Download Chemiosmotic systems in bioenergetics

Bioscience Reports, Vol. 11, No. 6, 1991
Chemiosmotic Systems in Bioenergetics: H + _
cycles and Na+-cycles
Vladimir P. Skulachev
The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated
the validity of the Mitchellian chemiosmotic H + cycle concept. The circulation of H + ions was shown
to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and
performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria,
tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the
direct chemiosmotic mechanism, in which H § potential formation is a simple consequence of the
chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre
complexes.
Recent progress in the studies on chemiosmotic systems has made it possible to extend the
coupling-ion principle to an ion other than H +. It was found that, in certain bacteria, as well as in the
outer membrane of the animal cell, Na + effectively substitutes for H + as the coupling ion (the
chemiosmotic Na + cycle). A precedent is set when the Na + cycle appears to be the only mechanism of
energy production in the bacterial cell. In the more typical case, however, the H + and Na + cycles
coexist in one and the same membrane (bacteria) or in two different membranes of one and the same
cell (animals). The sets of A/~H + and A/~Na + generators as well as A~H + and A/~Na + consumers
found in different types of biomembranes, are listed and discussed.
KEY WORDS: chemiosmotic action; H + cycle; Na § cycle; coupling ion.
INTRODUCTION
Definition of Chemiosmotic Systems
The word "chemiosmotic" was invented by Peter Mitchell at the end of the 'fifties
(Mitchell and Moyle, 1958; Mitchell, 1959) to define a primary chemical action
which results in an asymmetric distribution of the products across the osmotic
(solute-impermeable) membrane barrier.
Any enzyme that catalyzes a chemiosmotic action can, in principle, be
r e g a r d e d as a n e n e r g y - t r a n s d u c i n g d e v i c e w h i c h c o n v e r t s t h e c h e m i c a l a c t i o n released energy into the osmotic potential energy of the action-product gradient.
This kind of process, in fact, predicted by Mitchell three years before publication
Department of Bioenergetics, A. N. Belozersky Laboratory of Molecular Biology and Bioorganic
Chemistry, Moscow State University, Moscow 119899, USSR.
387
0144-8463/91/1200-0387506.50/0 ~) 1991 Plenum Publishing Corporation
388
Skulachev
of his most famous Nature paper on coupling of phosphorylation to electron and
hydrogen transfer (Mitchell, 1961), is nicely illustrated by the phosphotransferase
system (PTS) which was later found in many bacterial species. The system in
question: (a) imports extracellular glucose into the bacterium; and (b) phosphorylates it using phosphoenolpyruvate (PEP) to form intracellular glucose
6-phosphate (gl-6-P). The energy difference between the high energy phosphate
bond in the substrate (PEP) and low energy phosphate bond in the product
(gl-6-P) does not dissipate as heat, but is used to accumulate sugar in the form of
gl-6-P inside the cell (Fig. 1). Thus, the PTS system solves simultaneously two
problems: (i) it carries out the uphill transport of sugar from the medium to the
cytoplasm and (ii) it initiates glycolysis by phosphorylating the sugar.
The reversibility of chemiosmotic enzyme action allows two chemical
processes, exergonic and endergonic, to be coupled to each other provided that:
(a) they are localized in one and the same membrane; and (b) a product of the
exergonic reaction serves as a substrate of the endergonic one. In 1961 Mitchell
suggested that just this mechanism is responsible for energy coupling of
respiration and ATP synthesis (Mitchell, 1961). A slightly modified version of
such a coupling is given in Fig. 2.
It was assumed (Fig. 2A) that a hydrogen donor AH2 is oxidized by a
membrane-linked respiratory chain electron carrier on the outer surface of a
membrane. As a result, the oxidized donor (A) and reduced electron carrier are
formed. Two H + ions, which appear when two electrons are removed from AH2,
are released to the outer bulk water space. At the next step, the reduced electron
carrier is oxidized by a hydrogen acceptor B on the opposite (inner) membrane
surface, and B 2- is formed. The B 2- binds two H + ions originating from the inner
water bulk phase, giving BH2. Thus, a transmembrane electrochemical H +
periplasm
glucose
~
~
p cytoplasm
/ ~ / j
pgruvate
~
gl- G-P
Fig. 1 An example of the chemiosmotic process--glucose
transport into the bacterial cell by the phosphotransferase
system (PTS). Energy released in the membrane-linkedchemical reaction (glucoseout + PEPin~ gl-6-Pi. + pyruvatein) is utilized to accumulatethe product, gl-6-P, in the cytoplasm.
