ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1504-1512 © Pleiades Publishing, Ltd., 2023.
Published in Russian in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1818-1828.
1504
REVIEW
Generation of Membrane Potential by Cytochrome bd
Vitaliy B. Borisov
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia
e-mail: bor@belozersky.msu.ru
Received June 10, 2023
Revised July 8, 2023
Accepted July 11, 2023
Abstract— An overview of current notions on the mechanism of generation of a transmembrane electric potential differ-
ence(Δψ) during the catalytic cycle of a bd-type triheme terminal quinol oxidase is presented in this work. It is suggested
that the main contribution to Δψ formation is made by the movement of H
+
across the membrane along the intra-protein
hydrophilic proton-conducting pathway from the cytoplasm to the active site for oxygen reduction of this bacterial enzyme.
DOI: 10.1134/S0006297923100073
Keywords: respiratory chain, terminal oxidase, cytochromebd, heme, protonmotive force, membrane potential
Abbreviations: Δψ,transmembrane electrical potential difference; Δp,protonmotive force; τ,time constant, reciprocal of rate
constant(t
1/e
); H
+
/e
–
,proton/electron stoichiometry which in the case of Escherichia coli respiratory chain means number of pro-
tons released into the periplasm per electron used to reduce oxygen to water; RuBpy,tris(2,2′-bipyridyl)ruthenium(II) chloride.
INTRODUCTION
In 1974, L. A. Drachev and co-authors at the
Belozersky Institute of Physico-Chemical Biology of
Lomonosov Moscow State University developed an
electrometric method for direct measurement of elec-
trical activity of coupling membranes, which provided a
unique opportunity to track intra-protein movements of
electrical charges within one molecular turnover of the
enzyme [1]. Using this method, it was possible to ob-
serve in real time generation of the transmembrane elec-
tric potential difference (Δψ) by bacteriorhodopsin [2],
reaction centers [3], and cytochrome bc
1
[4] from photo-
synthetic bacteria, as well as terminal cytochrome c oxi-
dase [5-7] and non-canonical retinal-containing bacte-
rial proteins [8, 9]. Two different approaches are used
to study electrogenic mechanism of cytochromec oxi-
dases. The first approach uses photochemical injection
of a single electron into the enzyme embedded in a li-
posome. In this case, tris(2,2′-bipyridyl)ruthenium(II)
chloride (RuBpy) acts as a direct photoactivated reduc-
ing agent, which forms a complex with the cytochrome c
binding site in the oxidase near the input Cu
A
redox cen-
ter due to electrostatic interactions [5, 6]. As a result of
photoexcitation of RuBpy by a pulsed laser, an electron
is transferred from RuBpy* to Cu
A
. The oxidized RuBpy
is re-reduced by aniline. This approach makes it possi-
ble to record time-resolved electrogenic charge transfer
during individual one-electron transitions in the cat-
alytic cycle of cytochrome c oxidase [7]. In the second
approach, laser flash photolysis of a complex of car-
bon monoxide (CO) with the oxygen-binding high-spin
hemea
3
is used to initiate enzymatic reaction in the sin-
gle-turnover mode. Formation of the CO complex with
the partially or completely reduced enzyme occurs un-
der anaerobic conditions. Oxygen(O
2
) dissolved in water
is then added to the anaerobic cell with the CO-bound
oxidase using rapid mixing technique. During decompo-
sition of the CO-oxidase complex triggered by photolysis,
O
2
binds to hemea
3
and is reduced by electrons pres-
ent in the oxidase that is accompanied by generation of
Δψ[10]. Thus, the second approach uses combination
of the direct electrometric method [1] and the flow-
flash method [11]. Terminal quinol oxidases including
cytochrome bd, which is the subject of this review, do
not have a cytochromec binding site. For this reason,
the first approach is inapplicable for tracking movement
of electric charges inside their protein molecule.
GENERAL CHARACTERISTICS
OF CYTOCHROME bd
Membrane-bound terminal oxidases of the aerobic
respiratory chains of organisms are classified as translo-
cases (enzymes in class EC7). They catalyze four-electron
ELECTROGENIC CYTOCHROME bd 1505
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
reduction of molecular oxygen to water by ferrocyto-
chromec or quinol (ubiquinol, menaquinol, and possi-
bly plastoquinol) [12,13]. The catalyzed redox reaction
is coupled with generation of Δp (protonmotive force).
