Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1513-1527 © Pleiades Publishing, Ltd., 2023.

Russian Text © The Author(s), 2023, published in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1829-1846.

1513

REVIEW

Investigation of the Mechanism of Membrane Potential

Generation by Heme-Copper Respiratory Oxidases

in a Real Time Mode

S e r g e i A . S i l e t s k y

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

119991 Moscow, Russia

e-mail: siletsky@belozersky.msu.ru

Received June 29, 2023

Revised August 15, 2023

Accepted August 15, 2023

Abstract— Heme-copper respiratory oxidases are highly efficient molecular machines. These membrane enzymes catalyze

the final step of cellular respiration in eukaryotes and many prokaryotes: the transfer of electrons from cytochromes or

quinols to molecular oxygen and oxygen reduction to water. The free energy released in this redox reaction is converted

by heme-copper respiratory oxidases into the transmembrane gradient of the electrochemical potential of hydrogen ions

ΔμH

). Heme-copper respiratory oxidases have a unique mechanism for generating ΔμH

, namely, a redox-coupled proton

pump. A combination of direct electrometric method for measuring the kinetics of membrane potential generation with the

methods of prestationary kinetics and site-directed mutagenesis in the studies of heme-copper oxidases allows to obtain

a unique information on the translocation of protons inside the proteins in real time. The review summarizes the data of

studies employing time-resolved electrometry to decipher the mechanisms of functioning of these important bioenergetic

enzymes.

DOI: 10.1134/S0006297923100085

Keywords: bioenergetics, cytochrome oxidase, proteoliposomes, electrogenic proton transfer, ΔΨ generation, proton

pump, photoreduction, cytochrome aa

, kinetics, direct electrometric method, capacitive potentiometry, time resolution,

zinc ions

Abbreviations: ΔΨ, transmembrane electrical potential difference; ΔμH

,transmembrane gradient of the electrochemical poten-

tial of hydrogen ions; BNC,binuclear center; COX,cytochrome oxidase; F, O, O

, P, E, E

, and R,cytochrome c oxidase states;

P and N sides of the membrane, positively and negatively charged, respectively, aqueous phases separated by the coupling mem-

brane; PLS,proton loading site; Rubpy,tris(bipyridine)ruthenium complex.

INTRODUCTION.

HEME-COPPER RESPIRATORY OXIDASES

Terminal respiratory oxidases are key components

of respiratory chains of the mitochondria and most aer-

obic bacteria [1, 2]. These ubiquitous enzymes are at

the end of the respiratory chain and catalyze reduction

of molecular oxygen by cytochromec or ubiquinol. This

process is coupled with the formation of proton motive

force (PMF), a transmembrane gradient of the electro-

chemical potential of hydrogen ions (ΔμH

) [3-7] used as

an energy source for various energy-demanding cellular

processes, such as membrane transport, synthesis of bio-

molecules, movement of bacterial cells, etc.

Terminal respiratory oxidases are typically classified

into two superfamilies: heme-copper oxidases and cyto-

chromes bd [7]. A characteristic feature of heme-copper

oxidases is the presence of the oxygen-reducing catalyt-

ic site (the so-called binuclear center, BNC) formed by

closely located iron ion of the heme group and copper

ion. Mitochondrial cytochromec oxidase contains four

redox centers (Fig. 1). Its catalytic site is located inside

the enzyme hydrophobic core and consists of two redox

centers: high-spin heme a

iron and copper ion Cu

Four electrons supplied by cytochrome c through two

other redox centers (Cu

and low-spin heme a) sequen-

tially enter the BNC, which ultimately results in the re-

duction of one oxygen molecule to two water molecules.

SILETSKY1514

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 1. Scheme of cytochrome oxidase (COX) activity. Negatively and positively charged sides of the membrane face the mitochondrial matrix

and the intermembrane space, respectively. In the case of prokaryotic enzymes, the corresponding sides of the membrane face the cytoplasm and

the periplasmic space. Red and blue arrows indicate the transfer of electrons and protons, respectively.

In bacterial oxidases, the low-spin heme a can be re-

placed by heme b, and the high-spin heme a

can be re-

placed by hemes o

orb

. Some bacterial oxidases have

additional hemec centers.

The superfamily of heme-copper oxidases includes

three large groups: familiesA, B, and C [8]. Structur-

al modeling and comparative analysis of genomes have

identified genes of five new families of terminal oxidases

(D, E, F, G, and H) in archaebacteria [5]. FamilyA has

the largest number of representatives and contains the

most studied enzymes, including cytochrome oxidases

(COXs) from the bovine cardiac muscle mitochondria

and yeast, aa

-type COXs from Paracoccus denitrificans

and Rhodobacter sphaeroides, caa

oxidase from Thermus

thermophiles, and bo-type quinol oxidase from Esche-

richia coli [9-17]. A typical representative of the much

less studied familyB is ba

-type COX from T. thermo-

philus, which shows low amino acid sequence homology

with family A enzymes [18, 19]. The most evolutionary

distant family C includes cbb

oxidases from various or-

ganisms [20,21].

COX generates the PMF due to the vectorial (across

the membrane) delivery of electrons and protons neces-

sary for the catalytic reduction of oxygen to water in the

BNC. The so-called chemical (or substrate) protons are

transferred to the BNC from the internal aqueous phase

(negatively charged N side of the membrane), while elec-

trons are transferred from cytochromec from the posi-

tive outer side of the membrane (P side) (Fig. 1, [22]).

