870

and quinone

acceptors, but disrupts transmembrane electric field

generation. Thus, these observations confirm the

conclusion drawn from the experiments with direct

measurements of electrogenesis in chromatophores.

However, the mechanism of this inhibitory effect in

the experiments on chromatophores could not be

established, so we studied the action of antiseptics

on preparations of RCs isolated from C. sphaeroides

chromatophores, which are a convenient object for

assessing the efficiency of the functioning of quinone

acceptors Q

and Q

. In the absence of an electron

donor in the medium, the kinetics of dark reduction

of P

870

after a flash reflects the degree of electron

occupancy of the primary and secondary acceptors.

In RC preparations with two quinone acceptors, Q

and Q

, after an activating flash of light, a slow recov-

ery due to dark recombination of P

870

−

is observed

Fig. 3. Kinetic curves of dark decay of absorption changes (normalized by amplitude and expressed as a positive value) in a

suspension of C. sphaeroides RCs, recorded in the IR (790 and 865nm) and visible (430, 550, and 600nm) spectral regions.

LUKASHEV et al.888

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 4. Kinetic curves of dark decay of absorption changes for a C. sphaeroides RC suspension recorded at different wave-

lengths. a)790nm, curves 1 and2– control without and in the presence of 5mM o-phenanthroline, respectively, curves3

and4 – after adding 150 μM miramistin to these samples; b-d)430, 460, and 550nm, respectively; curves: 1) in the pres-

ence of 150 μM miramistin, 2) normalized kinetic curves at 790 nm in the presence of 150 μM miramistin, 3) differential

curves “2 minus 1”. An approximation of the differential curve by a sum of three exponentials is also shown in panel c

(description in the text). d) curves 4 and 5: the kinetics at 550 nm in the presence of 5 mM o-phenanthroline in the con-

trol (4) and after the addition of 150 μM miramistin(5).

with a characteristic half-time of about 1 s (the rate

of direct electron transfer from Q

−

to Q

is 3 orders

of magnitude higher than that of the electron return

to P

870

, therefore, all electrons that hit the primary

quinone will then go to the secondary). And in prepa-

rations where the direct transfer is blocked in some

RCs, the recovery of P

870

will occur from Q

−

with a

characteristic time of about 0.1s. Since the RC prepa-

rations used in this work contained both quinone ac-

ceptors and there was no exogenous electron donor,

after a flash of light we observed P

870

recovery from

the secondary acceptor Q

−

with a characteristic time

of ~1 s. The kinetics of the dark decay of absorption

changes in the visible spectrum (400-650  nm) and

near IR range (700-900nm) were almost identical. The

characteristic curves for a number of wavelengths are

shown in Fig. 3.

However, when antiseptics are added, the kinetic

curves begin to diverge. While in the IR region, only

a slight acceleration of the decay kinetics is observed

for absorption changes reflecting the transformations

of the P

870

dimer, in the visible region of the spectrum

the discrepancies are more significant (Fig. 4).

Fig. 4a demonstrates the kinetics of absorption

changes at 790 nm in the control (curve 1, τ  ≈  1.1  s)

and in the presence of 5mM o-phenatroline (curve2,

τ  ≈  85  ms). It also shows the kinetic curves for

these samples in the presence of 150  μM miramistin

(curves 3 and 4). It should be emphasized that even

at very high concentrations of miramistin or octeni-

dine (400μM), the reduction in recovery time was no

more than 10% of this value in the control. In other

words, the effect of antiseptics is fundamentally dif-

ferent from the action of o-phenanthroline, which is

well known to replace the secondary acceptor at the

site of its localization in moderate concentrations, and

even displace the primary quinone in high concentra-

tions[26]. It should also be noted that in the presence

of o-phenanthroline, the kinetics of photoinduced ab-

sorption changes in the visible and IR regions coin-

cided for all the studied concentrations of antiseptics.