Δp is an “energy currency” and is used by the cell to syn-
thesize ATP through the mechanism of oxidative phos-
phorylation [14]. Terminal oxidases are divided into two
evolutionarily unrelated superfamilies: heme/Cu-con-
taining oxidases and bd-type oxidases also called cyto-
chromes bd [15-18]. Unlike the heme-copper oxidases,
all biochemically characterized cytochromes bd are qui-
nol oxidases, do not contain Cu, and are found only
in bacteria and archaea, including pathogens [19-21].
The latter circumstance makes it possible to consider
bd enzymes as promising therapeutic targets [21, 22].
Since generation of Δψ in the single-turnover mode has
been studied so far only in the terminal bd oxidases from
Escherichia coli, we should consider these enzymes in
more detail.
Like the electron transport chains of many bac-
teria, aerobic respiratory chain of E. coli is branched.
Its terminal region is generally represented by three qui-
nol oxidases: heme-copper cytochromebo
3
and two cy-
tochromes bd, bd-I and bd-II [23,24]. Unlike bd-type
oxidases, cytochrome bo
3
forms Δp by the proton pump
mechanism that makes it possible to double pump-
ing stoichiometry: proton/electron (H
+
/e
–
) [25, 26].
Cytochromes bo
3
, bd-I, and bd-II are encoded by the
cyoABCDE, cydABX, and appCBX operons, respectively.
cyoABCDE is predominantly expressed under high O
2
partial pressure, while cydABX is predominantly ex-
pressed under microaerobic conditions. The appCBX
expression is induced during anaerobic growth of E. coli,
entry of the culture into the stationary phase of growth,
and phosphate starvation [24]. Recently, there has been
more and more evidence reported that, in addition
to functioning as molecular energy transducers, bd-type
oxidases are involved in other vital processes in the bac-
terial cell [27-30]. Cytochromebd-I is involved in for-
mation of disulfide bonds during protein folding [31],
heme biosynthesis [32], and mechanisms of bacterial
resistance to antibiotics [33], peroxynitrite [34], nitro-
gen monoxide [35-41], and ammonia [42]. Both bd oxi-
dases (bd-I and bd-II) also endow E. coli with resis-
tance to cyanide [43], sulfide [43-45], and hydrogen
peroxide [46-54].
Three-dimensional structures of both E. coli bd en-
zymes have recently been published (Figs. 1 and2) [55-58].
Cytochrome bd-I was found to contain four subunits
(CydA, CydB, CydX, CydY), while cytochrome bd-II
only three (AppC, AppB, AppX). The CydA, CydB, and
CydX subunits are homologous to the AppC, AppB, and
AppX subunits, respectively. Two large subunits, CydA/
AppC and CydB/AppB, form structural core of the pro-
tein. Of the other structural differences between the two
bd oxidases, it should be noted that the bd-II protein in-
corporated into amphipoles is mainly in the form of a
dimer (Fig. 2), while the bd-I enzyme exists only as a
monomer [57] (Fig. 1). Oxygen channel in cytochrome
bd-II has a smaller diameter compared to that of cyto-
chrome bd-I [57]. In addition, the putative proton-con-
ducting pathway in the bd-II enzyme is shorter than in
the bd-I oxidase [57]. CydA/AppC contains three dif-
ferent hemes acting as redox cofactors: one low-spin
hexacoordinate, b
558
, and two high-spin pentacoordi-
nate, b
595
and d. The heme axial ligands are amino acid
Fig. 1. Three-dimensional structure of E. coli cytochrome bd-I with
3.3 Å resolution (pdb ID 6RX4). In addition to hemesb
558
, b
595
, andd
associated with the CydA subunit, ubiquinone-8 (Q8) and glycero-
phospholipid (shown as spherical symbols) associated with the CydB
subunit are found in the protein structure. Reprinted from Theßeling
et al. [55] under the terms of the Creative Commons Attribution4.0
International Public License.
Fig. 2. Three-dimensional structure of E. coli cytochrome bd-II dimer
with 3.0Å resolution (pdbID 7OSE). In addition to hemesb
558
, b
595
,
andd associated with the AppC subunit, ubiquinone-8 (shown in red)
associated with the AppB subunit is found in the protein structure.