COX also transfers an average of 4 protons (pumped pro-

tons) from the N side to the P side per each reduced oxy-

gen molecule, i.e., functions as a proton pump [23, 24].

Therefore, the reaction catalyzed by COX is associated

with a charge separation corresponding to the directed

transmembrane transfer of 8 charges per each reduced

oxygen molecule and can be described by equation(1):

4 c

+O

+8H

→4c

+2H

O+4H

, (1)

where n is the mitochondrial matrix and p is the inter-

membrane space.

Generation of the membrane potential in a station-

ary mode (i.e., with multiple enzyme turnovers) by the

mitochondrial COX integrated into proteoliposomes was

demonstrated in the laboratory of Drachev in the early

experiments on the development of direct electrometric

method [25]. However, these studies failed to reveal the

electrogenic mechanism of membrane potential genera-

tion and formation of ΔμH

in the enzyme catalytic cycle.

Later advancements in the development of direct elec-

trometric method with the time resolution adequate for

studying the events of the catalytic cycle of photoactive

proteins [26] have been successfully used to investigate

the activity mechanisms of heme-copper oxidases. Since

these enzymes are not photoactivatable proteins, a new

methodological approach has been created to study their

electrogenic mechanisms with a high time resolution

(see below) based on a combination of direct electrom-

etry and special photoactivatable chemical reactions in-

duced by nanosecond laser pulses to trigger electron and

proton transfer in COX. This method allows to obtain

unique information on the structure and functioning

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BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

of the redox-dependent COX proton pump, as well as

on the coupling mechanism and stages of proton trans-

location in the protein in real time (prestationary mode)

[27-31].

THREE-DIMENSIONAL STRUCTURE OF COX

X-ray diffraction analysis has been used to estab-

lish the three-dimensional structures of several typical

heme-copper terminal oxidases of the A family, including

COX from the bovine heart mitochondria [32-35], bac-

terial COXs of the aa

type from P. denitrificans [36-38]

and R. sphaeroides [39, 40], and quinol oxidase bo

from

E. coli [41] and COX caa

from T. thermophiles [42].

Atomic structures have also been obtained for the mem-

bers of the B and C families, such as cytochrome ba

from T. thermophilus [19, 43] and COX cbb

from Pseudo-

monas shutzeri [21], respectively.

Heme-copper oxidases are characterized by the

largest central subunitI consisting of 12 transmembrane

α-helices. While bacterial COXs contain no more than

4subunits, COX from mammalian mitochondria is com-

posed of 13 subunits; its molecular weight (~200 kDa)

is approximately twice as large as that of bacterial en-

zymes. The three largest subunits of mitochondrial COX

are encoded by the mitochondrial genome. They form

the catalytic core of the enzyme and are homologous

to the three main subunits found in most typical pro-

karyotic family A aa

type COXs of the superfamily of

heme-copper oxidases.

All family A COXs contain two potential proton

pathways (from the internal aqueous phase towards the

BNC) that involve conserved proton-exchanging amino

acid residues and bound water molecules [19, 32, 36,

39, 42]. Proton pathway D (D channel) [30-32] leads

from the D132 residue through a chain of water mole-

cules and amino acid residues bound by hydrogen bonds

to the conserved E286 residue located 10-12 Å from

the BNC and 24-26 Å from D132 [24-26]. Water mole-

cules in the channel are stabilized through the hydrogen

bonds formed with highly conserved hydrophilic amino

acid residues (N139, N121, N207, S142, S200, S201, and

S197) [33]. Proton pathwayK (K channel) [44-46] is lo-

cated directly below the BNC and leads from the protein

surface on the membrane N side through T352, T359,

K362, and bound water to Y288 located near the BNC.

The entrance to the K channel is formed with the in-

volvement of the E101 residue of the second subunit I

[38, 47]. The chain of hydrogen bonds is interrupted be-

tween the K362 and T359 residues. It is believed that

changes in the K362 conformation restore the chain of

hydrogen bonds, ensuring controlled proton transfer

[38,48].

The hydrophilic domain, which is located above

the heme groups and Cu

, can theoretically provide the

exit of the pumped proton from the membrane outer

side to the aqueous phase [49]. This domain contains

a cluster of negatively charged amino acids (including

conserved D399 and D404 residues), several arginine

residues, propionate substituents of the heme, water

molecules, and bound redox-inactive metal atom (Mg

in the mitochondrial COX that is partially replaced by

in prokaryotic enzymes) [38]. The transfer of mo-

lecular oxygen in the BNC toward the active site occurs

through several hydrophobic channels in the middle part

of the membrane bilayer [32, 39, 50, 51]. The release of

water molecules formed in the BNC presumably takes

place at the membrane P side, above the heme groups,

in the area of contact between subunitsI andII, near the

/Mn

site [52].

COX CATALYTIC CYCLE INTERMEDIATES

The catalytic cycle of family A COXs is character-

ized by two phases, namely, oxidation and reduction

half-reactions (Fig. 2), each including two single-elec-

tron transitions [53]. During the reduction phase (O → E

and E → R transitions), the first two electrons are trans-

ferred to the BNC; as a result, it acquires the ability to

bind molecular oxygen. The oxidative phase begins with

the interaction of reduced BNC (stateR; Fig. 2) with an

oxygen molecule with the formation of primary diatom-

ic oxygen adduct (state A; not shown in Fig. 2) [54].

