ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1544-1554 © The Author(s) 2023. This article is an open access publication.
Russian Text © The Author(s), 2023, published in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1867-1879.
1544
REVIEW
Features of the Mechanism of Proton Transport in ESR,
Retinal Protein from Exiguobacterium sibiricum
L a d a E . P e t r o v s k a y a
1,a
*, Sergei A. Siletsky
2
, Mahir D. Mamedov
2
, Eugene P. Lukashev
3
,
Sergei P. Balashov
4
, Dmitry A. Dolgikh
1,3
, and Mikhail P. Kirpichnikov
1,3
1
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia
2
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
3
Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia
4
Department of Physiology and Biophysics, University of California, 92697 Irvine CA
a
e-mail: lpetr65@yahoo.com
Received June 6, 2023
Revised July 11, 2023
Accepted July 11, 2023
Abstract— Retinal-containing light-sensitive proteins– rhodopsins– are found in many microorganisms. Interest in them
is largely explained by their role in light energy storage and photoregulation in microorganisms, as well as the prospects for
their use in optogenetics to control neuronal activity, including treatment of various diseases. One of the representatives
of microbial rhodopsins is ESR, the retinal protein of Exiguobacterium sibiricum. What distinguishes ESR from homol-
ogous proteins is the presence of a lysine residue (Lys96) as a proton donor for the Schiff base. This feature, along with
the hydrogen bond of the proton acceptor Asp85 with the His57 residue, determines functional characteristics of ESR
as a proton pump. This review examines the results of ESR studies conducted using various methods, including direct elec-
trometry. Comparison of the obtained data with the results of structural studies and with other retinal proteins allows us
to draw conclusions about the mechanisms of transport of hydrogen ions in ESR and similar retinal proteins.
DOI: 10.1134/S0006297923100103
Keywords: retinal protein, proteorhodopsin, Schiff base, proton acceptor, proton donor, photocycle, direct electrometric
method
Abbreviations: BR,bacteriorhodopsin from Halobacterium sa-
linarum; ESR,retinal protein from Exiguobacterium sibiricum;
PR,proteorhodopsin; PRG,proton-releasing group.
* To whom correspondence should be addressed.
INTRODUCTION
Microbial rhodopsins belong to the family of reti-
nal-containing proteins that perform light-dependent ion
transport, sensory and other functions [1-3]. The mole-
cules of these proteins have similar structure, including
seven or eight (in the case of enzyme rhodopsins) trans-
membrane alpha-helical segments and a retinal covalent-
ly linked through a Schiff base to a lysine residue[3, 4].
Absorption of a light quantum initiates the isomerization
of the retinal from all-trans to the 13-cis configuration.
Relaxation of the retinal to its original state is accom-
panied by a number of conformational transformations
in the protein molecule associated with proton transport
(in rhodopsins, which perform the function of a pro-
ton pump) [5, 6]. The details of this process have been
most extensively studied for the bacteriorhodopsin from
Halobacterium salinarum(BR), the first member of the
microbial rhodopsin family, which was discovered over
50years ago [7-9]. It has been established that during the
photocycle, the Schiff base formed by retinal and a lysine
residue is deprotonated and a proton is transferred to the
acceptor residue Asp85. At the same time, a proton is re-
leased on the extracellular surface of the protein with the
participation of residues Glu194 and Glu204 (the so-
called PRG– proton-releasing group [10]). The Schiff
base is then reprotonated by the donor residue Asp96,
which then receives a proton from the cytoplasm. At the
end of the photocycle, the proton is transferred from the
acceptor to the residues of the PRG[7, 10]. These mo-
lecular events correspond to the emergence and decay
of spectrally identified intermediate states (intermedi-
ates) of the photocycle BR
→
K
→
L
↔
M1
↔
M2
↔
N1
↔
hν
ERS, RETINAL PROTEIN FROM Exiguobacterium sibiricum 1545
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 1. Comparison of structure of BR(a) and ESR(b). Regions near the Schiff base are shown. The figures were prepared in PyMol using pdb
structures 1C3W and 4HYJ, respectively. Ret,all-trans retinal; CP,cytoplasmic surface of the protein; EC,extracellular protein surface.
↔N2↔O→BR[7, 10]. Data from the functional studies
of BR are in good agreement with its structural changes
in various states [11-13].
Subsequently, in the course of various genomic and
metagenomic studies, many new proteins were discov-
ered that were homologous to BR and performed sim-
ilar functions [3, 14, 15]. In particular, proteorhodop-
sins(PR) were found in many members of Proteobacteria.
They play an important role in the survival of microor-
ganisms under unfavorable conditions. Studies of PRs
have revealed that, like BRs, they have acceptor and
donor residues (aspartate and glutamate, respectively),
but there is no PRG in their molecules [16, 17]. An im-
portant feature of PR is the hydrogen bond between the
acceptor residue (aspartate) and the side group of the
histidine residue; it regulates the degree of protonation
and the functional state of the acceptor (depending on
pH) and is involved in proton release [18,19].
ESR, the retinal protein from soil bacterium Ex-
iguobacterium sibiricum, also belongs to the proteorho-
dopsins, but exhibits a significant structural difference
from typical representatives of the family. The amino
acid sequence of ESR has Lys96 residue in the position
corresponding to the carboxyl residues with the function
of proton donor for the Schiff base in BR and PR mol-
ecules (Asp96 and Glu108, respectively) [20] (Fig. 1).
This feature, along with others, sparked interest in re-
search into the structure and mechanism of ESR func-
tioning, which we have been conducting for more than
10 years. In 2015, we published a review of these works
[21], but since then a number of important new results
have been obtained, largely thanks to the use of the di-
rect electrometry method developed by L. A. Drachev
and his colleagues [22,23].
We employed the version of this method which is
based on the use of proteoliposomes containing the pro-
tein under study and the macroscopic flat phospholipid
membrane reinforced with a collodion film for greater
stability. Prior to the measurement, proteoliposomes are
adsorbed on the surface of the macroscopic membrane
separating the compartments of the measuring cell [23].
In response to the flash, the transmembrane transfer of
protons, as well as the movement of charged groups of
the protein perpendicular to the membrane plane, gen-
erate the electrical potential difference at the proteoli-
posome membrane (ΔΨp) and, proportionally, at the
macroscopic membrane(ΔΨm) which is recorded using
electrometric technology with high time resolution.
BR was one of the first proteins for which di-
rect electrometric measurements were applied [22, 24].
PETROVSKAYA et al.1546
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Subsequently, the method was widely used in studies of
various retinal proteins [25-27], components of photo-
systemsI and II [28-30], cytochrome oxidase [31-35],
and other objects. This review discusses our recent re-
sults of the studies of ESR and its mutant variants, in-
cluding those obtained using direct electrometry, and
compares them with those previously published for BR
and other retinal proteins.