Reprinted from Grauel et al. [57] under the terms of the Creative Com-
mons Attribution4.0 International Public License.
BORISOV1506
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 3. Triangular arrangement of hemesb
558
, b
595
, andd in the CydA
subunit of E.coli cytochromebd-I. Periplasm: top of the picture, cy-
toplasm: bottom of the picture. Reprinted from Theßeling et al. [55]
under the terms of the Creative Commons Attribution4.0 International
Public License.
residues of the CydA/AppC subunit. These are His186
and Met393 for heme b
558
and Glu445 for heme b
595
[55-58]. In cytochromebd-II, the axial ligand of hemed
is His19 [57-58]. In the case of cytochrome bd-I, data
on the nature of the hemed axial ligand are contradic-
tory. Safarian etal. [56] state that this is also His19, but
according to the model of Theßeling etal. [55], such a
ligand is Glu99. The hemes in the protein are arranged
in a triangle (Fig. 3). Additional structural element in
CydA/AppC is the so-called Q-loop. It is located near
heme b
558
and is directly involved in binding of quinol,
a lipophilic electron donor. The other large subunit,
CydB/AppB, does not contain any metal-containing co-
factors. Instead, it carries a tightly bound ubiquinone-8
or demethylmenaquinone-8. This quinone occupies posi-
tion equivalent to the heme-binding site in CydA/AppC
and, probably, plays a role in the protein structure sta-
bilization. Heme b
558
is the primary electron acceptor
upon quinol oxidation. Heme d serves as a site for O
2
binding and its subsequent reduction to 2H
2
O [59,60].
Hemed in cytochromebd-I has an unusually high affin-
ity for O
2
, and the resulting oxygenated complex is very
stable [61-64]. Functions of hemeb
595
are poorly under-
stood. Excessive distance between the central Fe atoms
of hemesb
595
andd (10.9-11.3Å) most likely does not
allow them to form a structural binuclear center similar
to that of heme-copper oxidases. However, van der Waals
contacts between these hemes are possible since the dis-
tance between their edges is much smaller (3.5-3.8 Å)
[55-58]. The latter circumstance suggests the possibility
of a very fast electron transfer between hemesb
595
andd
that was experimentally confirmed [65,66]. Therefore,
these hemes could potentially form a functional diheme
center. This assumption is consistent with the data of a
number of studies [67-79]. An electron that came from
quinol to hemeb
558
is apparently transferred to hemeb
595
and next to hemed.
ELECTROGENIC REACTIONS
AND CATALYTIC CYCLE
OF CYTOCHROME bd-I FROM E. coli
Use of optical and electrometric methods in com-
bination with flow-flash method made it possible to
observe in real time transient formation and decay of
the individual intermediates of the catalytic cycle of
E. coli cytochrome bd-I at 21°C [26, 80-83]. The pro-
posed scheme of the catalytic cycle is shown in Fig.4.
In spectroscopic studies, hemoprotein was in detergent
micelles, and in electrometric studies, it was incorporated
into liposomes. In the experiment, the enzyme was ini-
tially converted into a completely reduced state in which
heme d was bound to CO (R
3
–CO, b
558
2+
b
595
2+
d
2+
–CO).
Photolysis of CO from this state of the oxidase leads to
the transient appearance of the form of cytochromebd-I
not bound to CO (R
3
, b
558
2+
b
595
2+
d
2+
). This transition
(R
3
–CO→R
3
) is not resolved in time in both spectro-
photometric and electrometric measurements. In the
presence of O
2
, a molecule of this diatomic gas binds
to heme d. As a result, the oxygenated complex, inter-
mediate A
3
(b
558
2+
b
595
2+
d
2+
–O
2
), is formed. The rate of
formation of A
3
is directly proportional to concentra-
tion of O
2
, with the second-order rate constant to be
about 2 × 10
9
M
–1
s
–1
[64, 82]. The R
3
→A
3
transition
is not accompanied by generation of Δψ [26, 82, 83].