The oxygen molecule is transferred through Cu

to the

central iron atom of the high-spin heme a

with the gen-

eration of the oxycomplex [55], which is a mixture of

the Fe

–O

and Fe

–O

–

states [56]. At the next stage,

the interatomic O–O bond is broken and the P

state is

formed (Fig. 2), which requires the transfer to O

of at

least one proton and four electrons from the active site

[57,58]. Two electrons are supplied during oxidation of

the heme a

ion to the oxoferryl state Fe

2−

One electron comes from Cu

that is oxidized to Cu

The fourth electron and the proton come from the closely

located conserved Y288 residue [59] that forms a cova-

lent bond with the histidine ligand of Cu

[33, 60, 61].

By donating an electron and a proton, Y288 is converted

into a radical [62].

Single-electron reduction of the P

intermediate

converts COX to the F state (transfer of the third electron

to Y288). Oxidation of the fourth cytochromec molecule

and transfer of the fourth electron to the BNC completes

the catalytic cycle with the formation of fully oxidized

state (Fig. 2). In the absence of electron donors, the

oxidized “unrelaxed” O

state spontaneously converts to

the oxidized stable state(O) within a few seconds. The O

and O

states differ in the electron affinity of their redox

centers, as well as in the ability for the transmembrane

proton transfer [11, 29, 63-66]. Using the treatment

with hydrogen peroxide or carbon monoxide, almost

SILETSKY1516

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 2. Catalytic cycle of familyA COX enzymes. Transitions R→O

and O

→R form, respectively, the oxidative and reduction phases of the

catalytic cycle. During the R→P

transition (red arrow), O

molecule binds to hemea

of the BNC in the completely reduced Fe

Cu

state,

the O–O bond breaks, and the hemea

oxoferryl complex Fe

2−

and Cu

-O

–

is formed. Transitions P

→F, F→O

, O

→E

, and E

→R

(blue and blue-green arrows) involve sequential transfer of four electrons into the BNC and pumping of protons.

entire enzyme population can be transferred to the sta-

tionary states P

or F that correspond to the partial re-

duction of molecular oxygen in the BNC with two or

three electrons.

TIME-RESOLVED METHODS

FOR STUDYING CHARGE TRANSFER IN COX

In direct electrometry, proteoliposomes with a

protein under study are attached to an artificial mac-

roscopic membrane (collodium film impregnated with

a phospholipid solution in decane) and the transmem-

brane potential generation kinetics is recorded with a

submicrosecond time resolution using macroscopic AgCl

electrodes. The difference of the transmembrane elec-

trical potentials (ΔΨ) on the measured macroscopic

membrane increases proportionally to the generation

of electrical potential difference on the proteoliposome

membrane, which makes it possible to monitor the ki-

netics of electrogenic translocation of charges in the

studied protein with an adequate time resolution in the

prestationary mode [26, 67, 68].

The catalytic cycle of COX occurs on a millisec-

ond time scale and involves a sequence of intermediate

states of the oxygen-reducing BNC, as well as individual

electron and proton transfer events within transitions be-

tween the BNC intermediate states. All of these reactions

occur faster than the time resolution limits of fast mix-

ing methods. To study the catalytic cycle of COX with

a sufficient time resolution, oxidation and reduction re-

actions were induced with a nanosecond laser flash that

synchronizes the entire ensemble of enzyme molecules

(i.e., triggers simultaneous electron transfer by all mol-

ecules of the enzyme population). The first option (the

flow-flash method) involves studying the kinetics of ox-

idation of the completely reduced enzyme (R state) by

molecular oxygen in a single-turnover mode. To avoid

the stage of mixing with oxygen becoming the limiting

stage of the reaction, interaction with oxygen is triggered

by photolysis of the preformed COX complex with the

reduced BNC [69]. In the second case, electron is “in-

jected” using photoactivated tris(bipyridine) ruthenium

complex (Rubpy) into the enzyme that has previously

been transferred to a state characterized by a particular

extent of oxygen reduction in the BNC (O, P, or F) [70].

Although the second approach has been developed

more recently than the flow-flash method, it was the

first to be used in a combination with direct electrometry.

Itshould be noted that electron injection into COX offers

an important advantage, which is an ability to separate

the sets of elementary stages of charge transfer associated

with individual single-electron transitions in the catalyt-

ic cycle. In addition, COX oxidation by the oxygen mol-

ecule in the first approach occurs from the completely

reduced state R (in which, in addition to BNC, the input

redox centers are also in the reduced state), which is not

observed under normal conditions. This review presents

a detailed description of results obtained using electron

injection by the photoactivated Rubpy complex (results

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BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

of the studies using the flow-flash method can be found

in the brilliant reviews [71, 72]).