In the RC preparations without o-phenanthroline,

negative changes appeared in the kinetics of photoin-

duced absorption changes in the 400-650  nm region

a few milliseconds after the activating flash, which

distorted the “normal” form of the kinetics, if we con-

sider that the kinetics of transformations at 790 nm

adequately reflects the charge separation in RCs be-

tween P

870

and Q

−

with the formation of a local elec-

tric field and the subsequent charge recombination

(Fig. 4, b-d). The strongest effect was observed in the

presence of miramistin (then, in the descending or-

der – octenidine, chlorhexidine, picloxydine), so we

studied the effect of this preparation in most detail.

ANTISEPTICS BLOCK Δψ GENERATION 889

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 5. Single-flash-induced differential absorption “light minus dark” spectra in a suspension of C. sphaeroides RCs. a)Bythe

amplitude of absorption changes 1 ms after the flash (1) and by the amplitude of negative absorption changes appearing

with a delay(2), shown, in particular, in Fig. 4, b-d by curves 3; b) the same (2) dependence on an enlarged scale.

Let us recall that the recorded absorption changes in

the visible spectrum region reflect a number of pro-

cesses: oxidation of the BChl dimer, formation of the

semiquinone radical, as well as electrochromic shifts

of the absorption bands of carotenoids and BPh.

For illustration, Fig. 4, b-d shows photoinduced

absorption changes for wavelengths of 430, 460, and

440 nm (curves 1) and absorption changes at 790  nm

normalized by amplitude, which we considered “un-

distorted” (curves 2), as well as differential kinetic

curves (curves 3). Fig. 4c shows the fitting of such

kinetics by two growth exponents with characteristic

times τ

  ≈  25.6 ms (~30%) and τ

  ≈  191.2 ms (~70%)

and one decay exponent characterized by a time

  ≈  3s. The kinetic parameters differed little, but the

amplitude of the resulting curves depends significantly

on the wavelength.

Fig. 5a shows the differential absorption spec-

trum “light minus dark” in the visible part of the

spectrum (1), constructed from the signal amplitude

1 ms after the light flash until the moment of neg-

ative changes appearing, as well as the dependence

of the amplitude of the discussed negative absorption

changes on the wavelength (2). Fig. 5b presents this

dependence on an enlarged scale. We recall that in

the 400-600 nm spectral region, multiple absorption

changes are observed associated with redox transfor-

mations and electrochromic shifts of various RC com-

ponents: RC BChl (about 430 and 600 nm), BPh (about

540  nm), RC carotenoid (500-600 nm), and semiqui-

none forms of Q

and Q

acceptors (in the 450-460  nm

region) [27].

It has been previously shown that the formation of

the semiquinone anion Q

−

in the RC of Rb. sphaeroi-

des is accompanied by the appearance of a broad ab-

sorption band with a maximum at 450nm[28]. Asfol-

lows from Fig. 5b, the amplitude of the absorption

changes occurring ~200 ms after the flash reaches a

maximum of about 460 nm, which indicates a possi-

ble connection with a change in the absorption of the

semiquinone anion.

Despite long-term studies, the mechanisms of Q

reduction, proton binding and its migration in various

redox states are not yet fully understood [29]. It was

previously suggested that electrostatics plays a deci-

sive role in the interaction of various redox states

LUKASHEV et al.890

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

of Q

with the surrounding amino acid residues [30].

Indeed, all models of stabilization of the anionic form

of Q

suggest that this requires a significant rear-

rangement of charges and dipoles at the Q

site [31].

At the same time, it is known that in RCs from wild-

type Rb. sphaeroides bacteria, in which ubiquinone

is located at the site of the secondary acceptor

, the formation of its semiquinone anion form is

not accompanied by direct protonation, which is ex-

plained by the low value of its pK

 ~ 4.5 [13]. Since

the environment of Q

is rich in protons and polar

groups, the appearance of a negative charge on Q

affects the equilibrium of protons in the system of

hydrogen bonds involved in stabilizing the electron

on the acceptor. In particular, the pK of the amino

acid Glu L212 changes [32]. This amino acid, linked

through a network of hydrogen bonds with other

protonated amino acids and water molecules in the

RC structure, plays a key role in donating the first

proton in the process of forming hydroquinone Q

during two-electron reduction of the secondary accep-

tor[28]. It is proposed that in response to the electron

transfer to Q

, changes occur in the hydrogen bond

network formed by the surrounding water molecules

and the polar groups of the amino acids. Calculations

show that even small changes in the orientation of

these molecular groups can induce shifts in their pK,

ensuring the directional proton transfer[33].