A
3
is quickly (τ ~ 4.5 μs) converted into an intermediate,
which Belevich et al. first discovered, described, and
named as compoundP [82]. It was found that the A
3
→P
transition is also not coupled with generation of Δψ [26,
82, 83]. In contrast to the production of A
3
from R
3
,
the rate of formation of compoundP does not depend
on concentration of O
2
. During the A
3
→P transition,
hemeb
595
undergoes oxidation, hemeb
558
remains in the
reduced state, and new oxygen intermediate of heme d
reveals an unusual absorption maximum at 635 nm[82].
There is still no consensus on chemical structure of
the compound P. In the original work [82], Belevich
et al. suggested that P is a true peroxide complex, ei-
ther a ferryl intermediate with an amino acid radical or
a cation radical of the porphyrin ring. According to the
more recent report by Paulus etal. [84], compound P
is a ferryl form of hemed with π-cation radical on the
porphyrin ring, which is in magnetic interaction with
heme b
595
. It is important to emphasize that Paulus etal.
[84] observed formation of compound P at 1°C, i.e.,
under non-physiological conditions. It is possible that the
spectral intermediateP, appearance of which was regis-
tered in real time by Belevich et al. [82], is a mixture
of a true peroxide complex (b
558
2+
b
595
3+
d
3+
–O–O–(H))
and a ferryl π-cation radical (b
558
2+
b
595
3+
d*
4+
=O
2–
), pro-
vided that they have similar absorption spectra. At the
next stage, P is converted (with τ ~ 47 μs) into a non-rad-
ical form of the ferryl complex of heme d (compound F)
that is accompanied by oxidation of heme b
558
. Cat-
ELECTROGENIC CYTOCHROME bd 1507
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Fig. 4. Catalytic cycle of the E.coli cytochromebd-I. Compounds A
3
,
P, F, O
1
, A
1
are catalytic intermediates of the enzyme. Compounds
R
3
and R
3
–CO are not part of the oxidase catalytic cycle, but can be
obtained artificially. Red “arrows of coupling” indicate generation of
Δψ at P→F and F→A
1
transitions. Structures of the compounds are dis-
cussed in the main text.
Fig. 5. Proposed proton-conducting pathway in E.coli cytochrome bd-I.
The pathway is lined with side chains of several hydrophilic amino
acids and allows transfer of protons from the cytoplasmic side of the
membrane to hemed propionate. It also contains numerous water mol-
ecules (indicated by blue spherical symbols). Reprinted from Friedrich
et al. [22] under the terms of the Creative Commons Attribution4.0
International Public License.
alytic intermediate F, most likely, has the structure of
b
558
3+
b
595
3+
d
4+
= O
2–
. The P→F transition is associated
with generation of Δψ [26, 82, 83].
The bd-type oxidase contains three hemes. There-
fore, if the isolated enzyme does not contain a bound
quinol, then it can be expected that in the completely
reduced state it carries three electrons(R
3
). In this case,
the reaction of R
3
with O
2
stops at formation of com-
pound F [80]. If cytochrome bd-I contains a molecule
of bound quinol, which is a two-electron donor, its oxi-
dation in the presence of O
2
makes it possible to convert
F into intermediate A
1
with τ ~ 0.6-1.1 ms [81, 82]. A
1
is,
probably, the one-electron form of the oxidase with the
heme d oxycomplex (b
558
3+
b
595
3+
d
2+
–O
2
). The F→A
1
tran-
sition, like the previous P→F transition, is accompanied
by generation of Δψ [81, 82]. Whether Δψ is formed at
one more particular stage of the catalytic cycle, in the
A
1
→A
3
transition (Fig.4), is still unknown.
It was found that under the steady-state conditions
in the presence of O
2
and ubiquinol-1, F and A
1
are main
catalytic intermediates of the cytochrome bd-I (about
40% each) [60]. About 20% of the oxidase is probably in
the O
1
state that is a one-electron form with the oxidized
heme d (b
558
2+
b
595
3+
d
3+
–OH). The O
1
state has not been
observed in the single-turnover experiments using the
flow-flash method [80, 82, 83]. However, it is currently
recognized that O
1
is apparently also a catalytic interme-
diate of the cytochrome bd-I (Fig.4). It should also be
noted that the R
3
form of the enzyme is most likely not
its catalytic intermediate [59,60], however, for the needs
of experiment, it can be easily produced artificially.