KINETICS OF MEMBRANE POTENTIAL

GENERATION BY MITOCHONDRIAL COX

For the first time, the stages of charge translocation

in the F → O transition of COX from bovine heart mito-

chondria were recorded by the joint efforts of the labo-

ratories headed by A. D. Kaulen and A. A. Konstantin-

ov [73, 74]. Electron transfer in the COX molecule was

triggered by a single electron injection from the Rubpy

complex induced with a laser flash. Later, the use of this

technique has been extended to investigation of the COX

molecules fixed in the P

state (the third electron in

the catalytic cycle; Fig. 2) and fully oxidized O state [63,

75, 76]. Several years after the time-resolved kinetics

of the membrane potential generation by COX had been

recorded at the Belozersky Institute of Physico-Chemi-

cal Biology, the Helsinki Bioenergetics Group developed

an approach that used a combination of time-resolved

electrometry and flow-flash method [77].

Electron injection into the F state (F → O

tran-

sition) allowed to resolve three main components of

the membrane potential generation by family A COXs

[73,74]. The “fast” microsecond phase (~40 μs) reflect-

ed electron transfer from Cu

to heme a. The “medium”

(~1 ms) and “slow” (~4 ms) components corresponded

to the stages of vectorial proton transfer in the enzyme

caused by the electron transfer from heme a to the BNC.

Electron transfer between heme a and BNC normally

occurs along the membrane, i.e., is non-electrogen-

ic[22]. Electron injection from Rubpy into the P state

of mitochondrial COX (P → F transition) was accompa-

nied by a set of the ΔΨ generation proton components

that had a similar amplitude, but were somewhat fast-

er [63], while photoreduction of oxidized COX (injec-

tion in the O state) was limited to the hemea reduction

by Cu

[63, 70, 76].

The total amplitudes of millisecond electrogenic

components in the kinetics of potential generation for

the P → F and F → O stages were similar (~80%), which

led to the initial estimate of ~3 protons pumped through

the membrane. Or ~1.5 pumped protons at each of the

single-electron stages of the oxidative phase of the cata-

lytic cycle [63, 73]. The amplitudes in the presteady-state

measurements were estimated using the effect of mem-

brane potential on the steady-state redox equilibrium

between cytochrome c and heme a [78]. Based on this

effect, the transfer of an electron from Cu

to heme a

(20% of photoelectric response; the “fast” component) is

equivalent to the translocation of an elementary charge

by ½ the value of the membrane dielectric barrier. Hence,

the total amplitude of the millisecond proton phases

(80%) is equivalent to the transmembrane transloca-

tion of two total charges, which would correspond to the

transfer from the internal aqueous phase to the BNC of

one proton by ½ of membrane thickness for protonation

of the reduced oxygen atom (“substrate” proton) and

coupled translocation of two “pumped” protons: one

through the entire membrane and one more through ½

of its thickness [63, 73]. This assessment was consistent

with the earlier conclusions obtained in the studies of the

steady-state quasi-equilibrium reversal of the COX-cat-

alyzed reaction in the coupled mitochondria. It was as-

sumed that all four pumped protons can be transferred

through the membrane during the catalytic cycle oxida-

tive phase [79]. It was assumed that the reducing stage

of the photocycle does not possess a sufficient supply of

free energy and is not associated with the pumping of

protons, which also followed from relatively low values

of the BNC redox potentials under stationary conditions

(in the O state) [80].

KINETICS OF MEMBRANE POTENTIAL

GENERATION BY FAMILYA

BACTERIAL COX ENZYMES

The use of direct electrometric method for studying

the mechanism of COX activity has received further de-

velopment due to site-directed mutagenesis of family A

bacterial COXs, which are homologous to mitochondrial

COXs. The most studied enzymes of this group are bac-

terial COXs of the aa

type from R. sphaeroides [15, 28,

30, 31, 45, 81, 82] and P. denitrificans [76, 83-86].

The studies of the F → O transition in the mutant

-type COX from R. sphaeroides with the uncoupled

phenotype (N139D substitution [30, 87, 88]) have re-

sulted in the overestimation of the electrogenicity of

the single-electron transitions in the oxidative phase of

the COX catalytic cycle. The enzyme with the N139D

mutation in the D channel fully retained its oxygen re-

ductase activity in the steady-state measurements, but

lost the ability to acidify the outer medium in the prote-

oliposome suspension (i.e., to pump protons across the

membrane in the steady-state measurements). Gener-

ation of the membrane potential at the F → O stage in

response to the electron injection in the mutant enzyme

reflected translocation of one elementary charge through

the membrane, namely, the transfer of an electron and

a proton from the opposite sides of the membrane to

the heme a

oxoferryl complex [30].

The ratio of the amplitudes of electrogenic compo-

nents of the electron transfer from the outer side of the

membrane [30, 31] and of the substrate proton trans-

fer from the internal aqueous phase to the BNC in the

N139D mutant allowed to estimate the electrogenic dis-

tance of heme a and BNC from the external aqueous

phase (~0.4 of the membrane dielectric thickness), which

was in good agreement with the structural data [32,36].

SILETSKY1518

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Accordingly, the relative amplitude of the electrogenic

phase, which reflects the transfer of the substrate proton

in the N139D mutant from the internal aqueous phase to

the BNC, was ~0.6 of the membrane dielectric thickness.