The protonation of Q

−

in RC preparations was

successfully recorded in an experiment when ubiqui-

noneQ

was replaced with rhodoquinone, for which

the pK

value was much higher (about 7.3) [13].

At acidic pH values, a decrease in the amplitude of

the photoinduced absorption band of the semiquinone

anion Q

−

was observed. As noted in the Introduction,

in contrast to purified RC preparations, in chromato-

phores, this stage of protonation of the secondary

acceptor after its one-electron reduction was experi-

mentally observed [12]. This result was attributed to

an increase in the pK

of the secondary quinone Q

by more than 3units due to RC immersion in the lipid

bilayer of the chromatophore membranes.

RC protein relaxation during electron transfer

to quinone acceptors is sensitive to protein-bound

lipids. It has been shown that electrostatic changes

on the protein surface or in the hydrogen bond net-

work surrounding Q

can lead to changes in pK

amino acids near Q

and the pK of the semiquinone

itself [34]. It can be assumed that the absorption

changes we detected reflect the partial disappearance

of the absorption band of the semiquinone anion Q

−

and the appearance of the absorption band of the

electroneutral form Q

H due to the displacement of

protons in the immediate environment or upon direct

binding with a proton in the medium[12,13]. The ob-

served effect of amphiphilic cationic antiseptics in RC

preparations could be associated with their influence

on the pK

value of the secondary quinone, as it oc-

curs in the membrane lipid matrix of chromatophores.

The rate of proton binding in our experiments was

quite low; this process takes 100-200ms. At the same

time, the calculations performed for Q

−

protonation

with rhodoquinone give a value of several microsec-

onds[13], since this process must precede the transfer

of the second electron from Q

, which occurs on a

microsecond time scale. Apparently, the increase in

in our case is not very large, and therefore, when

the measurements were performed with a medium pH

of 7.5, the rate constant of the direct protonation re-

action is close to the deprotonation constant. Never-

theless, the results obtained indicate that cationic an-

tiseptics (mainly miramistin and octenidine) promote

the direct protonation of Q

−

with the formation of a

neutral complex Q

H. It should be noted that in the

structure of the resulting “negative” absorption chang-

es, a certain constant value is present for the entire

spectral range of 400-650 nm, which clearly does not

belong to the semiquinone anion spectrum (Fig. 5b).

Apparently, neutralization of the charge on the sec-

ondary quinone partially reduces the local electric

field, which reduces the magnitude of the electrochro-

mic shifts of carotenoids and BPh, making a signifi-

cant contribution to the absorption changes in this

wavelength range. Indeed, it has been shown that an

increase in the yield of the neutral form of semiqui-

none Q

H correlates with a decrease in the amplitude

of the electrochromic shift of the BPh absorption band

at 760 nm [12].

It should be noted that the analyzed cationic

antiseptics, according to various data, exhibit a de-

tergent-like effect in high concentrations [35, 36].

Moreover, in a number of studies, the ability of these

compounds to disrupt the integrity, i.e. barrier prop-

erties of membranes is considered the basis of their

antimicrobial action[37, 38]. Considering the acceler-

ation of the decline in the photoelectric response of

chromatophores to single light flashes in the presence

of 100 μM octenidine which indicates a marked in-

crease in the passive ionic conductivity of the mem-

brane (Fig. 1), as well as a sharp drop in the ampli-

tude of the carotenoid shift under similar conditions

(Fig.3), the possibility of a significant contribution of

the detergent-like effect to the observed effects of cat-

ionic antiseptics cannot be excluded.