Chemiosmotic Bioenergetics
389
A
~,~-21t*
21"!*
i!ii!i!',l//',t!llilli/!i!illlliilli_arp
/
; 2 ~
~.,,,,.,,,' ~,, ,
,*
5B~
i,
ADPO"@03
+H=O
A / ) P - O ~ - ~ - 3-
ADPO~ HoPO? )~,,
2 "4 2/"/*
B
A + 2H +-
-~2H +
Fig. 2 Chemiosmotic coupling of oxidation and phosphorylation; the original
concrete version of the mechanism. AH2 and A, hydrogen atom donor in its
reduced and oxidized forms, respectively. B and BH2, hydrosen atom acceptor
in the oxidized and reduced forms, respectively. ADPO - PG ~- stands for ATP
(Adapted from Mitchell, 1961).
potential difference (A/~H +) arises, the interior of the vesicle being positively
charged and alkalized.
It was postulated that a reversible H+-motive ATPase was localized in the
same membrane. The enzyme in question was assumed to catalyze ATP
hydrolysis coupled to H + pumping. The specific version of this mechanism
formulated in 1961 (Mitchell, 1961) suggested that the H § ions pumped by the
ATPase originate from the water molecule which hydrolyzes the terminal
anhydride bond in ATP. Essentially, the direction of the ATPase-produced
A/~H § was assumed to be the same as the A/~H + formed by the respiratory
chain-catalyzed AH2:B oxidoreduction (Fig. 2A). This means that A/~H+ can be
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Skulachev
considered as a common product of respiration and ATP hydrolysis. The most
important consequence of such a situation is that the respiration-produced A/~H +
can, in principle, reverse the H+-motive ATPase and force the latter to synthesize
ATP from ADP and phosphate (Fig. 2B).
Two aspects of the original presentation of the chemiosmotic hypothesis
should be distinguished:
(i) The general principle of energy coupling was based on the transmembrane
circulation of an ion (H+). This principle, illustrated in Fig. 3, simplifies the initial
Mitchellian scheme to the following two statements: (a) in the membrane of a
topologically closed vesicle, there is an H § pump (A~H § generator) which uses
an energy source to carry out the uphill translocation of H § and (b) the A/~H +
formed is utilized by A/~H § consumer(s) to perform useful work.
(ii) Specific versions of the A/~H+-generating and A/~H+-consuming mechanisms
were suggested (see Fig. 2). As later mentioned by Racker (1976), an advantage
of Mitchell's (1961) presentation was that it formulated both the general idea and
some mechanistic versions of this idea so that one could attack both the principle
and the specific scheme; and this fact greatly extended the circle of bioenergeticists attempting to prove or (much more often!) to falsify the hypothesis.
Below I summarize the present state of the chemiosmotic concept in both general
and specific versions.
Verification of the Proton Cycle Principle
Three major lines of evidence should be noted in connection with the
experimental verification of the general chemiosmotic principle shown in Fig. 3.
(i) Respiratory and photosynthetic redox chain complexes as well as mitochondrial, chloroplast and some bacterial ATPases proved to be competent in the
formation of A/ill +.
H+
H+
Fig. 3 Chemiosmotic system of energy coupling: the
general principle ("the protonic cycle").
ChemiosmoticBioenergetics
391
(ii) ATP synthesis and some other kinds of membrane-linked work were shown
to be supported by an artificially-imposed A/~H §
(iii) Discharge of A/2H § produced by the H+-motive redox-chain complexes was
found to uncouple electron transfer from the performance of useful work (for
review, see Skulachev, 1988).
Thus, a consensus has been achieved that the proton cycle really operates in
mitochondria, chloroplasts and at least in certain respiring or photosynthetic
bacteria.
Such a statement does not mean that the proton cycle is the only
membrane-linked energy coupling mechanism. In fact, it has recently become
clear that in some cases Na + effectively substitutes for H + (the sodium cycle
systems). The general principle of the Na § cycle is quite similar to that of the H §
cycle. However, mechanistically, the two cycles seem to be different at least in
their &fill +- and AfiNa+-generating parts (see below).
A#H+-GENERATING SYSTEMS
In the great majority of living organisms, A/~H + generation can be described
by one of the three redox chain schemes, i.e. the cyclic photoredox chain (purple
bacteria), the non-cyclic photoredox chain of the chloroplast type (thylakoids in
chloroplasts and cyanobacteria) and the respiratory chain (mitochondria and
many respiring bacteria). Among them, the cyclic photoredox chain is the
simplest one.
Cyclic Photoredox Chain of Purple Bacteria
The General Scheme
The system in question is composed of two membrane-linked protein
complexes [the reaction centre complex and quinol: cytochrome Cz reductase
(homologous to the mitochondrial Complex III)] and two mobile electron carriers
(ubiquinone and water-soluble cytochrome c2 (Fig. 4)). The reaction starts with
photoexcitation of the bacteriochlorophyll dimer in the RC complex, which
results in its oxidation by the so-called primary (stable) electron acceptor, bound
ubiquinone or menaquinone (QA)- In this oxidoreduction, bacteriochlorophyll
monomer and bacteriopheophytin take part as intermediatory electron acceptors.