The total amplitude of the “medium” and “slow” elec-

trogenic phases in the P → F and F → O transitions in

the wild-type COX and mitochondrial COX is ~4 times

larger than the amplitude of the electrogenic phase of

electron transfer from the outer side of the membrane to

heme a [31, 63, 73], which is equivalent to the transfer of

~1.6 positive charge through the entire thickness of the

membrane dielectric. That is, in addition to the transfer

of one substrate proton from the internal aqueous phase

to the BNC (~0.6 of the membrane dielectric thickness),

additional electrogenic contribution to the single-elec-

tron transition in the wild-type COX, compared to the

N139D mutant, corresponds to the transfer of approx-

imately one proton through the membrane. In other

words, both single-electron transitions in the oxidative

phase of the COX catalytic cycle are associated with the

transfer of one substrate proton to the BNC and pump-

ing of no more than one proton through the membrane.

Similar estimates were obtained for the P → F and F → O

transitions in the reaction of fully reduced COX with ox-

ygen using the electrometric method [89] and by mea-

suring the protonation kinetics of a pH indicator [90].

Independently, Verkhovsky et al. [53] from the Hel-

sinki Bioenergetics Group demonstrated that the total

membrane potential generation in the reducing half-re-

action of the COX catalytic cycle can involve not only

the transfer of protons to the BNC, but also transmem-

brane pumping of protons. Later, the same group used a

variant of the flow-flash method to oxidize fully reduced

COX with an O

molecule in a single-turn mode followed

by the electron injection from Rubpy. This approach al-

lowed to study the kinetics of membrane potential gen-

eration associated with the single-electron reduction of

the metastable oxidized state O

of cytochrome oxidase.

It was shown with time resolution [85,86] that rapid oxi-

dation of completely reduced COX was immediately fol-

lowed by the short-lived oxidized state O

characterized

by a much more positive redox potential of BNC than the

O state. In contrast to the O state, electron injection into

COX in the O

state was accompanied by rapid electron

transfer to the BNC and associated stages of electrogen-

ic proton translocation [65, 85, 86, 91, 92]. The charac-

teristics of the electrogenic phases in the single-electron

→ E

transition [85, 86] generally resembled those

for the transitions in the oxidative phase and are consis-

tent with the transmembrane transfer of a single proton.

Proton pumping during the O

→ E

transition was also

confirmed in the case of natural (and not artificial, like

Rubpy) electron donor in the study of family A (subfam-

ily A2) heme-copper oxidase caa

from T. thermophilus,

which has an additional redox center (cytochrome c)

and, accordingly, five electrons in a completely reduced

state [11]. When the fully reduced caa

oxidase from

T. thermophilus was oxidized by an oxygen molecule in

the single-turnover mode, the final transition was the

→ E

transition [92]. The final electron acceptor in

the O

→ E

transition of this enzyme, as in the case of

the artificial electron donor Rubpy, was Cu

The E

→ R transition is presumably associated with

the pumping of one proton. However, the stage of the

second electron transfer in the COX catalytic cycle still

remains the least studied process, since it was not pos-

sible to obtain a homogeneous population of enzyme

molecules in the single-electron state (E

). This is pri-

marily due to the difficulty of fixing the enzyme in the

state and existence of multiple possible states of the

single-electron (E and E

) and oxidized (O and O

) en-

zyme differing in their functional properties. Treating

COX in the Fstate with carbon monoxide under steady-

state conditions converted it to the E state, which has

one electron equivalent more than the oxidized stateO.

Electron injection from Rubpy into this state led to a

photoelectric response similar in its characteristics to

the F → O transition [84]. However, electron distribution

between the redox centers in the resulting E state dif-

fered significantly from the distribution observed for the

state formed by the electron injection from Rubpy

into COX in the O

state [65, 86, 92].

FUNCTIONAL ROLE

OF PROTON-CONDUCTING PATHWAYS

IN THE CATALYTIC CYCLE

OF FAMILYA COX ENZYMES

The studies on the effect of mutations of proton-ex-

changing groups on the functional characteristics of the

coupled proton transfer demonstrated that electrogenic

translocation of protons by familyA COX enzymes oc-

curs through the proton-conducting structures (“chan-

nels” D and K) containing critical conserved proton-ex-

change residues [30, 31, 45, 81]. However, participation

of these channels in the conduction of different protons

at the individual stages of the catalytic cycle is organized

in a non-trivial way. Based on the effect of mutations of

particular amino acid residues [93, 94] and the three-

dimensional structure of COX [32, 36], it was assumed

that these two channels are specialized for conducting

substrate protons (K channel) and protons pumped

through the membrane (D channel).

If the D and K channels were responsible for the

transfer of protons of different types, then mutations

blocking their activity should have inhibited the F → O

transition. However, while amino acid substitutions in

the D channel suppressed the millisecond components

of the potential generation and, therefore, electrogenic

proton transfer at the F → O stage, the blockade of the

K channel did not inhibit electrogenic proton transfer

MECHANISM OF MEMBRANE POTENTIAL GENERATION 1519

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

at this stage [30, 31, 45, 81]. Moreover, in the presence

of excess respiration substrates, i.e., when the influx of

electrons was not the reaction rate-limiting stage, reduc-

tion of the BNC (O → R transition) in the K-channel

mutants was hindered due to the blockade of proton cap-

ture from the matrix into the BNC during the reducing

phase of the catalytic cycle. It was concluded that the

D and K channels differ not in the nature of conduct-

ed protons (substrate or pumped), but in the type of the

catalytic cycle half-reaction they serve [45, 81, 85].