ESR PHOTOCYCLE: REACTIONS COUPLING
WITH PROTON UPTAKE AND RELEASE
The study of light-induced absorption changes
of ESR using flash photolysis revealed the presence of
main intermediates also characteristic of BR [36] and
PR [37] photocycle. Following the decay of interme-
diates K and L, an increase in absorption is observed
at 410 nm on microsecond time scale, which corre-
sponds to the formation of the M-state (deprotonated
Schiff base). It is interesting to note that this interme-
diate is detected only at pH > 8 for ESR in micelles of
the detergent DDM (n-dodecyl-β-D-maltopyranoside),
while for the protein in micelles of lipid-like detergent
LPG (1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-1′-
rac-glycerol) or in proteoliposomes, the pK
a
of its for-
mation is ~6.5[38, 39]. Reprotonation of the Schiff base
is accompanied by a decrease in absorption at 410 nm
(reflecting the decay of the M state) and the appearance
of intermediates N1 and N2, which are characterized by
the absorption increase at 510 and 550 nm. As a result of
retinal reisomerization, intermediate O is formed, which
absorbs at 590 nm and decays to the initial ESR state.
The N-intermediate is present in the ESR photocycle
up to its completion [40], thus, the photocycle diagram
includes the following transitions: ESR
→
K,L
→
M1
↔
↔
M2↔N1↔N2/O→ESR[39].
Illumination of Escherichia coli cells suspension ex-
pressing ESR or ESR-containing proteoliposomes re-
sulted in acidification of the suspension, confirming
transmembrane proton transfer by this protein [20, 38].
Addition of protonophore CCCP to the cells eliminates
this effect, which confirms the proton transport function
of ESR [38]. It was also found that, in contrast to BR,
ESR releases protons at the end of the photocycle, which
correlates with the absence of residues homologous to
PRG in its molecule [20]. Measurements carried out in
the presence of the pH-sensitive dye pyranine showed
the temporal relationship of proton release with the de-
cay of long-wavelength photocycle intermediates and the
return to the initial state [41]. Kinetics comparison of the
proton uptake and intermediate M decay in wild-type
ESR demonstrated that, unlike in BR, proton uptake
in this protein precedes the reprotonation of the Schiff
base, which is especially noticeable when measurements
are performed in D
2
O [41].
hν
ELECTROGENIC STAGES
OF THE ESR PHOTOCYCLE
The time-resolved direct electrometry makes it pos-
sible to isolate and study individual electrogenic (i.e.,
directed perpendicularly to the membrane plane) stag-
es of charge transfer within the protein in response to
a single flash; simultaneous analysis of the absorption
changes kinetics allows us to relate them to the stages
of the retinal protein photocycle. Complimentary to
thedye-involving methods, (i) such measurements make
it possible to obtain directly the information about the
proton movement inside the protein, in contrast to just
an information on the disappearance or appearance of a
proton on its surface, and (ii) they are not limited to pH
region close to the pK
a
of the dye, which generally opens
up the possibilities for reconstructing the complete pic-
ture of the functioning of the proton pump at different
pH values.
For ESR-containing proteoliposomes measure-
ments were carried out at pH 5.1-9.5 [39]. The total
amplitude of the photoelectric response at neutral pH
values reached 50mV, which is comparable to the max-
imum response amplitude for BR [22]. However, the
directions of the response for these proteins were oppo-
site, due to the different orientations of their molecules
in proteoliposomes (ESR is incorporated in the same
orientation as in cells, with the N-terminus facing out
[20, 38], whereas BR is oriented in the opposite direc-
tion [42]). Accordingly, ESR transports protons from
proteoliposomes to the outside, while transport of pro-
tons into proteoliposomes is observed for BR due to the
reverse orientation as compared to that in cells.
It was previously demonstrated that the photoelec-
tric response of BR involves three main electrogenic
processes, with the fastest (<1 μs) having a negative sign
and corresponding to the stages BR → K → L(photoin-
duced isomerization of retinal and subsequent relaxation
of adjacent residues). The second stage (30-50 μs) re-
flects the proton movement from the Schiff base to the
Asp85 acceptor and the simultaneous proton release
into the external medium by the proton-releasing group.
Theamplitude of the latter phase (5-20 ms) accounts for
up to 80% of the total response and corresponds to pro-
ton transfer from the internal donor Asp96 to the Schiff
base and subsequent reprotonation of the donor, as well
as proton transfer from Asp85 to PRG[43](Fig.2a).
The kinetics of membrane potential formation for
ESR differs significantly from that described above
for BR. In particular, the photoelectric response of
ESR does not exhibit the fast negative phase character-
istic of BR (Fig. 2b). A detailed analysis of the kinetic
curves at pH 5.1, however, made it possible to detect
its presence, “hidden” by positive signals from electro-
genic phases coinciding with it in time. The relative to-
tal amplitudes of the microsecond electrogenic phases
ERS, RETINAL PROTEIN FROM Exiguobacterium sibiricum 1547
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 2. Kinetics of light-induced formation of membrane electric potential(ΔΨ) by proteoliposomes containing BR(a) and ESR(b), at pH7.5.
The arrows indicate the moment of laser flash. The relative amplitudes of the microsecond and millisecond electrogenic phases are shown,
indicating the corresponding stages of proton transfer. SB,Schiff base; PRG,proton releasing group; EC,extracellular surface of the protein;
CP,cytoplasmic surface of the protein; Group X is an unidentified residue/group of residues that release protons at the extracellular surface
ofESR. For convenience, the photoelectric responses of both proteins are displayed in the same direction.
of ESR (3, 24, and 100 μs at pH 7.5) were approximate-
ly three times smaller than those of BR, which may be
due to the lack of early proton release in ESR molecule.
The decay of intermediate M includes two electrogenic
phases, 0.6 and 3.4ms, with a total contribution of 75%,
presumably reflecting the transition M ↔ N1 ↔ N2/O.
The last electrogenic phase of ESR (~18.4 ms) is asso-
ciated with the return to the initial state (N2/O → ESR)
and reflects the deprotonation of Asp85 acceptor and
proton release by the protein molecule into the external
aqueous medium[39].
It should be noted that the photoelectric response
of ESR-containing proteoliposomes at pH 8.4 demon-
strates a 10-fold (as compared to pH 6.5) slowdown of
the electrogenic stages corresponding to the decay of the
intermediate M, resulting from redistribution of the rel-
ative contributions of kinetic components in favor of the
slower ones. In this case, a separate electrogenic phase
with τ ≈ 0.25 ms emerges, corresponding to the pro-
tonation of the initially neutral Lys96 [39]. These data
suggest that in ESR, Lys96 and/or the water molecule
interacting with it acquires a proton after formation of the
state M and transfers it to the Schiff base shortly there-
after. This confirmed the previously made conclusion
that the Lys96 residue is not protonated in the initial
state at neutral pH and is protonated in the M state with
pK
a
≈ 8.5 [41]. In ESR molecule, Lys96 is surrounded
predominantly by hydrophobic residues[44], which pre-
sumably contribute to the a reduced pK
a
of Lys96 in the
initial state. A similar large shift in pK
a
(but in opposite
direction) is observed for Asp96 residue in BR, which
is neutral, has a very high pK
a
in the initial state (~11.4)
and, accordingly, is protonated due to the highly hydro-
phobic environment [45].