Then successive electrons are transferred to the secondary acceptor (bound
ubiquinone, QB) which, when reduced, binds two H + to form ubiquinol (stages
1-4). Reduction of photooxidized (BChl)z is carried out by cytochrome c2 (stage
10).
All these processes are catalyzed by the RC complex whereas, after release
of QH2 from the QB site, electron transfer from QH2 to cytochrome c2 occurs in
Complex III. The latter system can be described in terms of the Q-cycle scheme
suggested by Mitchell (t975a, b; 1976). It is assumed that QH2, released from the
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Skulachev
4H +
periplasm or chromatophoee interior
.OH+
Fig. 4 Cyclicphotoredox chain in the cytoplasmicor chromatophore membrane of purple
bacteria. (BChl)a and BChl, bacteriochlorophylldimer and monomer, respectively: Pheo,
bacteriopheophitin; QA, primary quinone of the RC Complex (menaquinone or CoQ); QB,
secondary quinone of the RC Complex (COO); bh and bt, high and low potential haems of
cytochrome b; FeSm, iron-sulphur cluster similar to that in mitochondrial Complex II[; c~
and c2, correspondingcytochromes.Diffusionof CoQ is shown by wavy lines.
RC complex, crosses the hydrophobic membrane core to be oxidized by the
non-haem iron protein (FeSni, or Rieske protein) localized close to the opposite
(outer) membrane surface (stages 5-7). As a result, reduced FeSta and
ubisemiquinone anion are formed. The protons removed from ubiquinol are
assumed to be released from the closest (periplasmic) water phase, whereas the
electrons pass from FeSaii to cytochrome ca and then to cytochrome c2 (stages 8
and 9). Ubisemiquinone is oxidized by the low potential haem (bL) of cytochrome
b, which reduces the high potential haem (bH) of the same cytochrome in a
transmembrane fashion (stages 11 and 12). Ubiquinone formed from ubisemiquinone diffuses back to the cytoplasmic membrane surface (stage 13) to be
reduced by QA or by bH.
It should be stressed that the arrangement of the redox centres, and the
mechanism of A # H + generation in the RC Complex, are directly proved (see
below), while the actions in Complex III are supported by several pieces of
indirect evidence. The scheme seems to be already rather well substantiated even
in the case of Complex III. (For review, see Skulachev, 1988, and Rich, in this
volume).
Mechanism of AftH + Generation by the Bacterial RC Complex
Recently a specific Mitchellian version of the redox-linked A/IH + generator
("the electron transfer half-loop") was directly proved in studies on bacterial
ChemiosmoticBioenergetics
393
photosynthetic reaction centre complexes. As early as in 1961, Mitchell indicated
that the transmembrane electron flux can be responsible for A~H + formation not
only in the respiratory but also in the photosynthetic redox chain (Mitchell,
1961). In 1966 he specified this idea in a scheme assuming that photoexcited
chlorophyll donates electrons to the primary electron acceptor in a transmembrane fashion (Mitchell, 1966).
During the last 25 years, several indirect indications were obtained that the
electron flux from excited chlorophyll to the primary acceptor crosses the
hydrophobic membrane core.
The validity of this concept was directly demonstrated when: (i) the positions
of the electron-carrying prosthetic groups in the bacterial reaction centre complex
were elucidated using X-ray techniques of atomic resolution; and (ii) the
electrogenesis of the partial electron and proton transfer reactions was measured
by a voltmeter.
According to the X-ray analysis of Deisenhofer et al. (1984, 1985a, b), the
Rhodopseudomonas viridis reaction centre complex is plugged through the
membrane in such a manner that (BChl)2, the primary electron donor, and
menaquinone, the primary stable electron acceptor are localized close to the
outer and inner surface of the cytoplasmic membrane, respectively. Intermediately electron carriers (BChl and BPheo) were shown to be arranged between
(BChl)z and MQ, being immersed into the hydrophobic membrane core. The
tetrahaem c-type cytochrome [in Rps. viridis, it mediates electron transfer from
cytochrome c2 to the photooxidized (BChl)2 ] proved to be protruding into the
periplasm.
Later, a similar arrangement of the redox centres was revealed in another
photosynthetic bacterium, Rhodobacter sphaeroides (Allen et al., 1987).
As long as (BChl)2 and quinones are localized close to the opposite
membrane surfaces, the process of electron transfer must be electrogenic, and
hence, energy conserving, provided that the electric potential difference (AU2),
due to transmembrane electron transfer, is not compensated by any other charge
displacements in the reaction centre complex. Indications that this is the case
were obtained by several indirect methods. The final proof of the electrogenic
character of the partial electron transfer reactions in the reaction centre complex
was obtained in our group when photoelectrogenesis was measured by a
voltmeter, using the Rps. viridis proteoliposome-collodion film system (Dracheva
et al., 1988). The data of this study are summarized in Fig. 5.