Later experimental results indicated that the K

channel does not appear to be involved in the transfer of

pumped protons at all, while the D channel provides the

transfer of substrate protons in the oxidative part of the

cycle and all pumped protons in the catalytic cycle (as

in oxidative phase and in the reducing phase). Indeed,

if the transfer of pumped protons occurred through the

K channel, then the N139D mutation in the D chan-

nel (which preserves the oxygen reductase function, but

completely inhibits proton pumping through the mem-

brane [30,87]) should not affect the transfer of pumped

protons in the reducing phase of the catalytic cycle.

In accordance with this, no mutations in the K channel

have been identified that specifically inhibited the pump-

ing of protons across the membrane. It is currently ac-

cepted that the K channel transfers one or both substrate

protons during the reducing part of the cycle (Fig. 2) [12,

85, 95]. It was found that the hydrogen bond between

the hydroxyl group of the heme a

farnesyl substituent

and redox-active tyrosine residue (Y288), which is pres-

ent in the oxidized COX, was absent in the crystals of

reduced COX form [40] and was replaced by water mole-

cules (i.e., this bond can act as a “latch” in the upper part

of the K channel).

The signal to turn off the K channel can be forma-

tion of the oxoferryl state of heme a

during generation

of intermediate P in the A → P transition. The resulting

oxene state of the oxygen atom (as a strong axial ligand

of heme iron) can cause changes in the enzyme con-

formation, similar to the R → T transition in hemoglo-

bin [45]. A slight shift of charges inside the K channel,

which is closed in its upper part, can also occur during

the oxidative half-reaction of the catalytic cycle [31, 82].

The role of such charge shift inside the channel is not

clear; for example, it may lead to a decrease in the en-

ergy barrier for the electron transfer reactions inside the

COX hydrophobic core.

Beside the D and K channels, mitochondrial COX

might have the third proton pathway, the H channel,

whose role in the conduction of protons remains a mat-

ter of debates [34, 96]. According to the authors of the

study on the X-ray structural analysis of mammalian

COXs [34, 35, 96], who identified this hypothetical pro-

ton pathway, the H channel conducts pumped protons

into COX from the N side of the membrane through

the vicinity of heme a to the D51 residue located near

the P side of the membrane. The function of this chan-

nel and regulation of proton translocation through it, in-

cluding physiological aspects of this regulation, have been

poorly studied [14]. It was suggested that the H channel

is involved in the conduction of pumped protons across

the entire membrane [97]; according to other hypoth-

esis, only the upper part of this pathway is used [98].

Finally, the third hypothesis states that the H channel is

not involved in the proton pumping, but represents the

so-called dielectric “well”, whose role may be modula-

tion of the redox potential of the nearby electron trans-

fer, hemea [99, 100].

There are indications that mutations in the H chan-

nel inhibit proton translocation by the mitochondrial

COX [34, 96]. However, targeted mutagenesis of enzymes

from higher eukaryotes is technically difficult, which

complicates interpretation of the obtained results [101].

At the same time, the exit portion of the proton H chan-

nel, whose conformational changes are the key element

of the alternative hypothetical mechanism of proton

pumping by the mitochondrial COX, is not conserved

in homologous oxidases from bacteria [102, 103]. More-

over, mutations of homologous residues in the H chan-

nels of bacterial aa

-type COXs do not inhibit these

enzymes [103, 104]. The studies of COX proteins from

lower eukaryotes (yeast) do not support involvement of

the H channel in proton pumping [16].

MECHANISM OF MEMBRANE

POTENTIAL GENERATION DURING

THE SINGLE-ELECTRON TRANSITION F → O

Studying the effects of isotope substitution and zinc

ions has been instrumental in the identification of elec-

trogenic processes underlying the “medium” and “slow”

phases of the membrane potential generation kinetics

during the F → O transition. The effects of isotope sub-

stitution differ significantly between the electrogenic

phases, which allowed to identify analogs of these phases

in COX mutants and to correlate them with the transfer

of different types of protons [30]. The “medium” phase

was absent in the uncoupled mutant with the N139D

substitution in the D channel, which retained the oxy-

gen reductase activity but was unable to pump protons

through the membrane. This suggests the association of

the “medium” phase with the transfer of pumped pro-

ton, as well as that the pumped proton is translocated

before the substrate one. Accordingly, the “slow” phase,

which was retained in the mutant enzyme, is interpreted

as the substrate proton transfer to the BNC.

In the presence of zinc ions (proton transport in-

hibitors) added externally to proteoliposomes with the

mitochondrial COX or wild-type cytochrome aa

oxi-

dase from R. sphaeroides [15, 105], the “slow” electro-

genic phase decelerated. No such effect was observed

SILETSKY1520

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

for the “medium” electrogenic phase. Zinc ions also

had no impact on the protonic electrogenic phase in the

uncoupled N139D mutant. Taken together, these data

indicate that the “slow” electrogenic phase in the wild-

type enzyme includes a stage of the pumped proton re-

lease to the membrane outer side and that this process

is likely the rate-limiting reaction for the entire “slow”

electrogenic phase [15]. In contrast to the proton entry

pathways, the trajectory of proton exit to the outer mem-

brane has been poorly studied. There are indications that

the exit of water molecules is organized in a form of dis-

crete trajectory through two channel-like structures that

can also be involved in conducting the pumped proton

to the outer side of the membrane [52, 106]. Studying

the effects of different zinc concentrations on the ki-

netics of membrane potential generation in proteolipo-

somes with membrane-incorporated cytochrome aa

oxidase from R.sphaeroides indicated the presence of at

least two separate effective binding sites for Zn

ions on

the P side of the membrane. It was also shown that the

release of the pumped proton from the proton loading

site (PLS) of COX into the external aqueous phase can

occur along several trajectories [15].