A decrease of pH to 5.1 is accompanied by an almost
complete suppression of ESR photoelectric response and
the appearance of noticeable negative phases, presum-
ably as a result of protonation of Asp85 acceptor and/or
the associated His57 residue in the initial state.
Lys96 – EFFICIENT PROTON DONOR
FOR THE SCHIFF BASE IN ESR
To determine the functional role of the Lys96 res-
idue, we used a classical approach involving generation
and study of mutant ESR variants [41, 46]. At first, the
K96A mutant was constructed; characteristic features of
its photocycle included a significant slowdown (more
than 100-fold) of the intermediate M decay (Fig.3a) and
strong dependence of the rate of this process on pH [41].
E. coli cells expressing this mutant showed a significant
reduction in proton transport as compared to cells con-
taining the wild-type protein. Thus, it was shown that
the presence of the Lys96 residue accelerates the repro-
tonation of the Schiff base in the ESR molecule.
Measurements of the photoelectric response of
K96A-containing proteoliposomes demonstrate that this
mutation is accompanied by the almost complete disap-
pearance of the millisecond electrogenic phase, which is
associated with the reprotonation of the Schiff base (de-
cay of intermediate M) [47] (Fig. 3b). The slowdown of
this process, characteristic of the K96A mutant, in itself
should not have led to such an effect.
Approximation of the experimental kinetics from
K96A with a sum of kinetic components revealed the
presence of several electrogenic phases with a negative
sign in the millisecond part of the kinetics. The nega-
tive amplitude of the millisecond phases, corresponding
to the rapid decay of the intermediate M in the mutant
protein, indicates that the reprotonation of the Schiff
base occurs from an unusual direction– from the extra-
PETROVSKAYA et al.1548
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 3. Properties of mutant ESR variants with substitutions of the Lys96 residue. a)Kinetics of light-induced absorption changes at 410nm of wild
type ESR(WT) and its mutant variants in 0.06% lipid-like detergent LPG, 100 mM NaCl, pH 7.5; b)kinetics of the formation of transmembrane
potential difference(ΔΨ) at proteoliposomes containing wild type ESR and its mutant variants at pH 7.5. Adapted from [46], with modifications.
cellular surface of the protein. This is also substantiated
by the negligible amplitude of the positive component
of the electrogenic response, which apparently includes
both the diffusion of the proton from the cytoplasmic
surface of the protein and its movement in the oppo-
site direction. At low pH, the first of these mechanisms
dominates, while at pH 8.5 the second one is prevailing.
Theaddition of azide to this mutant led to an accelerated
delivery of protons from the cytoplasmic side of the pro-
tein and to an increase in the amplitude of the positive
components of the electrogenic response [47].
Thus, the experiments demonstrated that the re-
duced efficiency of proton transport in K96A mutant
can be explained not only by the slowdown in the photo-
cycle, but also by the decrease in forward transport and
the increase of contribution of back (reverse) reactions,
presumably from His57-Asp85 acceptor site to the Schiff
base. It should be noted that mutations of the donor res-
idue in BR (D96N and D96A) are accompanied by the
slowdown in the photoelectric response in the millisec-
ond region, but not by its disappearance, which results
from the different configurations of the proton acceptor
sites in BR and ESR (Fig. 1) and indicates differenc-
es in the mechanism of proton transport in these pro-
teins[48, 49]. Additional arguments in favor of the role
of His57 residue in the back reactions were obtained
with the mutation study of this residue (see below).
CARBOXYL AMINO ACID
RESIDUES IN PLACE OF Lys96 IN ESR
“Can carboxyl residues act as proton donors in ESR
molecule?” – was the question we asked in the previ-
ous review [21]. Now there is a definite answer: “In gen-
eral, yes, but there are differences as compared to how
they operate in BR.” We examined the kinetics of the
photocycle, proton transfer, and electrogenicity of ESR
mutants K96E and K96D/A47T [46]. In contrast to the
K96A mutant, the rates of proton uptake and proton
transfer to the Schiff base in these proteins are compara-
ble to those in the wild type protein. In K96E, interme-
diate M decays somewhat slower than in the wild type–
with a larger contribution of the slow component, and
in K96D/A47T– even faster than in the wild type (the
time constant of the fast component is ~0.7 ms versus
1 ms in the wild type) (Fig. 3a). Reprotonation of the
Schiff base and proton uptake from the medium in these
mutants occur almost simultaneously with the M to N
transition (as in wild-type ESR at neutral pH), whereas
in BR these two stages are well separated and coincide in
time with the M to N and N to O transitions, respective-
ly [48, 50]. This reflects the different mechanism of donor
involvement in the reprotonation of the Schiff base in BR
and ESR. It is interesting to note that in K96D/A47T
and K96E mutants, the formation of the M state is ac-
companied by partial proton release, presumably at the
cytoplasmic surface of the protein (i.e., by movement in
the opposite direction). The reasons for this phenome-
non will be discussed below.
The photoelectric responses of K96D/A47T and
K96E mutants include electrogenic millisecond phases
associated with Schiff base reprotonation that are simi-
lar to those of the wild type but are absent in the K96A
mutant. The main different feature of the electrogenic
kinetic of the K96E mutant is the much slower main
millisecond component of ΔΨ generation (Fig. 3b).
In proteoliposomes containing K96D/A47T, generation
ERS, RETINAL PROTEIN FROM Exiguobacterium sibiricum 1549
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
of ΔΨ occurs primarily during the decay of the inter-
mediate M, while in the K96E mutant it occurs during
the decay of N. This provides independent evidence that
carboxyl residues functioning as proton donors in the
ESR with the efficiency comparable to that of lysine, in
the case of the double mutant, and with a slightly lower
efficiency in K96E.
It should be noted that Japanese scientists published
a study of mutant PR variants, in which the donor aspar-
tate residue was replaced by lysine and glutamine (E108K
and E108Q), as well as ESR mutants with substitutions
K96D, K96Q, and K96E [51]. The K96D mutant ex-
hibited a fast decay rate of intermediate M as compared
to the wild type, while the K96E mutation exhibited a
slower M decay rate. It has also been shown that Lys can
replace native Glu in the donor region ofPR[51].
The absence of a significant delay between proton
uptake and reprotonation of the Schiff base in K96D/
A47T and K96E mutants, as well as the early release of
the proton from the opposite side of the membrane, in-
dicate that in ESR mutants the carboxyl residues at po-
sition 96 are protonated (uncharged) in the initial state,
similarly to BR. Presumably, after the formation of the
intermediate product M, they are in a more hydrophilic
environment as compared to the initial state, resulting in
a decrease in their pK
a
to 6.5-8.5. This results in partial
deprotonation of the donor residues and release of a pro-
ton into the medium, followed by reprotonation of the
Schiff base and almost simultaneous proton uptake [46].
Therefore, donor residues in the ESR are able to rapidly
switch from equilibrium with the medium to equilibri-
um with the Schiff base during the M to N transition,
which is consistent with data from structural studies.