In this scheme, the distances between the redox groups are given according
to X-ray data. It is shown that a single turnover of the photosynthetic centre
complex is accompanied by a transmembrane charge translocation resulting from
oppositely-directed electron and proton transfers. To reach the secondary
acceptor (ubiquinone, QB) an electron and a proton cover distances equal to
7.5 nm and 3.9nm respectively (charge displacements in the direction perpendicular to the membrane plane are taken into account). The total electrogenesis
(AW formation) by the reaction centre complex is found to be composed of five
components. Four of them are due to electron transfers and one is due to proton
transfer, greatly differing in their time constants. The electron transfer steps are
as follows: (BChl)2"-'~ BPheo, BPheo--~ QA, c556---~c559. The QA---~QB electron
394
Per
alm
35%A~, T~<4Ops
t
Skulachev
BChl
1.Snm
\
10% z~u?,T 4 0 0 ~ s
3.9nm
\
cytoplasm
H+
Fig. 5 Mechanism of A/~H+ generation by the Rps. viridis
photosynthetic reaction centre complex, c554, cs56, (7559, haems of
the tetraheme cytochrome c. The distances between prostetic redox
groups are given according to the X-ray data of Deisenhofer et al.
(1984). The 9 values of corresponding electrogenic steps and their
contributions to the overall electrogenesis (AW) are from Dracheva
et al. (1988), Deprez et al., (1986) and Semenov (1991). AqJ was
measured by a voltmeter in the proteoliposome-coUodion film
system (Dracheva et al., 1988) or, in the case of (BChl)2--->BPheo
and BPheo---~QA steps, by the light gradient method (Deprez et al.,
1986).
transfer directed parallel to the m e m b r a n e plane proved to be electrically silent
whereas protonation of the reduced QB was electrogenic.
Comparing contributions of partial charge transfer steps with distances
covered by the transferred charges, we may infer a rule that the electrogenesis is
the higher, the deeper the redox groups are immersed into the membrane. For
instance, (BChl)2---~BPheo oxidoreduction (distance, 1.5nm) contributes as
much as 35% to the total A ~ , whereas c559--->(BChl)2 oxidoreduction (distance,
2.1 nm) is responsible for only 15% of &qJ. According to the X-ray data, the
former process occurs closer to the membrane core than the latter.
Chemiosmotic Bioenergetics
395
The above-mentioned rule can easily be explained by differences in the
dielectric strength which should be lower in the core of the reaction centre
complex than on its periphery. In fact, all the charged hydrophilic amino acid
residues are localized, according to the X-ray analysis, on the periphery of the
membrane, the core being composed of hydrophobic residues. Many water
molecules are seen in the peripheral parts of the complex. But, in the core there
are only two bound water molecules.
In conclusion, the study showed that energy conservation by the Rps. viridis
reaction centre complex is mainly (90%) due to the transmembrane electron flux
whereas the rest (10%) originates from the transfer of protons in the direction
opposite to that of electrons.
Non-cyclic Photoredox Chain of Chloroplasts and Cyanobacteria
In thyalkoids of chloroplasts and cyanobacteria, the photoredox chain is
relatively complicated. Its operation results (besides AfiH + generation) in
reduction of NADP § by electrons removed from water molecules. The process is
carried out by three complexes: reaction centre (RC) Complex I (Photosystem I),
reaction centre Complex II (Photosystem II), and plastoquinol-plastocyanin (PC)
reductase (Complex III), interconnected by plastoquinone and plastocyanin. The
RC Complex I is supplemented with ferredoxin and FAD-containing flavoprotein.
The water-splitting system (WSS), containing Mn 2§ is associated with RC
Complex II (Fig. 6).
2H§
AH
Fig. 6. Non-cyclicphotoredox chain in the thylakoid membrane of chloroplasts or cyanobacteria.
ChlI and Chin, chlorophylls of RC Complex I and II, respectively; PQ, plastoquinone; PC,
plastocyanin; FeSx, FeSa, FeSA, iron-sulphur clusters of the RC Complex I; Fd, ferredoxin; f,
cytochromef.
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Skulachev
Electron transfer from the chloroplyll dimer of RC Complex I to N A D P H is
most probably mediated by chlorophyll monomer, vitamin K1, a series of FeS
centres and FAD. The N A D P H that is thus produced serves as donor of the
reducing equivalents utilized, first of all, in carbohydrate synthesis (steps 1-9).
The photooxidation of (Chl)2 in RC Complex II occurs by a process similar to
that in the bacterial RC Complex i.e. via pheophytin and two bound quinones (in
this case, plastoquinones) (steps 10-12).
The Q cycle (steps 13-16, 19-21) resembles very much its bacterial
homologue. The main difference is that the electrons from the cl-type cytochrome
f reduce plastocyanin (instead of cytochrome c2 in bacteria) which transfers
electrons to the photooxidized RC Complex I chlorophyll dimer (steps 17 and 18).