Analysis of the “medium” and “slow” phases us-

ing sequential reaction model revealed that the values of

their relative amplitudes were close [107, 108]. Thus, the

relative amplitudes of the “medium” and “slow” elec-

trogenic protonic phases for the F → O transition in the

mitochondrial enzyme were ~1.9-2.1 of the value for the

“fast” electrogenic phase, i.e., which together corre-

sponded to the transfer of two protons through most of

the membrane dielectric [108]. As mentioned above, the

D channel ensures the transfer of both pumped and sub-

strate protons during transitions of the oxidative phase

of the COX catalytic cycle [45, 81]. Therefore, the “me-

dium” and “slow” electrogenic protonic phases can be

interpreted as two sequential identical processes of re-

protonation of some key residue located in the vicinity

of the BNC in the D channel, with a minor contribu-

tion from other coupled charge transitions in the protein

[107, 109, 110].

The conserved E286 residue located in the upper

part of the D channel serves as an intermediate proton

donor that ensures conduction of the pumped and sub-

strate protons (Fig.3). E286 is a bifurcation point in the

translocation of protons through the D channel that can

occur either along the substrate proton pathway to the

heme a

/Cu

center or to the outer side of the mem-

brane. Replacement of E286 with a non-protonatable

analog (E286Q) inhibited the transfer of both pumped

and substrate protons in the single-electron F → O tran-

sition [45, 81]. Experiments with the enzyme carrying

the N139L mutation, which blocks the entrance to the

D channel, allowed to identify the electrogenic stage of

proton transfer from the putative primary proton donor

(residue E286) to the BNC and to establish the electro-

genic distance between them (~0.15 of thickness of the

membrane dielectric) [31]. This is in good agreement

with the structural data, according to which the distance

from the internal aqueous phase to E286 is ~0.56 of the

geometric thickness of the membrane [107]. In the case

of proton deficiency in the D channel (deprotonated

E286), the role of the proton donor for the BNC can be

performed by the Y35 residue in the middle part of the

channel [27].

Single-electron transition F → O in family A heme-

copper oxidases upon electron injection from Rubpy

begins with the reduction of Cu

and electrogenic trans-

fer of electron from Cu

to heme a (“fast” component

of the ΔΨ generation kinetics) [17] (Fig. 3). A possible

shift of the positive charge within the gated K channel

in response to the heme a reduction may facilitate this

process (step 2′ in Fig. 3) [31]. Electron transfer from

hemea to heme a

2–

occurs along the membrane

plane in almost electrically neutral manner. Proton

translocation begins with the transfer of the pumped

proton from E286 to the proton “trap” (PLS) located

upstream of the BNC. In different family A heme-cop-

per oxidases, heme a

propionate A and/or one of the

histidine ligands of Cu

can be considered as the PLS

[111, 112]. There are indications that the role of PLS can

belong to a cluster of proton-exchange groups, includ-

ing propionates A and D of heme a

and nearby residues

(D52 and K171 in mitochondrial COX) [113], or by the

hydrophilic domain located above hemea [98].

In the F → O transition, the substrate proton is also

transferred from E286 directly to the BNC, and E286

is reprotonated twice through the D channel during the

entire transition. The “medium” electrogenic phase in-

cludes electrogenic proton transfer from E286 to the

primary proton acceptor (PLS), as well as one of the

successive electrogenic reprotonations of E286 from

the internal aqueous N phase through the D channel

(Fig. 3). The “slow” electrogenic phase involves the sec-

ond reprotonation of E286 following the transfer from

E286–COOH to the BNC of the proton involved in the

oxygen chemical conversion.

There are indications that the proton transfer

from E286 to the BNC (stage 5) is a part of the “slow”

(in R. sphaeroides COX) or “medium” (in mitochon-

drial COX) electrogenic phases [28]. The transfer of the

substrate proton to the BNC leads to the neutralization

of transferred electron, the negative charge of which

could stabilize the pumped proton located in the PLS.

The transfer of a new proton deep into the membrane

dielectric upon E286 reprotonation should lead to the

electrostatic expulsion of the pumped proton from

thePLS [112, 114, 115]. Accordingly, the “slow” electro-

genic phase also involves proton release from the PLS

to the outer side of the membrane, which can be slowed

down by the addition of zinc ions from the P side of the

membrane (Fig.3).