The side chain of Lys96 has a certain mobility, and the
cavity around this residue is very close to the surface of
the protein and is separated from the aqueous medium
only by the polar side chain of Thr43, which distinguish-
es ESR from the more covered position of proton donor
groups in BR and xanthorhodopsin(XR) [44]. A similar
conclusion was reached by Sasaki etal. [51], who pro-
posed that protein conformational changes associated
with Schiff base reprotonation are smaller in ESR than
in BR mutants and may involve side chain movements
of the donor residue connecting it to the environment or
tothe Schiff base.
His57 REPLACEMENT ALLOWS PROTON
TRANSPORT BY ESR OVER A WIDE pH RANGE
The presence of the histidine residue with the hy-
drogen bond to the aspartate acceptor residue is a dis-
tinctive feature of proteins belonging to proteorhodop-
sin family, including ESR, as well as XR [38, 52, 53].
The interaction between the histidine side chain and
residues of the adjacent subunit plays an important role
in the oligomerization of PR and Gloeobacter rhodop-
sin (GR) [54-56]. The close interaction between His57
and Asp85 in the ESR molecule was revealed both in
functional studies of mutants of this residue [38] and
by determining the spatial structure of the protein[44]
(Fig. 1). It has been established that the His57 substitu-
tion has a radical effect on various properties of ESR, in-
cluding the pH dependence of the absorption maximum
and the pK
a
of the formation of the intermediate M [38].
Unlike wild type ESR, which absorption maximum
weakly depends on pH in the range of 3-8, the H57M
mutant is characterized by a significant (47 nm) blue
shift of the absorption maximum as pH increases from 5
to 8 from deprotonation of Asp85. At pH 5, the absorp-
tion maximum of H57M is at 565nm, which corresponds
to the absorption maximum of the D85N mutant. Thus,
interaction with His57 determines the degree of proton-
ation of the Asp85 residue, i.e., its ability to function as
a proton acceptor from a Schiff base. In order to clarify
the functional role of this interaction, we examined the
properties of ESR with the H57N substitution and the
double mutant H57N/K96A using direct electrometry.
In the studies of the photocycle of proteoliposomes
containing H57N mutant, it was established that this
substitution leads to an acceleration of the formation
of the M intermediate and elimination of slow spec-
tral changes in this process, previously found in the
photocycle of ESR and the K96A mutant (Fig. 4a).
A corresponding component is also absent in the pho-
toelectric response, which indicates a connection be-
tween the slow phase of M generation in ESR and the
His57 residue (a possible explanation for this connec-
tion is given in the next section). As compared to the
wild type ESR, the decay of the intermediate M in the
H57N mutant also occurs faster and consists of a single
phase, since it is not accompanied by the accumulation
of the N1 state.
Interestingly, in the double mutant H57N/K96A,
the decay of the M intermediate occurs 1000 times slow-
er than in the wild type and in the H57N mutant, and
10times slower than in K96A(Fig.4a). This phenom-
enon can be explained by the removal of the backward
proton movement to the Schiff base as a result of the
H57N substitution. Presumably, the replacement of the
His57 residue in the environment of the proton acceptor
Asp85 leads to changes in the charge or other properties
of the environment, resulting in the creation of a kinetic
barrier to such movement.
Previously, investigation of the pH dependence of
proton transport in E. coli cells with ESR expressed re-
vealed that the efficiency of the wild-type protein as a
proton pump is significantly reduced at pH below 5[38].
It turned out that under the same conditions the H57N
and H57N/K96A mutants demonstrate high transport
efficiency [57]. In accordance with these data, mu-
tants with the H57N substitution retain the amplitude
PETROVSKAYA et al.1550
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 4. Properties of mutant ESR variants with substitutions of the His57 residue. a)Kinetics of light-induced absorption changes at 410nm by
proteoliposomes containing wild-type(WT) ESR and its mutant variants at pH7.5; b)kinetics of the transmembrane potential difference(ΔΨ)
formation by proteoliposomes containing wild-type ESR and its mutant variants at pH4.5. Adapted from[57], with modifications.
and direction of the photoelectric response at pH 4.5,
in contrast to the wild type and the mutant with a sin-
gle K96A substitution(Fig.4b). Thus, the interaction of
His57 and Asp85 limits proton transport at low pH for
wild-type ESR. In the H57N mutant at pH4.5, the Lys96
residue is presumably protonated in the initial state. Due
to this, reprotonation of the Schiff base during the decay
of the M intermediate occurs at a high rate, after which
the donor receives a proton from the environment, sim-
ilarly to what happens at neutral pH in BR, where the
donor Asp96 is initially protonated. The data obtained
indicate that Lys96 can effectively function as a donor at
low pH values.
As mentioned above, based on the pH dependence
of the absorption maximum it was previously conclud-
ed that Asp85, the proton acceptor from the Schiff base
in the H57N mutant, should be in the protonated state
in the detergent DDM at pH < 6 [38]. However, subse-
quent experiments demonstrated the efficient function-
ing of this mutant as a proton pump at pH below 5 in
liposome membranes [57]. It can be assumed that thepK
a
of Asp85 in H57N decreases in more hydrophobic envi-
ronment of liposomes and after isomerization of retinal to
the 13-cis configuration, opening the possibility for pro-
ton transfer to the Schiff base during the decay ofinter-
mediateL to N without significant accumulation of M.
FEATURES OF ESR
FUNCTIONING AS A PROTON PUMP
Based on the obtained results, we proposed mech-
anisms for the functioning of ESR and its mutant variants
under various conditions [21, 39, 46, 47, 57]. The pre-
viously published sequence of proton transfer reactions
for the wild type ESR included: (i) proton transfer from
the Schiff base to the Asp85 acceptor during the forma-
tion of intermediate M; (ii) protonation of the Lys96
donor from the cytoplasmic surface of the protein;
(iii) reprotonation of the Schiff base involving Lys96,
and (iv) deprotonation of Asp85 and release of the pro-
ton at the extracellular surface of the protein involving
an unidentified group of residues [41]. The key steps of
this scheme were directly confirmed and characterized
by time-resolved measurements of electrogenic proton
transport events during the ESR photocycle. In addition,
the scheme was updated taking into account the data ob-
tained by the direct electrometric method. In particular,
the origin of the slow stages of intermediate M forma-
tion, which are observed in ESR at neutral pH values,
was clarified. Since in the H57N mutant this process is
accelerated and does not include slow components, same
as in the wild-type protein under alkaline conditions,
it was concluded that they are related to the presence of
a positive charge at the histidine residue, which prevents
the transfer of a proton from the Schiff base to the Asp85
acceptor.
The presence of the His57 residue and its interac-
tion with Asp85 also explains some other features of pro-
ton transport in the ESR, including reduced efficiency
of transport at low pH values and an increased contribu-
tion of back reactions in the K96A mutant as compared
to similar BR mutants. Back reactions, i.e., the return of
proton from the Asp85 acceptor to the Schiff base, may
be driven by the lower pK
a
of the ESR acceptor (Asp85-
His57) in the M state as compared to BR or by a lower
barrier of reverse transfer from Asp85 to the Schiff base.