CONCLUSION

The new experimental data presented in this

study complement the general picture of the effects

of cationic antiseptics on biomembranes. The present-

ed results indicate that these agents can affect the

ANTISEPTICS BLOCK Δψ GENERATION 891

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

cyclic electron transfer in bacterial chromatophores,

reducing the generation of the transmembrane pro-

ton gradient on the photosynthetic membrane. Ex-

periments on RC preparations showed the effect of

cationic antiseptics on local proton equilibria respon-

sible for stabilization of electrons on the secondary

quinone acceptor in protein-pigment complexes. The

discovered differences in the action of octenidine and

miramistin on membrane preparations and prepara-

tions of isolated RCs are apparently due to the specific

physico-chemical properties of these antiseptics– their

amphiphilicity, charge characteristics, and general mo-

lecular structure. As a result, octenidine has a great-

er effect on membrane preparations, influencing their

functional activity. Miramistin modifies protein–pig-

ment complexes isolated from membranes more ef-

fectively, interacting with the surface of the complex

and, possibly, partially penetrating the surface struc-

ture of the protein.

The main conclusion from the obtained results

is that cationic antiseptics, which are increasingly

entering the environment, disrupt the functioning of

the quinone acceptor side of the electron transport

chain, reducing the energization of the coupling pho-

tosynthetic membrane necessary for ATP synthesis.

Thus, they can have a negative impact on the global

processes of photosynthetic utilization of light energy.

Abbreviations. Δψ, transmembrane electrical

potential difference; BPh, bacteriopheophytin; BChl,

bacteriochlorophyll; P

870

, special pair of bacteriochlo-

rophyll, the primary donor of RC; Q

and Q

, the

primary and the secondary quinone acceptors, re-

spectively; RC, photosynthetic reaction center; TMPD,

N,N,N′N′-tetramethyl-p-phenylenediamine.

Contributions. E. P. Lukashev, M. D. Mamedov,

P. P. Knox, V. Z. Paschenko– setting the objectives, dis-

cussion of the results of the study, writing the text of

the paper; E. P. Lukashev, M. D. Mamedov, L. A. Vitukh-

novskaya, A. M. Mamedova– conducting experiments,

processing results; L. A. Vitukhnovskaya, A. M. Mame-

dova– preparing the samples for the study.

Funding. The study was carried out within the

framework of the scientific project of the State Assign-

ment of Moscow State University No.121032500058-7.

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.

REFERENCES

1. Strakhovskaya, M. G., Lukashev, E. P., Korvatovskiy,

B. N., Kholina, E. G., Seifullina, N. Kh., Knox, P. P.,

and Paschenko, V. Z. (2021) The effect of some an-

tiseptic drugs on the energy transfer in chromato-

phore photosynthetic membranes of purple non-sul-

fur bacteria Rhodobacter sphaeroides, Photosyn.

Res., 147, 197-209, https://doi.org/10.1007/s11120-

020-00807-x.

2. Knox, P. P., Lukashev, E. P., Korvatovskiy, B. N.,

Strakhovskaya, M. G., Makhneva, Z. K., Bol’shakov,

M. A., and Paschenko, V. Z. (2022) Disproportionate

effect of cationic antiseptics on the quantum yield

and fluorescence lifetime of bacteriochlorophyll mol-

ecules in the LH1-RC complex of R.rubrum chromato-

phores, Photosynth. Res., 153, 103-112, https://doi.org/

10.1007/s11120-022-00909-8.

3. Paschenko, V. Z., Lukashev, E. P., Mamedov, M. D.,

Korvatovskiy, B. N., and Knox, P. P. (2023) Influence

of the antiseptic octenidine on spectral character-

istics and energy migration processes in photosys-

temII core complexes, Photosynth. Res., 155, 93-105,

https://doi.org/10.1007/s11120-022-00972-1.

4. Paschenko, V. Z., Lukashev, E. P., Mamedov, M. D.,

Gvozdev, D.A., and Knox, P.P. (2024) Effect of cationic

antiseptics on fluorescent characteristics and electron

transfer in cyanobacterial photosystem I complexes,

Photosynth. Res., 159, 241-251, https://doi.org/10.1007/

s11120-024-01082-w.