As to the photooxidized (Chl)2 of RC Complex II, it is reduced by water via WSS
and a Tyr residue of RC Complex II, the water molecule being split to oxygen and
protons (steps 22 and 23).
According to Fig. 6, transfer of two electrons from H20 to N A D P H requires
two photons to be absorbed by RC Complex I and two photons by RC Complex
II, the process being coupled to translocation of 6H § through the thylakoid
membrane.
The mechanism of A~H + generation by RC Complex II seems to be
analogous to that by the RC Complex in purple bacteria since the structures of
these two complexes are very similar. RC Complex I also resembles the bacterial
RC complex but only in the initial steps of electron transfer (chlorophyll
dimer--~chlorophyll monomer---~quinone), whereas the terminal steps are catalyzed by FeS clusters absent from RC Complex II or bacterial RC Complex.
There is no doubt that in all three complexes, electrons traverse the hydrophobic
membrane core as originally postulated by Mitchell (1961). (For reviews, see
Murphy, 1986; Knaff, 1988; Skulachev, 1988; Rutherford, 1989).
The H+-motive Respiratory Chain of Mitochondria and Bacteria
Mitochondria and some aerobic bacteria possess a redox chain which is
composed of three A/~H+-generating complexes. The middle one (Complex III)
is functionally homologous to that in photosynthetic bacteria and chloroplasts
whereas the other two, NADH-quinone reductase (Complex I) and cytochrome
oxidase (Complex IV), serve as the Complex III reductant and oxidant,
respectively (Fig. 7). The respiratory Complex I resembles the terminal part of
RC Complex I in that the flavin and the series of FeS clusters are involved in
electron transfer which, however, occurs in the opposite direction [in mitochondria, from N A D H to quinone (ubiquinone), in chloroplasts, from quinol (vitamin
K1) to NADP+]. The mechanism of A/~H + generation, and even the sequence of
electron transfer steps in Complex I remain obscure. Nevertheless it is already
clear that more than one H § ion is translocated across the membrane per electron
passing through Complex I. Usually, the H§ - stoichiometry is assumed to be 2.
As to the respiratory Complex III, it is larger in mitochondria than in
bacteria and chloroplasts due to the presence of two large polypeptides, so-called
core proteins, which apparently participate in Complex III assembly and, maybe,
ChemiosmoticBioenergetics
mitoc~
' " .
.
.
.
or per
AHz
H-
NADh
~,,
~H +
Fig. 7 Respiratorychain in the inner mitochondrialmembrane or in the cytoplasmicmembrane of
some bacteria. FeSrla,lb,2,3,4, correspondingiron-sulphur clusters of Complex II; c x, c, a and a3,
haems of corresponding cytochromes; CUA and CuB, copper atoms of cytochromes a and a3,
respectively.
in its regulation. The additional domains formed by these proteins protrude into
the mitochondrial matrix. Like Complex III of the bacterial photoredox chain,
the respiratory Complex III employs ubiquinol and water-soluble cytochrome c as
mobile donor and acceptor of reducing equivalents, respectively.
The terminal steps of the respiratory chain in mitochondria and some
bacteria are catalyzed by cytochrome c oxidase (Complex IV) containing two iron
and two copper atoms directly involved in electron transfer from cytochrome c to
Oz. The H+/e - stoichiometry for Complex IV oxidizing a hydrogen donor is
equal to 2 as was originally indicated by Wikstr6m (1977). The mechanism of
A/IH + generation is yet unknown. (For reviews on the H+-motive respiratory
chain, see Wikstr6m e t a l . , 1981; Ragan, 1987; Skulachev, 1988; Van Belzen and
Albracht, 1989; Sone, 1990; Konstantinov, 1990; Wikstr6m and Babcock, 1990).
Other R e d o x Generators of AOH +
In green anaerobic bacteria, a non-cyclic photoredox chain was described
which oxidizes H2S and reduces NAD +. Apparently, it represents a version of
RC Complex ! equipped with an HzS oxidizing system, (quinone, b- and c-type
cytochromes) and a NAD+-reducing flavoprotein (reviewed in Okamura et a l . ,
1982; Skulachev, 1988). In some aerobic bacteria, e.g. in E . c o l i , quinol produced
by Complex I is oxidized by quinol oxidases with no Complex III and Complex
IV involved. Two quinol oxidases are described i.e. cytochrome o and cyto-
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Skulachev
chrome d complexes. In the former and in the latter cases, the H+/e - ratios are
assumed to be 2 and 1, respectively.
Certain anaerobic bacteria, as well as mitochondria isolated from the
parasitic helminth Ascaris, which lives under near anaerobic conditions in the
intestines of its host, are found to employ NADH---~ fumarate electron transfer to
produce A/~H +. In the Ascaris mitochondria, this is done by the usual respiratory
Complex I oxidizing N A D H by quinone (in this specific case, rhodoquinone) in
an H+-motive fashion. The resulting rhodoquinol is oxidized by fumarate, the
process being mediated by an enzyme homologous to the respiratory chain
Complex II (succinate dehydrogenase) which per se is not involved in energy
conservation [reviewed in (Takamiya et al., 1986; Skulachev, 1988)].