The high pK value of Asp85 in BR at this stage (~11)
ERS, RETINAL PROTEIN FROM Exiguobacterium sibiricum 1551
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
iscaused by the separation of the positively charged ar-
ginine residue away from Asp85, increased hydropho-
bicity of Asp85 environment, and early proton release
from PRG [10, 58]. In the ESR molecule, proton release
occurs at the end of the photocycle [20, 38], as a result
of which the extracellular part of the protein can retain
a positive charge for quite a long time. This presumably
leads to an increase in the probability of reverse pro-
ton transfer from the acceptor to the Schiff base [47].
Inwild-type ESR, due to the presence of the Lys96 do-
nor and the rapid reprotonation of the Schiff base, back-
ward reactions are insignificant, but in the K96A mutant
their effect is dramatic, leading to a significant decrease
in the efficiency of proton transport [41,47].
Thus, an important function of Lys96 in the ESR
molecule, in addition to direct reprotonation of the
Schiff base, is the prevention of back reactions that re-
duce the efficiency of the pump. Unlike BR and PR,
in which the function of a proton donor is performed
by initially protonated carboxyl residues, the lysine resi-
due in the ESR molecule acquires a proton immediately
before transferring it to the Schiff base. This sequence
of events was previously established in experiments us-
ing a pH-sensitive dye [41] and then confirmed using
direct electrometry [39]. The discovery of a separate
electrogenic stage, corresponding to the protonation of
the donor during the M1 ↔ M2 transition as a result of
an increase in pH to 8.4 and a corresponding slowdown
in the reprotonation of the Schiff base, further demon-
strated the capabilities of this method, since this stage
did not manifest in any way in optical measurements.
The described patterns may be important for under-
standing the mechanism of functioning of the retinal
proteins that were discovered recently and which also
contain a lysine residue as a proton donor for the Schiff
base [59, 60].
As noted above, the lack of PRG and no early re-
lease of protons make an important difference between
ESR and BR. However, during the study of mutant
protein variants, partial release of the proton at earlier
stages of the photocycle (at the time of intermediate M
formation) was revealed. Thus, the relative amplitude
of the corresponding electrogenic phase in the H57N
mutant turned out to be significantly greater than in the
wild type ESR (28 and 5%, respectively). Presumably,
in the mutant it also involves movement of a proton or
a positive charge to the extracellular side of the protein,
similar to what occurs in BR [57]. Early proton release
was also observed in the mutants containing substitutions
of lysine for carboxyl residues, K96D/A47T and K96E.
In this case, it apparently occurs at the cytoplasmic sur-
face of the protein as a result of an increase in the hydration
of the cavity containing donor residues during the for-
mation of intermediate M[46]. To clarify the mechanisms
of these processes, additional studies will be carried out
inthefuture.
CONCLUSION
In recent years, numerous studies in the field of ret-
inal proteins have demonstrated their impressive natural
diversity, as well as their wide abundance among micro-
organisms inhabiting various ecological niches. Inpartic-
ular, it has been established that more than half of the
representatives of the microbial communities of the world
ocean have rhodopsin genes, which make a significant
contribution to the absorption of solar energy by micro-
organisms [61, 62]. The general structure of the mole-
cule and the basic mechanisms of functioning of these
proteins are universal for the entire family, however, evo-
lutionary adaptation to specific living conditions leads to
the emergence of fine tuning of these mechanisms [5].
Using the example of ESR, a retinal protein from soil
bacterium, we demonstrated how changes in the primary
structure are reflected in the features of proton trans-
port in its molecule. An invaluable role in these studies
is played by biophysical methods that make it possible to
observe the processes occurring in the protein molecule
in response to the absorption of a light quantum with
high time resolution. In this regard, the method of direct
electrometry occupies a special niche, providing unique
information about the movement of the proton inside the
molecules of proton pumps. Investigation of the details
of the structural and functional characteristics of micro-
bial rhodopsins contributes to a more complete under-
standing of the principles of their function, and opens
up the possibility of developing approaches for targeted
changes in their properties, for example, with the aim of
creating new tools for optogenetics[6, 63-65].
Contributions. L.E.P., S.A.S., S.P.B., D.A.D., M.P.K.
concept and management of the study, editing the text of
the article; L.E.P., S.A.S., M.D.M., E.P.L., S.P.B. writing
the text of the article.
Funding. This work was financially supported by the
Russian Science Foundation (project no.22-14-00104).
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
oranimals performed by any of the authors.
Open access. This article is licensed under a Cre-
ative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution, and
reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license,
and indicate if changes were made. The images or other
third-party material in this article are included in the ar-
ticle’s Creative Commons license, unless indicated oth-
erwise in a credit line to the material. If material is not
included in the article’s Creative Commons license and
your intended use is not permitted by statutory regula-
tion or exceeds the permitted use, you will need to obtain
PETROVSKAYA et al.1552
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
permission directly from the copyright holder. To view
a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/.
REFERENCES
1. Engelhard, M. (2022) Molecular Biology of Microbi-
al Rhodopsins, in Rhodopsin: Methods and Protocols
(Gordeliy,V., ed) Springer US, New York, NY, pp.53-69,
doi:10.1007/978-1-0716-2329-9_2.
2. Govorunova, E. G., Sineshchekov, O. A., Li, H., and
Spudich, J. L. (2017) Microbial rhodopsins: diversity,
mechanisms, and optogenetic applications, Annu. Rev.
Biochem., 86, 845-872, doi: 10.1146/annurev-biochem-
101910-144233.
3. Rozenberg, A., Inoue, K., Kandori, H., and Béjà, O.
(2021) Microbial rhodopsins: the last two decades, Annu.
Rev. Microbiol., 75, 427-447, doi: 10.1146/annurev-
micro-031721-020452.
4. Gushchin, I., and Gordeliy, V. (2018) Microbial Rho-
dopsins, in Membrane Protein Complexes: Structure
and Function (Harris, J. R., and Boekema, E. J., eds)
Springer Singapore, Singapore. pp 19-56, doi: 10.1007/
978-981-10-7757-9_2.
5. Brown, L. S. (2022) Light-driven proton transfers and
proton transport by microbial rhodopsins – A biophys-
ical perspective, Biochim. Biophys. Acta Biomembranes,
1864, 183867, doi:10.1016/j.bbamem.2022.183867.
6. Kandori, H. (2020) Biophysics of rhodopsins and op-
togenetics, Biophys. Rev., 12, 355-361, doi: 10.1007/
s12551-020-00645-0.
7. Lanyi, J. K. (2006) Proton transfers in the bacteriorho-
dopsin photocycle, Biochim. Biophys. Acta, 1757, 1012-
1018, doi:10.1016/j.bbabio.2005.11.003.
8. Skulachev, V. P., Bogachev, A. V., and Kasparinsky,
F.O. (2013) Bacteriorhodopsin, in Principles of Bioener-
getics, Springer-Verlag. Berlin, Heidelberg, pp. 139-156,
doi:10.1007/978-3-642-33430-6_6.
9. Oesterhelt, D., and Stoeckenius, W. (1971) Rhodop-
sin-like protein from the purple membrane of Halobacte-
rium halobium, Nat. New Biol., 233, 149-152, doi:10.1038/
newbio233149a0.