5. Šlouf,V., Chábera,P., Olsen, J.D., Martin, E.C., Qiac,P.,

Huntec, C. N., and Polívka, T. (2012) Photoprotection

in a purple phototrophic bacterium mediated by ox-

ygen-dependent alteration of carotenoid excited-state

properties, Proc. Natl. Acad. Sci. USA, 109, 8570,

https://doi.org/10.1073/pnas.1201413109.

6. Kirmaier, C., Holten, D., and Parson, W. W. (1985)

Temperature and detection-wavelength dependence

of the picosecond electron transfer kinetics measured

in Rhodopseudomonas sphaeroides reaction centers–

resolution of new spectral and kinetic components in

the primary charge-separation process, Biochim. Bio-

phys. Acta, 810, 33-48, https://doi.org/10.1016/0005-

2728(85)90204-X.

7. Holzapfel, W., Finkele, U., Kaiser, W., Oesterhelt, D.,

Scheer, H., Stilz, H. U., and Zinth, W. (1989) Obser-

vation of a bacteriochlorophyll anion radical during

the primary charge separation in a reaction center,

Chem. Phys. Lett., 160, 1, https://doi.org/10.1016/0009-

2614(89)87543-8.

8. Holzwarth, A. R., and Muller, M. G. (1996) Energet-

ics and kinetics of radical pairs in reaction centers

from Rhodobacter sphaeroides. A femtosecond tran-

sient absorption study, Biochemistry, 35, 11820-11831,

https://doi.org/10.1021/bi9607012.

9. Camara-Artigas, A., Brune, D., and Allen, J. P. (2002)

Interactions between lipids and bacterial reaction

centers determined by protein crystallography,

Proc. Natl. Acad. Sci. USA, 99, 11055-11060, https://

doi.org/10.1073/pnas.162368399.

LUKASHEV et al.892

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

10. Milano, F., Dorogi, M., Szebenyi, K., Nagy, L.,

Maroti,P., Varo,G., Giotta,L., Agostiano,A., and Trot-

ta, M. (2007) Enthalpy/entropy driven activation of

the first interquinone electron transfer in bacterial

photosynthetic reaction centers embedded in vesi-

cles of physiologically important phospholipids, Bio-

electrochemistry, 70, 18-22, https://doi.org/10.1016/

j.bioelechem.2006.03.024.

11. Agostiano, A., Milano, F., and Trotta,M. (2005) Trap-

ping of a long-living charge separated state of pho-

tosynthetic reaction centers in proteoliposomes of

negatively charged phospholipids, Photosynth. Res.,

83, 53-61, https://doi.org/10.1007/s11120-004-3197-6.

12. Lavergne,J., Matthews, C., and Ginet, N. (1999) Elec-

tron and proton transfer on the acceptor side of the

reaction center in chromatophores of Rhodobacter

capsulatus: evidence for direct protonation of the

semiquinone state of Q

, Biochemistry, 38, 4542-4552,

https://doi.org/10.1021/bi9827621.

13. Graige, M.S., Paddock, M.I., Feher,G., and Okamura,

M. Y. (1999) Observation of the protonated semiqui-

none intermediate in isolated reaction centers from

Rhodobacter sphaeroides: implications for the mech-

anism of electron and proton transfer in proteins,

Biochemistry, 38, 11465-11473, https://doi.org/10.1021/

bi990708u.

14. Woronowicz,K., Sha,D., Frese, R.N., and Niederman,

R.A. (2011) The accumulation of the light-harvesting

2 complex during remodeling of the Rhodobacter

sphaeroides intracytoplasmic membrane results in a

slowing of the electron transfer turnover rate of pho-

tochemical reaction centers, Biochemistry, 50, 4819-

4829, https://doi.org/10.1021/bi101667e.

15. Clayton, R. K. (1966) Spectroscopic analysis of bacte-

riochlorophylls invitro and invivo, Photochem. Pho-

tobiol., 5, 669-677, https://doi.org/10.1111/j.1751-1097.

1966.tb05813.x.