In fumarate-reducing anaerobic bacteria, A/2H + generation between a
substrate (e.g., formate) and quinone (usually menaquinone) is assumed to be
catalyzed by quite another protein complex containing molybdenum, FeS and a
low potential haem b as prostetic groups. The menaquinol that is formed reduces
fumarate by a respiratory Complex II analogue (Unden et al., 1980: see also for
review, Skulachev, 1988).
Anaerobic methanogenic bacteria are shown to be competent in an electron
transfer-linked A # H § generation (Miiller et al., 1987). The enzymes involved in
this process are not yet characterized.
Bacteriorhodopsin
Bacterjorhodopsin, the light-driven H + pump from Halobacterium halobium,
translocates one H + ion per photon causing the a l l - t r a n s ~ 1 3 - c i s
photoisomerization of the chromophore, retinal. At least two H+-acceptor groups
are shown to be directly involved in H § transfer by bacteriorhodopsin, namely:
(a) the Schiff base forming the link between retinal and the e-amino group of
Lys-216, and (b) the Asp-96 carboxylic group. Involvement of the Schiff base is
confirmed by many independent pieces of evidence (e.g., the electrogenic H §
transfer disappears at a pH below 3.5, the pK value of the Schiff base in the
M-intermediate of the bacteriorhodopsin photocycle). As to Asp-96, its participation in the H + transfer relay was recenty demonstrated by site-directed
mutagenesis studies (Holz et al., 1989; Drachev et al., 1989a; Butt et al., 1989;
Gerwert et al., 1989; Otto et al., 1989; Tittor et al., 1990; Engelgard et al., 1990).
Three partial electrogenic reactions have been shown to contribute to the
total electrogenesis directly measured in the proteoliposome-collodion film
system.
(i) The transfer of H + from the protonated Schiff base to the periplasm.
(ii) Reprotonation of the Schiff base by the H § ion belonging to the Asp-96
carboxylic group.
(iii) The transfer of H § from the cytoplasm to the Asp-96 carboxyl (Danshina et
al., 1991b; see also Otto et al., 1989).
The exact locations of the Schiff base and the Asp-96 carboxyl remain
unknown since the bacteriorhodopsin three-dimensional structure of atomic
ChemiosmoticBioenergetics
399
per'iplasm
H+
T=,40tu.s
=N+H
T M
1
4C%A~, ~=3ms
Asp96 - C00H
40?ooAY, T=60m,s \
--)
cytoplasm
H§
Fig. 8 Three steps of H+ transfer in bacteriorhodopsin. The v
values are given for pH 8.4, 23~ (From Danshina et al.,
1991b).
resolution is not yet done. Electron microscopy of two-dimensional bacteriorhodopsin crystals indicates that the Schiff base is localized in the middle of the
protein molecule while Asp-96 is somewhere between the Schiff base and the
cytoplasmic surface of the membrane (Henderson et al., 1990) (Fig. 9). It was
also shown that the protein regions separating the Schiff base from the
periplasmic and cytoplasmic membrane surfaces strongly differ in hydrophobicity,
which is low in the former case and high in the latter case. Above the Schiff base
(Fig. 8) there are four charged amino acids and no valine, leucine and isoleucine.
Below the Schiff base five leucines, valine and only one charged amino acid
(Asp-96) seem to be localized (Henderson et al., 1990). Thus, the dielectric
strength in regions of the outer H+-conducting pathway is high and that of the
inner pathway is low. This may explain the observation that the electrogenic
contribution of the outer H + pathway is much lower (20%) than the contribution
of the inner one (80%) (Drachev et al., 1978, 1984).
The outer and inner H + pathways were shown to operate in/~s and ms time
scales so that their contributions to the total AU2 formation can be easily
measured. On the other hand, the rate of H + transfer from the Asp-96 carboxylic
group to the Schiff base is of the same order of magnitude as the rate of
reprotonation of this carboxylate by cytoplasmic H + ions. To measure A ~
contributions of these two steps separately, one may specifically decelerate the
Asp-96 carboxylate reprotonation by decreasing the H + concentration in the
medium. Under such conditions, the two steps in question seem to contribute
almost equally to energy conservation (Danshina et al., 1991b).
A tentative scheme of the bacteriorhodopsin H + pump is shown in Fig. 9.
The following chain of events is assumed.