10. Balashov, S. P. (2000) Protonation reactions and their
coupling in bacteriorhodopsin, Biochim. Biophys. Acta
Bioenergetics, 1460, 75-94, doi: 10.1016/S0005-2728
(00)00131-6.
11. Neutze, R., Pebay-Peyroula, E., Edman, K., Royant, A.,
Navarro, J., and Landau, E.M. (2002) Bacteriorhodop-
sin: a high-resolution structural view of vectorial proton
transport, Biochim. Biophys. Acta Biomembranes, 1565,
144-167, doi:10.1016/S0005-2736(02)00566-7.
12. Luecke, H., Schobert, B., Richter, H.-T., Cartailler, J.-P.,
and Lanyi, J.K. (1999) Structural changes in bacterior-
hodopsin during ion transport at 2 angstrom resolution,
Science, 286, 255-260, doi:10.1126/science.286.5438.255.
13. Nango, E., Royant, A., Kubo, M., Nakane, T., Wick-
strand, C., Kimura, T., Tanaka, T., Tono, K., Song, C.,
Tanaka, R., Arima, T., Yamashita, A., Kobayashi, J.,
Hosaka, T., Mizohata, E., Nogly, P., Sugahara, M., Nam,
D., Nomura, T., Shimamura, T., etal. (2016) Athree-di-
mensional movie of structural changes in bacteriorhodop-
sin, Science, 354, 1552-1557, doi: 10.1126/science.aah3497.
14. Pushkarev, A., and Béjà, O. (2016) Functional metage-
nomic screen reveals new and diverse microbial rhodop-
sins, ISMEJ., 10, 2331-2335, doi:10.1038/ismej.2016.7.
15. Inoue, K. (2021) Diversity, Mechanism, and Optogenetic
Application of Light-Driven Ion Pump Rhodopsins, in
Optogenetics: Light-Sensing Proteins and Their Applications
in Neuroscience and Beyond (Yawo, H., Kandori, H., Koi-
zumi, A., and Kageyama, R., eds) Springer Singapore,
Singapore, pp.89-126, doi:10.1007/978-981-15-8763-4_6.
16. Bamann, C., Bamberg, E., Wachtveitl, J., and Glaubitz,C.
(2014) Proteorhodopsin, Biochim. Biophys. Acta Bioener-
getics, 1837, 614-625, doi:10.1016/j.bbabio.2013.09.010.
17. Brown, L. S. (2014) Eubacterial rhodopsins– unique pho-
tosensors and diverse ion pumps, Biochim. Biophys. Acta Bio-
energetics, 1837, 553-561, doi:10.1016/j.bbabio.2013.05.006.
18. Bergo, V. B., Sineshchekov, O. A., Kralj, J. M., Par-
tha, R., Spudich, E. N., Rothschild, K.J., and Spudich,
J. L. (2009) His-75 in proteorhodopsin, a novel com-
ponent in light-driven proton translocation by prima-
ry pumps, J.Biol. Chem., 284, 2836-2843, doi: 10.1074/
jbc.M803792200.
19. Hempelmann, F., Holper, S., Verhoefen, M. K., Woerner,
A. C., Kohler, T., Fiedler, S. A., Pfleger, N., Wacht-
veitl, J., and Glaubitz, C. (2011) His75-Asp97 cluster in
green proteorhodopsin, J. Am. Chem. Soc., 133, 4645-
4654, doi:10.1021/Ja111116a .
20. Petrovskaya, L. E., Lukashev, E. P., Chupin, V. V., Sychev,
S. V., Lyukmanova, E. N., Kryukova, E. A., Ziganshin,
R. H., Spirina, E. V., Rivkina, E. M., Khatypov, R. A.,
Erokhina, L. G., Gilichinsky, D. A., Shuvalov, V. A.,
and Kirpichnikov, M. P. (2010) Predicted bacteriorho-
dopsin from Exiguobacterium sibiricum is a functional
proton pump, FEBS Lett., 584, 4193-4196, doi:10.1016/
j.febslet.2010.09.005.
21. Petrovskaya, L., Balashov, S., Lukashev, E., Imasheva,E.,
Gushchin, I.Y., Dioumaev, A., Rubin, A., Dolgikh,D.,
Gordeliy, V., Lanyi, J., and Kirpichnikov, M. (2015)
ESR – A retinal protein with unusual properties from
Exiguobacterium sibiricum, Biochemistry (Moscow), 80,
688-700, doi:10.1134/S000629791506005X.
22. Drachev, L. A., Jasaitis, A. A., Kaulen, A. D., Kondrash-
in, A. A., Liberman, E.A., Nemecek, I.B., Ostroumov,
S.A., Semenov, A.Y., and Skulachev, V.P. (1974) Direct
measurement of electric current generation by cytochrome
oxidase, H
+
-ATPase and bacteriorhodopsin, Nature,
249, 321-324, doi:10.1038/249321a0.
23. Drachev, L. A., Kaulen, A. D., Semenov, A. Y., Severi-
na, I. I., and Skulachev, V.P. (1979) Lipid-impregnated
filters as a tool for studying the electric current-generat-
ERS, RETINAL PROTEIN FROM Exiguobacterium sibiricum 1553
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
ing proteins, Anal. Biochem., 96, 250-262, doi: 10.1016/
0003-2697(79)90580-3.
24. Drachev, L. A., Kaulen, A. D., Khitrina, L. V., and
Skulachev, V. P. (1981) Fast stages of photoelectric pro-
cesses in biological membranes. I. Bacteriorhodopsin,
Eur.J. Biochem., 117, 461-470, doi:10.1111/j.1432-1033.
1981.tb06361.x.
25. Siletsky, S. A., Mamedov, M. D., Lukashev, E. P., Bal-
ashov, S. P., and Petrovskaya, L. E. (2022) Application
of direct electrometry in studies of microbial rhodop-
sins reconstituted in proteoliposomes, Biophys. Rev., 14,
771-778, doi:10.1007/s12551-022-00986-y.
26. Kovalev, K., Volkov, D., Astashkin, R., Alekseev, A.,
Gushchin, I., Haro-Moreno, J. M., Chizhov, I., Si-
letsky, S., Mamedov, M., and Rogachev, A. (2020)
High-resolution structural insights into the heliorhodop-
sin family, Proc. Natl. Acad. Sci. USA, 117, 4131-4141,
doi:10.1073/pnas.1915888117.
27. Rokitskaya, T. I., Maliar, N. L., Siletsky, S. A., Gor-
deliy,V., and Antonenko, Y.N. (2022) Electrophysiolog-
ical Characterization of Microbial Rhodopsin Transport
Properties: Electrometric and ΔpH Measurements Using
Planar Lipid Bilayer, Collodion Film, and Fluorescent
Probe Approaches, in Rhodopsin: Methods and Protocols
(Gordeliy, V., ed) Springer US, New York, NY, pp.259-
275, doi:10.1007/978-1-0716-2329-9_12.
28. Mamedov, M. D., Beshta, O. E., Samuilov, V. D., and Se-
menov, A.Y. (1994) Electrogenicity at the secondary qui-
none acceptor site of cyanobacterial photosystemII, FEBS
Lett., 350, 96-98, doi:10.1016/0014-5793(94)00742-X.