In 2005, Belevich et al. [81] postulated based on
the obtained results existence of an intra-protein pro-
ton-conducting pathway for the transfer of H
+
from cyto-
plasm to the oxygen reductase center of cytochrome bd-I.
The authors suggested that such movement of H
+
across
the membrane, associated with the transfer of an electron
from hemeb
558
to hemesb
595
andd, is accompanied by
generation of Δψ observed in the experiments [26, 80-83].
Release of H
+
into the periplasmic space during oxida-
tion of quinol by the enzyme also contributes to cre-
ation ofΔp. 3D structures of the bd-I oxidase published
in 2019, with resolution of 2.68Å (pdb ID 6RKO) [56]
and 3.3Å (pdbID 6RX4) [55], confirmed the hypothesis
suggested by Belevich etal. [81]. The structures show a
chain of water molecules stretching along a hydrophilic
proton-conducting pathway that starts at the cytoplasmic
interface between the CydA and CydB subunits and runs
perpendicular to the membrane plane towards heme d.
This proton-conducting pathway includes several hydro-
philic amino acid residues (Fig. 5). From the cytoplas-
mic side, the pathway begins with Asp119
CydA
, then, ap-
parently, it includes Lys57
CydA
, Lys109
CydA
, Asp105
CydA
,
Tyr379
CydB
, and, finally, Asp58
CydB
, from which protons
are likely delivered to the propionate group of heme d [55].
It has been suggested that the conserved hydrophilic resi-
dues Ser108
CydA
, Glu107
CydA
, and Ser140
CydA
also belong
to this pathway, facilitating transfer of H
+
from Asp58
CydB
to the hemed propionate [56].
BORISOV1508
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Belevich et al. [81] also hypothesized that the bd-I
molecule contains two amino acid residues with pro-
tonated groups, which are sensitive to the redox state of
the high-spin pentacoordinate hemesb
595
andd. Studies
of the Glu445Ala and Glu107Leu mutant forms of the
E. coli cytochrome bd-I by electrometry and absorp-
tion spectroscopy with microsecond resolution indi-
cate that exactly these highly conserved Glu445
CydA
and
Glu107
CydA
residues are functionally important [81,83].
As noted above, Glu445
CydA
is the axial ligand of the
hemeb
595
iron [55, 56]. Its replacement with Ala in the
CydA subunit leads to the enzyme inactivation. At the
same time, hemeb
595
is retained in the protein but loses
its ability to be reduced even in the presence of a strong
electron donor, dithionite, added in excess [81]. As in
the case of the wild-type cytochrome bd-I, the reaction
of the reduced Glu445Ala mutant enzyme with oxygen
exhibits an initial non-electrogenic stage consisting of
the R
3
→A
3
and A
3
→P transitions. However, in contrast
to the wild-type enzyme, the microsecond phase is not
detected in the mutant during Δψ generation. Instead,
the mutant form exhibits a slower, smaller electrogen-
ic phase (τ ~ 1.3 ms; amplitude –0.36 mV) followed by
a much larger electrogenic transition (τ ~ 12.5 ms; am-
plitude –1.7 mV). Both electrogenic phases, most likely,
reflect the P→F transition in different subpopulations
of the Glu445Ala enzyme [81]. Thus, the substitu-
tion of Glu445 for Ala strongly inhibits the transmem-
brane charge transfer associated with oxidation of cyto-
chromebd-I by oxygen.
Similarly, substitution of Glu107 for Leu in the
CydA subunit leads to the loss of quinol oxidase ac-
tivity by cytochrome bd-I. The fully reduced mutant
form of the Glu107Leu protein (R
3
) binds O
2
at about
the same rate as the wild-type enzyme. However, for-
mation of the ferryl intermediate(F) in the mutant ox-
idase is believed to be significantly slower compared to
the wild-type enzyme. This is evidenced by the results
of spectrophotometric experiments, according to which
production of the compound F has not been observed
within the 100-μs time interval in the mutant oxidase,
in contrast to the wild-type protein [83]. This conclu-
sion is consistent with the fact that the rate of generation
ofΔψ (the main phase) by the mutant oxidase is about
350times slower than the rate measured for the wild-type
enzyme [83].