16. Zakharova, N.I., and Churbanova, I.Yu. (2000) Meth-

ods of isolation of reaction center preparations from

photosynthetic purple bacteria, Biochemistry (Mos-

cow), 65, 149-181.

17. Drachev, L.A., Kaminskaya, O.P., Konstantinov, A.A.,

Mamedov, M.D., Samuilov, V.D., Semenov, A.Yu., and

Skulachev, V.P. (1986) Effects of electron donors and

acceptors on the kinetics of the photoelectric respons-

es in Rhodospirillum rubrum and Rhodopseudomas

sphaeroides chromatophores, Biochim. Biophys. Acta,

850, 1-9, https://doi.org/10.1016/0005-2728(86)90002-2.

18. Drachev, L. A., Kaurov, B. S., Mamedov, M. D.,

Mulkidjanian, A. Ya., Semenov, A. Yu., Shinkarev,

V. P., Skulachev, V. P., and Verkhovsky, M. I. (1989)

Flash-induced electrogenic events in the photosyn-

thetic reaction center and bc

complexes of Rhodo-

bacter sphaeroides chromatophores, Biochim. Bio-

phys. Acta, 973, 189-197, https://doi.org/10.1016/

S0005-2728(89)80421-9.

19. Jackson, J. B., and Crofts, A. R. (1971) The kinetics

of light induced carotenoid changes in Rhodopseu-

domonas sphaeroides and their relation to elec-

trical field generation across the chromatophore

membrane, Eur. J. Biochem., 18, 120-130, https://

doi.org/10.1111/j.1432-1033.1971.tb01222.x.

20. Drachev, L.A., Kaulen, A.D., Semenov, A.Y., Severina,

I.I., and Skulachev, V.P. (1979) Lipid-impregnated fil-

ters as a tool for studying the electric current-gen-

erating proteins, Anal. Biochem., 96, 250-262, https://

doi.org/10.1016/0003-2697(79)90580-3.

21. Skulachev, V. P. (1982) A single turnover study of

photoelectric current-generating proteins, Methods

Enzymol., 88, 35-45, https://doi.org/10.1016/0076-

6879(82)88010-5.

22. Jackson, J. B., and Crofts, A. R. (1969) Bromothy-

mol blue and bromocresol purple as indicators

of pH changes in chromatophores of Rhodospiril-

lum rubrum, Eur. J. Biochem., 10, 226-237, https://

doi.org/10.1111/j.1432-1033.1969.tb00678.x.

23. Witt, H.T. (1979) Energy conversion in the functional

membrane of photosynthesis. Analysis by light pulse

and electric pulse methods. The central role of the

electric field, Biochim. Biophys. Acta, 505, 355-427,

https://doi.org/10.1016/0304-4173(79)90008-9.

24. Malferrari, M., Malferrari, D., Francia, F., Galletti, P.,

Tagliavini, E., and Venturoli, G. (2015) Ionic liq-

uids effects on the permeability of photosynthetic

membranes probed by the electrochromic shift of

endogenous carotenoids, Biochim. Biophys. Acta,

1848, 2898-2909, https://doi.org/10.1016/j.bbamem.

2015.09.006.

25. Saphon,S., Jackson, J. B., and Witt, H. T. (1975) Elec-

trical potential changes, H

translocation and phos-

phorylation induced by short flash excitation in Rho-

dopseudomonas sphaeroides chromatophores, Bio-

chim. Biophys. Acta, 408, 67-82, https://doi.org/10.1016/

0005-2728(75)90159-0.

26. Gibasiewicz, K., and Pajzderska, M. (2008) Primary

radical pair P

–

lifetime in Rhodobacter sphaeroi-

des with blocked electron transfer to Q

. Effect of

o-phenanthroline, J. Phys. Chem. B, 112, 1858-1865,

https://doi.org/10.1021/jp075184j.

27. Okamura, M. Y., Feher, G., and Nelson, N. (1982) Re-

action centers, in Photosynthesis Energy conversion

by plants and bacteria, (Govindjee, ed.) Vol. 1, Acad.

Press, N-Y – London – Paris, pp.197-254.