400
Skulachev
bP,
L
H|
=
--
-~H\
(i)
_.Z.
moo
-COOH
-COOH
M
=
=
=N /
\
-COOH
m
N(P)
0
-
|
~/NH'-
~
-
~
=I~H /
Fig. 9. A tentative scheme of the bacteriorhodopsin H § pump. bR, the bacteriorhodopsin
ground state. L, M, N(P) and O, corresponding intermediates of the photocycle. //NH\ , = N /
and =NH / , protonated Schiffbase of the all-trans retinal residue, deprotonated and protonated
Schiffbases of 13-c/s retinal residues, respectively.--COOH and --CO0 e, protonated and deprotonated Asp-96 carboxylic group, respectively. The outward hydrophilic H+-conducting pathway
(the proton well) is shaded. (Adapted from Danshina et al., 1991a).
(i) bR---~L. The all-trans---~13-cis photoisomerization of the retinal residue
results in translocation of the protonated Schiff base from a more hydrophobic to
a less hydrophobic environment inside the protein. This causes a strong shift in
the Schiff base pK i.e. from >10 to 3.5. In the L state, the Schiff base is supposed
to be situated close to the bottom of the outward H § well.
(ii) L---~ M. The proton leaves the Schiff base and appears in the periplasm, being
translocated along the outer H + pathway. When moving in this direction, the
proton meets with rather low resistance since the hydrophilic amino acid residues
dominate in this region of the protein.
(iii) M---~N. The protein changes its conformation in such a way that (a) the
Asp-96 carboxylic group moves in the direction of the Schiff base in order to
reprotonate it, and (b) a cleft is formed in the hydrophobic region below to the
carboxylic group, allowing the cytoplasmic solutes to reach this group. As a
result, the N560 intermediate (other names: P, R) is formed. In this intermediate
(a) the Schiff base is protonated, (b) the Asp-96 carboxylate is deprotonated and
(c) retinal is still in the 13-cis conformation.
(iv) N---~ O. The Asp-96 carboxylate is reprotonated by the cytoplasmic H § and
the retinal 13-cis ~ all-trans isomerization takes place.
(v) O ~ bR. The cleft disappears and the Asp-96 carboxylic group returns to its
starting position. The former event is electrogenic due to an increase in the
Chemiosmotic Bioenergetics
401
distance between the protonated Schiff base and a cytoplasmic counter-ion (under
natural conditions, CI-).
The great dielectric strength asymmetry of the two half-membrane charge
transfer pathways in bacteriorhodopsin is in contrast with the situation in the
photosynthetic reaction centre complex. In the latter case, both halves of the
membrane protein are very hydrophobic and contribute equally to AqJ formation
(as discussed above). The scheme is also in agreement with the findings that: (i)
the N intermediate decomposition (i.e. Asp-96 reprotonation) depends upon the
bulk phase pH whereas the M decomposition does not; and (ii) in Asp-96---> Asn
mutant, the M decomposition becomes pH dependent (Holz et al., 1989; Drachev
et al., 1989a; Gerwert et al., 1989; Otto et al., 1989).
An essential feature of the above scheme is that there is practically no inward
H+-conducting pathway in the "resting" bacteriorhodopsin molecule. This
pathway is organized only for a short period of time in the "working" protein at
the N and O stages. In these intermediates, no direct contact of the outer and the
inner H § pathways is assumed. Such a mechanism excludes any passive H + leak
via bacteriorhodopsin, a property which seems to be important for the protein
which can occupy up to 50% of the membrane in the halobacterial cell (Danshina
et al., 1991a).
The scheme shown in Fig. 9 differs from that suggested by Henderson et aL
(1990) who assumed that in the "resting" bacteriorhodopsin, two H§
half-channels pre-exist, being separated by the protonated Schiff base. The latter
scheme fails to answer the question of how H § is translocated through the very
hydrophobic part of the bacteriorhodopsin molecule, separating the Schiff base
and the cytoplasmic water phase, the process being assisted with the only
protolytic group i.e. Asp-96 (the inward H § pathway). If this pathway is of low
conductivity for H +, it remains obscure how it can satisfy the high rate of the
bacteriorhodopsin turnover. If it is of high I-I§ conductivity, it seems unclear how
the electric break-down of the membrane is prevented since two H § wells oppose
each other, and are connected with a proton-acceptor group, i.e. the Schiff base.
Our scheme suggests that the inner H § pathway is absent in the bacteriorhodopsin ground state and is temporarily organized at some step of the
photocycle i.e. when the N intermediate is formed. In this context, it should be
mentioned that the formation and decomposition of N were found to be
accompanied by large light-scattering changes indicating conformational transitions of the protein (Drachev et al., 1989b; Danshina et al. 1991b). Moreover,
these two stages are characterized by high activation energy, a fact consistent with
the assumption that the conformation change occurs in this part of the photocycle
(Danshina et al., 1991a).