Glu445
CydA
appears to be protonated during the
transition of heme b
595
from the oxidized to the re-
duced form, i.e., it serves to compensate for the nega-
tive charge of the electron that came to the heme. In the
case of heme d, Glu107
CydA
probably plays the same role.
According to the published three-dimensional struc-
ture of the E.coli cytochrome bd-I, heme b
595
is located
near the periplasmic surface [55, 56]. Therefore, if H
+
is
transferred to Glu445
CydA
from the periplasmic side of
the membrane during the heme b
595
reduction, this pro-
ton is unlikely to be used in the oxygen reductase reac-
tion catalyzed by the enzyme.
CAN CYTOCHROME bd-II
FROM E. coli GENERATE Δψ?
Functional studies of the E.coli cytochromebd-II
are still at the very early stages. Becker etal. [85] report-
ed that the H
+
/e
–
ratio for the bd-II enzyme is 0, i.e.,
it is an uncoupled quinol oxidase. It is known that cy-
tochromes bd-I and bo
3
from E.coli are coupled quinol
oxidases, with the H
+
/e
–
values of 1 and 2, respective-
ly[25, 26]. Becker et al. constructed the mutant strain
of E. coli, MB37, in the respiratory chain of which cy-
tochrome bd-II was present but all the primary proton
potential generators known at that time, NADH de-
hydrogenase 1 (NDH-1), cytochrome bo
3
, and cyto-
chrome bd-I, were absent. The authors calculated H
+
/e
–
by comparing the values for the specific rates of oxygen
consumption and ATP synthesis observed for the MB37
strain and other E. coli mutant strains for which the
H
+
/e
–
values had already been measured [85]. Bacterial
cells of all the strains were grown under the same condi-
tions, and the rate of ATP synthesis was calculated taking
into account the rates of formation of metabolic prod-
ucts– CO
2
, acetate, ethanol, and lactate. Unexpectedly,
the MB37 strain, which, according to the authors’ sug-
gestion, should have the completely uncoupled aerobic
respiratory chain, was capable of growing under aerobic
conditions on non-fermentable substrates. However, the
question arises: how is ATP synthesis ensured in this
case? Becker et al. hypothesized [85] that ATP in the
MB37 strain is produced exclusively via substrate phos-
phorylation. In the more recent work, Shepherd et al.
[86] suggested that in this mutant strain the proton po-
tential is formed due to functioning of an electrogenic
antiporter, which transports an anion of glutamic acid
(glutamate) into the cell in exchange for the release of
a neutral γ-aminobutyric acid (GABA) from the cell.
In this case, GABA in the cell is synthesized from glu-
tamate, and an intracellular proton is consumed during
the process.
Borisov et al. [26] found the conclusions of the
authors of those two studies [85, 86] unconvincing and
experimentally tested whether cytochrome bd-II gen-
erates Δp. It was found that, under the steady state
conditions, both components of Δp – Δψ and ΔpH –
are formed due to quinol oxidase activity of cyto-
chromebd-II [26]. The H
+
/e
–
ratio was measured. As in
the case of cytochrome bd-I, it turned out tobe1[26].
It was also shown that the bd-II oxidase is able to gen-
erate Δψ during a single molecular turnover of the en-
zyme, presumably, during the P→F and F→A
1
tran-
sitions [26]. Thus, one can conclude that the E. coli
cytochrome bd-II is the primary generator of Δp.
ELECTROGENIC CYTOCHROME bd 1509
BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023
Therefore, to explain the growth of the E. coli MB37
cells under aerobic conditions, alternative mechanisms
of ATP formation proposed in [85,86] are not required.
Funding. This work was financially supported by the
Russian Science Foundation (project no.22-24-00045,
https://rscf.ru/en/project/22-24-00045/).
Acknowledgments. The author would like to express
his deepest gratitude to M. I. Verkhovsky (passed away),
I. N. Belevich, N. P. Belevich, D. A. Bloch (passed away),
and A. Jasaitis for wonderful time spent measuring elec-
trogenic activity of the enigmatic cytochromebd.
Ethics declarations. The author declares no conflict
of interest in financial or any other sphere. This article
does not contain any studies involving animals or hu-
man participants performed by author.
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