28. Li,J., Gilroy,D., Tiede, D.M., and Gunner, M.R. (1998)

Kinetic phases in the electron transfer from P

–

to P

–

and the associated processes in Rhodo-

bacter sphaeroides R-26 reaction centers, Biochemis-

try, 37, 2818-2829, https://doi.org/10.1021/bi971699x.

29. Pingale, S.S., Ware, A.P., and Gadre, S.R. (2018) Un-

veiling electrostatic portraits of quinones in reduction

and protonation states, J.Chem. Sci., 130, 50, https://

doi.org/10.1007/s12039-018-1450-3.

ANTISEPTICS BLOCK Δψ GENERATION 893

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

30. Takahashi, E., and Wraight, C. A. (1996) Potentia-

tion of proton transfer function by electrostatic in-

teractions in photosynthetic reaction centers from

Rhodobacter sphaeroides: first results from site-di-

rected mutation of the H subunit, Proc. Natl. Acad.

Sci. USA, 93, 2640-2645, https://doi.org/10.1073/pnas.

93.7.2640.

31. Gunner, M. R., Madeo, J., and Zhu, Z. (2008) Modifi-

cation of quinone electrochemistry by the proteins

in the biological electron transfer chains: exam-

ples from photosynthetic reaction centers, J. Bioen-

erg. Biomembr., 40, 509-519, https://doi.org/10.1007/

s10863-008-9179-1.

32. Okamura, M. Y., Paddock, M. L., Graige, M. S., and

Feher, G. (2000) Proton and electron transfer in

bacterial reaction centers, Biochim. Biophys. Acta,

1458, 148-163, https://doi.org/10.1016/S0005-2728(00)

00065-7.

33. Scheiner, S., and Hillenbrand, E. A. (1985) Modifica-

tion of pK values caused by change in H-bond ge-

ometry, Proc. Natl. Acad. Sci. USA, 82, 2741-2745,

https://doi.org/10.1073/pnas.82.9.274.

34. Nagy, L., Milano, F., Dorogi, M., Agostiano, A.,

Laczkó, G., Szebényi, K., Váró, G., Trotta, M., and

Maróti,P. (2004) Protein/lipid interaction in the bacte-

rial photosynthetic reaction center: phosphatidylcho-

line and phosphatidylglycerol modify the free energy

levels of the quinones, Biochemistry, 43, 12913-12923,

https://doi.org/10.1021/bi0489356.

35. Chernysheva, M. G., Shnitko, A. V., Skrabkova, H. S.,

and Badun, G. A. (2021) Peculiarities of alkylami-

dopropyldimethylbenzylammonium (miramistin)

in the relationship to lysozyme in comparison with

quaternary ammonium surfactants: coadsorption

at the interfaces, enzymatic activity and molecular

docking, Colloids Surf.A, 629, 127503, https://doi.org/

10.1016/j.colsurfa.2021.127503.

36. Rzycki, M., Drabik, D., Szostak-Paluch, K., Hanus-

Lorenz, B., and Kraszewski, S. (2021) Unraveling the

mechanism of octenidine and chlorhexidine on mem-

branes: does electrostatics matter? Biophys. J., 120,

3392-3408, https://doi.org/10.1016/j.bpj.2021.06.027.

37. Malanovic, N., Ön, A., Pabst, G., Zellner, A., and

Lohner, K. (2020) Octenidine: novel insights into the

detailed killing mechanism of Gram-negative bac-

teria at a cellular and molecular level, Int. J. Anti-

microb. Agents, 56, 10646, https://doi.org/10.1016/

j.ijantimicag.2020.106146.

38. Osmanov, A., Farooq, Z., Richardson, M. D., and

Denning, D. W. (2020) The antiseptic miramistin: a

review of its comparative in vitro and clinical ac-

tivity, FEMS Microbiol. Rev., 44, 399-417, https://

doi.org/10.1093/femsre/fuaa012.

Publisher’s Note. Pleiades Publishing remains

neutral with regard to jurisdictional claims in published

maps and institutional affiliations. AI tools may have

been used in the translation or editing of this article.