29. Mamedov, M., Gadzhieva, R., Gourovskaya, K., Dra-
chev,L., and Semenov, A.Y. (1996) Electrogenicity at the
donor/acceptor sides of cyanobacterial photosystemI, J.Bio-
energ. Biomembr., 28, 517-522, doi:10.1007/BF02110441.
30. Mamedov, M. D., Tyunyatkina, A. A., Siletsky, S. A., and
Semenov, A. Y. (2006) Voltage changes involving pho-
tosystem II quinone–iron complex turnover, Eur. Bio-
phys.J., 35, 647-654, doi:10.1007/s00249-006-0069-3.
31. Siletsky, S. A., and Konstantinov, A. A. (2012) Cyto-
chrome c oxidase: charge translocation coupled to sin-
gle-electron partial steps of the catalytic cycle, Biochim.
Biophys. Acta Bioenergetics, 1817, 476-488, doi:10.1016/
j.bbabio.2011.08.003.
32. Siletsky, S. A., Soulimane, T., Belevich, I., Gennis, R.B.,
and Wikström, M. (2021) Specific inhibition of proton
pumping by the T315V mutation in the K channel of cy-
tochrome ba
3
from Thermus thermophilus, Biochim. Bio-
phys. Acta Bioenergetics, 1862, 148450, doi: 10.1016/
j.bbabio.2021.148450.
33. Siletsky, S. A., and Gennis, R. B. (2021) Time-resolved
electrometric study of the F→O transition in cytochromec
oxidase. The effect of Zn
2+
ions on the positive side of
the membrane, Biochemistry (Moscow), 86, 105-122,
doi:10.1134/S0006297921010107.
34. Siletsky, S. A., Belevich, I., Belevich, N. P., Soulimane,
T., and Wikström, M. (2017) Time-resolved generation
of membrane potential by ba
3
cytochromec oxidase from
Thermus thermophilus coupled to single electron injection
into the O and O
H
states, Biochim. Biophys. Acta Bioener-
getics, 1858, 915-926, doi:10.1016/j.bbabio.2017.08.007.
35. Siletsky, S. A., Belevich, I., Belevich, N. P., Souli-
mane, T., and Verkhovsky, M. I. (2011) Time-resolved
single-turnover of caa
3
oxidase from Thermus thermophi-
lus. Fifth electron of the fully reduced enzyme converts O
H
into E
H
state, Biochim. Biophys. Acta Bioenergetics, 1807,
1162-1169, doi:10.1016/j.bbabio.2011.05.006.
36. Lozier, R. H., Bogomolni, R. A., and Stoeckenius, W.
(1975) Bacteriorhodopsin: A light-driven proton pump
in Halobacterium halobium, Biophys. J., 15, 955-963,
doi:10.1016/S0006-3495(75)85875-9.
37. Dioumaev, A. K., Brown, L. S., Shih, J., Spudich, E. N.,
Spudich, J. L., and Lanyi, J.K. (2002) Proton transfers
in the photochemical reaction cycle of proteorhodopsin,
Biochemistry, 41, 5348-5358, doi:10.1021/bi025563x.
38. Balashov, S. P., Petrovskaya, L. E., Lukashev, E. P., Ima-
sheva, E. S., Dioumaev, A. K., Wang, J. M., Sychev, S.V.,
Dolgikh, D. A., Rubin, A. B., Kirpichnikov, M. P., and
Lanyi, J. K. (2012) Aspartate-histidine interaction in the
retinal Schiff base counterion of the light-driven proton
pump of Exiguobacterium sibiricum, Biochemistry, 51,
5748-5762, doi:10.1021/bi300409m.
39. Siletsky, S. A., Mamedov, M. D., Lukashev, E. P., Bal-
ashov, S. P., Dolgikh, D. A., Rubin, A. B., Kirpichnikov,
M. P., and Petrovskaya, L. E. (2016) Electrogenic steps of
light-driven proton transport in ESR, a retinal protein from
Exiguobacterium sibiricum, Biochim. Biophys. Acta Bioener-
getics, 1857, 1741-1750, doi:10.1016/j.bbabio.2016.08.004.
40. Dioumaev, A. K., Petrovskaya, L. E., Wang, J. M., Bal-
ashov, S. P., Dolgikh, D. A., Kirpichnikov, M. P., and
Lanyi, J.K. (2013) Photocycle of Exiguobacterium sibiri-
cum rhodopsin characterized by low-temperature trapping
in the IR and time-resolved studies in the visible, J.Phys.
Chem.B, 117, 7235-7253, doi:10.1021/jp402430w.
41. Balashov, S. P., Petrovskaya, L. E., Imasheva, E. S., Luka-
shev, E. P., Dioumaev, A. K., Wang, J. M., Sychev, S.V.,
Dolgikh, D. A., Rubin, A. B., Kirpichnikov, M.P., and
Lanyi, J.K. (2013) Breaking the carboxyl rule: lysine96 fa-
cilitates reprotonation of the Schiff base in the photocycle
of a retinal protein from Exiguobacterium sibiricum, J.Biol.
Chem., 288, 21254-21265, doi:10.1074/jbc.M113.465138.
42. Huang, K.-S., Bayley, H., and Khorana, H. G. (1980)
Delipidation of bacteriorhodopsin and reconstitution with
exogenous phospholipid, Proc. Nat. Acad. Sci. USA, 77,
323-327, doi:10.1073/pnas.77.1.323.
43. Kaulen, A. D. (2000) Electrogenic processes and protein
conformational changes accompanying the bacteriorho-
dopsin photocycle, Biochim. Biophys. Acta Bioenergetics,
1460, 204-219, doi:10.1016/S0005-2728(00)00140-7.
44. Gushchin, I., Chervakov, P., Kuzmichev, P., Popov, A. N.,
Round, E., Borshchevskiy, V., Ishchenko, A., Petrovska-
ya, L., Chupin, V., Dolgikh, D.A., Arseniev, A. S., Kir-
pichnikov, M., and Gordeliy, V. (2013) Structural insights
PETROVSKAYA et al.1554
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
into the proton pumping by unusual proteorhodopsin
from nonmarine bacteria, Proc. Natl. Acad. Sci. USA, 110,
12631-12636, doi:10.1073/pnas.1221629110.
45. Zscherp, C., Schlesinger, R., Tittor, J., Oesterhelt, D.,
and Heberle, J. (1999) Insitu determination of transient
pKa changes of internal amino acids of bacteriorhodopsin
by using time-resolved attenuated total reflection Fouri-
er-transform infrared spectroscopy, Proc. Natl. Acad. Sci.
USA, 96, 5498-5503, doi:10.1073/pnas.96.10.5498.
46. Petrovskaya, L. E., Lukashev, E. P., Siletsky, S. A., Ima-
sheva, E. S., Wang, J. M., Mamedov, M. D., Kryukova,
E. A., Dolgikh, D. A., Rubin, A. B., Kirpichnikov, M. P.,
Balashov, S.P., and Lanyi, J.K. (2022) Proton transfer
reactions in donor site mutants of ESR, a retinal protein
from Exiguobacterium sibiricum, J. Photochem. Photobi-
ol.B, 234, 112529, doi:10.1016/j.jphotobiol.2022.112529.