MECHANISM OF MEMBRANE POTENTIAL GENERATION 1521

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 3. The figure shows the electrogenic stages resolved by single-electron photoinjection using Rubpy in the F → O transition (transfer of the

fourth electron in the COX catalytic cycle). Before the reaction begins, hemea

is in the oxoferryl state (not shown). Red arrows indicate direc-

tions of electron transfer; blue arrows indicate the stages of proton translocation. Two proton pathways (D and K channels) and their conserved

amino acid residues are shown. Steps1 and3 reflect electron transfer from Cu

to hemea and from hemea to the BNC, respectively. During the

F → O transition, both protons (substrate and pumped) are transferred through the D channel. It is assumed that isomerization of E286 (transition

between the E286

and E286

conformations) precedes proton transfer from E286 to the PLS. Individual stages of proton transfer are components

of the “medium” (2 and 4) or the “slow” (6 and 7) electrogenic phases. An additional pathway for the pumped proton release from the PLS

(step8) was revealed in the presence of zinc ions [15]. Proton transfer from E286 to the BNC (step5) is presumably a component of the “medium”

(in mitochondrial COX) or “slow” (in R.sphaeroides COX) electrogenic phases. Stage 2′ is a possible charge shift in the K channel in response to

the hemea reduction [31].

THE USE OF DIRECT ELECTROMETRIC

METHOD IN THE STUDIES OF FAMILYB

HEME-COPPER OXIDASES

To understand the mechanism of coupled proton

pumping by COX, it is important to study the diversity of

heme-copper terminal oxidases [8], including enzymes

of much less investigated and evolutionarily distant

families B and C [19, 116, 117]. Although the studies of

family B and C oxidases have started relatively recently,

they have attracted a lot interest [29, 95, 118, 119]. Toa

great extent this is due to the common occurrence of

these enzymes (along with copper-free bd-type oxidases)

in pathogenic microorganisms and, therefore, their bio-

medical significance [120].

The use of direct electrometric method allowed

to discover an interesting feature of the BNC from the

-type COX from T. thermophilus, a typical repre-

sentative of family B [121]. In the initial oxidized state,

this enzyme does not react with exogenous ligands and,

therefore, cannot be converted and stabilized in the P or F

states by the treatment with hydrogen peroxide or carbon

monoxide [121], as it was done for typical aa

-type oxi-

dases [63]. However, when an electron from Rubpy was

injected into the fully oxidized (state O) COX ba

from

T. thermophilus in the presence of hydrogen peroxide, an

additional electrogenic reaction was observed, the rate

of which was directly proportional to the concentration

of the added peroxide. In other words, the BNC of this

enzyme, which was inactive in the oxidized state toward

external ligands, acquired the ability to bind ligands (and

transit to the active/open state) in response to the sin-

gle-electron injection and reduction of hemeb [121].

An important aspect in the context of uniqueness

and usefulness of direct electrometric method in the

studies of the ba

-type COX from T. thermophilus is that

the average parameters of the catalytic cycle of this en-

zyme assessed by the degree of acidification of the outer

compartment of proteoliposomes in the stationary oxy-

gen reductase reaction, indicated the variability in the

stoichiometry of proton pumping. Unlike family A oxi-

dases, the stoichiometry of proton pumping by family B

and C oxidases in the stationary measurements can vary

within a significant range: from 0.5 to ~0.85 protons per

electron (H

–

) entering the BNC [66, 118, 121-124].

Application of direct electrometric method makes it

possible to obtain and compare electrogenic parameters

of individual single-electron transitions and to obtain

SILETSKY1522

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

unique information about the features of the coupling

mechanism.

In the ba

-type COX from T. thermophilus (a typical

representative of family B proteins), the stages of elec-

trogenic proton transfer are resolved during the oxida-

tive phase [122] and the first transition of the reducing

phase of the catalytic cycle [66]. The stages during which

electron transfer is not associated with the proton pump-

ing across the membrane have been identified, which

explained the decrease in the effective stoichiometry of

proton pumping and its significant variability in family B

oxidases [66,122]. In particular, it was shown that in con-

trast to family A enzymes, one proton is pumped through

the membrane instead of two in the oxidative phase of

the catalytic cycle of ba

-type COX from T. thermophilus

[122]. At the same time, the transfer of the 1st electron in

the reducing phase (transition O

→ E

) is also not asso-

ciated with the proton pumping through the membrane,

which can be caused by the membrane potential forma-

tion during the oxidative phase [66].

Apparently, family B enzymes have preserved only

one functional proton entry channel, which is homolo-

gous to the K channel of familyA oxidases [5, 29]. Using

direct electrometric method, it was shown that in con-

trast to familyA enzymes, familyB oxidases use the K

channel for the transfer of both substrate and pumped

protons in the oxidative phase. The T315V mutation in

the K-channel of ba

-type COX from T. thermophilus

causes a slowdown of the F → O

transition due to the

deceleration of the substrate proton transfer to the BNC,

as well as uncoupling, i.e., complete absence of proton

pumping in the oxidative phase of the catalytic cycle [29].

Funding. The study was supported by the Russian

Science Foundation (project no.23-24-00143).

Acknowledgments. The author would like to ex-

press his deepest gratitude to the untimely deceased

A. A. Konstantinov, A. D. Kaulen, M. I. Verkhovsky,

L. A. Drachev, and V. P. Skulachev. Collaboration with

these outstanding scientists at different times has made

an indelible impression on the author. The author is

grateful to D. L. Zaslavsky, I. A. Smirnova, I. N. Be-

levich, Prof. M. Wikström and Prof. R. Gennis for col-

laboration in the studies of heme-copper oxidases by

direct electrometric method.

Ethics declarations. The author declares no conflict

of interest in financial or any other area. This article

does not contain description of studies with human par-

ticipants or animals performed by the author.

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