47. Siletsky, S. A., Mamedov, M. D., Lukashev, E. P., Bal-
ashov, S. P., Dolgikh, D.A., Rubin, A.B., Kirpichnikov,
M. P., and Petrovskaya, L.E. (2019) Elimination of pro-
ton donor strongly affects directionality and efficiency of
proton transport in ESR, a light-driven proton pump from
Exiguobacterium sibiricum, Biochim. Biophys. Acta Bioener-
getics, 1860, 1-11, doi:10.1016/j.bbabio.2018.09.365.
48. Otto, H., Marti, T., Holtz, M., Mogi, T., Lindau, M.,
Khorana, H.G., and Heyn, M.P. (1989) Aspartic acid-96
is the internal proton donor in the reprotonaion of the
Schiff base of bacteriorhodopsin, Proc. Natl. Acad. Sci.
USA, 86, 9228-9232, doi:10.1073/pnas.86.23.9228.
49. Holz, M., Drachev, L. A., Mogi, T., Otto, H., Kaulen,
A.D., Heyn, M.P., Skulachev, V.P., and Khorana, H.G.
(1989) Replacement of aspartic acid-96 by asparagine in
bacteriorhodopsin slows both the decay of the M inter-
mediate and the associated proton movement, Proc. Natl.
Acad. Sci. USA, 86, 2167-2171, doi:10.1073/pnas.86.7.2167.
50. Dioumaev, A. K., Brown, L. S., Needleman, R., and
Lanyi, J.K. (2001) Coupling of the reisomerization of the
retinal, proton uptake, and reprotonation of Asp-96 in the
N photointermediate of bacteriorhodopsin, Biochemistry,
40, 11308-11317, doi:10.1021/bi011027d.
51. Sasaki, S., Tamogami, J., Nishiya, K., Demura, M., and
Kikukawa, T. (2021) Replaceability of Schiff base proton
donors in light-driven proton pump rhodopsins, J. Biol.
Chem., 297, 101013, doi:10.1016/j.jbc.2021.101013.
52. Balashov, S. P., Imasheva, E. S., Boichenko, V. A.,
Antón,J., Wang, J.M., and Lanyi, J.K. (2005) Xanthorho-
dopsin: a proton pump with a light-harvesting carot-
enoid antenna, Science, 309, 2061-2064, doi:10.1126/sci-
ence.1118046.
53. Luecke, H., Schobert, B., Stagno, J., Imasheva, E. S.,
Wang, J. M., Balashov, S. P., and Lanyi, J. K. (2008)
Crystallographic structure of xanthorhodopsin, the
light-driven proton pump with a dual chromophore, Proc.
Natl. Acad. Sci. USA, 105, 16561-16565, doi: 10.1073/
pnas.0807162105.
54. Ran, T., Ozorowski, G., Gao, Y., Sineshchekov, O. A.,
Wang, W., Spudich, J.L., and Luecke, H. (2013) Cross-
protomer interaction with the photoactive site in oligo-
meric proteorhodopsin complexes, Acta Cryst., D69, 1965-
1980, doi:10.1107/S0907444913017575.
55. Maciejko, J., Kaur, J., Becker-Baldus, J., and Glaubitz, C.
(2019) Photocycle-dependent conformational changes in
the proteorhodopsin cross-protomer Asp–His–Trp triad
revealed by DNP-enhanced MAS-NMR, Proc. Nat. Acad.
Sci. USA, 116, 8342-8349, doi:10.1073/pnas.1817665116.
56. Morizumi, T., Ou, W.-L., Van Eps, N., Inoue, K., Kan-
dori, H., Brown, L. S., and Ernst, O. P. (2019) X-ray
crystallographic structure and oligomerization of Gloeo-
bacter rhodopsin, Sci. Rep., 9, 1-14, doi:10.1038/s41598-
019-47445-5.
57. Siletsky, S. A., Lukashev, E. P., Mamedov, M. D., Bor-
isov, V. B., Balashov, S. P., Dolgikh, D.A., Rubin, A.B.,
Kirpichnikov, M.P., and Petrovskaya, L.E. (2021) His57
controls the efficiency of ESR, a light-driven proton
pump from Exiguobacterium sibiricum at low and high
pH, Biochim. Biophys. Acta Bioenergetics, 1862, 148328,
doi:10.1016/j.bbabio.2020.148328.
58. Luecke, H., Schobert, B., Richter, H.-T., Cartailler, J.-P.,
and Lanyi, J.K. (1999) Structure of bacteriorhodopsin at
1.55Å resolution, J.Mol. Biol., 291, 899-911, doi:10.1006/
jmbi.1999.3027.
59. Iverson, V., Morris, R. M., Frazar, C. D., Berthiaume,
C. T., Morales, R.L., and Armbrust, E.V. (2012) Untan-
gling genomes from metagenomes: revealing an uncultured
class of marine Euryarchaeota, Science, 335, 587-590,
doi:10.1126/science.1212665.
60. Martin-Cuadrado, A. B., Garcia-Heredia, I., Molto,
A. G., Lopez-Ubeda, R., Kimes, N., Lopez-Garcia, P.,
Moreira, D., and Rodriguez-Valera, F. (2015) Anew class
of marine Euryarchaeota group II from the Mediterra-
nean deep chlorophyll maximum, ISMEJ., 9, 1619-1634,
doi:10.1038/ismej.2014.249.
61. Finkel, O. M., Béjà, O., and Belkin, S. (2013) Global
abundance of microbial rhodopsins, ISMEJ., 7, 448-451,
doi:10.1038/ismej.2012.112.
62. Gómez-Consarnau, L., Raven, J. A., Levine, N. M.,
Cutter, L.S., Wang, D., Seegers, B., Arístegui, J., Fuhr-
man, J. A., Gasol, J. M., and Sañudo-Wilhelmy, S. A.
(2019) Microbial rhodopsins are major contributors to the
solar energy captured in the sea, Sci. Adv., 5, eaaw8855,
doi:10.1126/sciadv.aaw8855.
63. Kojima, K., Shibukawa, A., and Sudo, Y. (2020) The unlim-
ited potential of microbial rhodopsins as optical tools, Bio-
chemistry, 59, 218-229, doi:10.1021/acs.biochem.9b00768.
64. De Grip, W. J., and Ganapathy, S. (2022) Rhodopsins: an
excitingly versatile protein species for research, develop-
ment and creative engineering, Front. Chem., 10, 879609,
doi:10.3389/fchem.2022.879609.
65. Emiliani, V., Entcheva, E., Hedrich, R., Hegemann, P.,
Konrad, K.R., Lüscher, C., Mahn, M., Pan, Z.-H., Sims,
R.R., Vierock, J., and Yizhar, O. (2022) Optogenetics for
light control of biological systems, Nat. Rev. Meth. Prim-
ers, 2, 55, doi:10.1038/s43586-022-00136-4.