The idea of the cleft formation in the hydrophobic region of the protein
originates from Mitchell's suggestion of a mobile membrane barrier, proposed to
explain the mechanism of operation of porters that translocate hydrophilic
metabolites (Mitchell, 1957, 1959). Recently Mitchell (1990) has presented a
specific version of the mobile membrane barrier principle. He assumes that slight
relative movement of o~-helical transmembrane columns (e.g. canting of the
402
Skulachev
columns at a small angle) may result in formation of a cleft allowing solutes to
reach the membrane core. We shall return to this intriguing idea when the
mechanism of the fatty acid-mediated uncoupling is discussed later in this paper.
H+-Motive ATPases and H+-Driven ATP Synthases
All the A/IH + generators described above utilize light energy or energy of
respiratory substrates to form a protonic potential. ATP is not involved in the
process. In some cases, however, the way from the energy source to A/~H +
appears to be more complex: the energy is first converted to ATP and only then is
utilized to generate A/~H + by a H+-motive ATPase.
A H+-motive ATPase seems to be necessary in those cases when a
A~H+-bearing membrane possesses neither a respiratory nor a photoredox chain.
From a formal standpoint, any enzyme competent in A/2H + generation at the
expense of ATP energy may be regarded as a H+-motive ATPase. The H+-driven
ATP synthase, usually employed to form ATP in a A#H+-driven fashion, can,
under certain conditions, operate in the opposite direction, hydrolyzing ATP and
generating A~H +. Therefore this enzyme is often called H+-motive ATPase. In
our opinion, however, it would be more appropriate to use such a name when
dealing with enzymes, the main biological function of which consists in ATP
hydrolysis rather than synthesis. Three types of H+-motive ATPase have been
described.
(i) In anaerobic eubacteria, there is an H+-motive ATPase closely related to the
H+-driven ATP synthases of eubacteria, mitochondria and chloroplasts i.e.
enzymes composed of typical H+-conducting (F0) and hydrolytic (F1) subcomplexes. The Streptococcus faecalis H+-motive ATPase is a well-known
example of such a system (Harold et al., 1970; Harold, 1977). In this facultative
anaerobe, the FoF1 complex functions as a H+-motive ATPase, producing AfiH +
at the expense of energy of glycolytically-formed ATP under anaerobic conditions. It seems possible that the S. faecalis ATPase had been initially employed as
a H+-driven ATP synthase before the microorganism occupied the anaerobic
niche. According to our data, the H+-motive ATPase of a strictly anaerobic
eubacterium, Lactobacillus casei is also of the FoFx type (Muntyan et al., 1990).
(ii) In plant and fungal vacuolar membranes (tonoplasts) as well as in secretory
granules, endosomes and in some other animal intracellular vesicles, a H+-motive
ATPase was found which seems to be similar to the H+-driven ATP synthase of
archaebacteria (see below). The arrangement of the enzyme resembles that of
FoF1, being composed of protonophorous and catalytic complexes (the former,
integral with the lipid membrane, and the latter peripheral). The protein
sequence is more homologous to archaebacterial FoF1 than to eubacterial,
mitochondrial or chloroplast FoF1 (Ihara and Mukohata, 1991; Mukohata et al.,
1990). The F1 part of the enzyme is found to be composed of six major subunits
(3A and 3B) showing moderate sequence homology to the eubacterial o~ and fl
subunits of F~ whereas minor subunits C, D and E have no such a homology
(Nelson et al., 1990). The F0 part includes several (probably six) 16 kDa subunits
403
Chemiosmotic Bioenergetics
(each of them represent a doubled c subunit of eubacterial Fo) and at least one
more subunit of similar molecular mass but of a different sequence (Mandel et al.,
1988, Lai et al., 1988). The function of this H+-motive ATPase consists in
pumping H + into vacuoles or other intracellular vesicles. The A/~H + produced by
this enzyme is utilized to support transport of metabolytes across tonoplast or
vesicular membranes. (For reviews, see Skulachev, 1988; Nelson, 1988).
(iii) A quite different type of H+-ATPase was described in the outer (plasma)
membrane of plant and fungal cells. The enzymes of this kind cannot be
dissociated into membrane and peripheral complexes. Instead they are composed
of a single very large (about 100 kDa) polypeptide. The structure and catalytic
mechanism of these H+-ATPases are quite similar to those of cation-transporting
ATPases such as the Na+/K+-motive ATPase or Ca+-motive ATPase. In contrast
to the FoFa-type ATPases, all of these form a phosphorylated intermediate (a
carboxylic group of an aspartatic acid residue in the polypeptide is phosphorylated by ATP). The H + translocation is accompanied by de-energization of the
phosphorylated intermediate i.e. by a strong decrease in the hydrolysis energy of
aspartyl phosphate. Such an effect is due to a conformation change in the
enzyme, designated as the E1--~ E2 transition (this is why ATPases of this type are
called EIE2 ATPases).
The bioenergetic role of the plasma membrane H+-ATPase consists: (a) in
the formation of A/ill + which is required to transport metabolytes, and (b) in the
maintenance of intracellular pH homeostasis. (For a review, see Serrano, 1988).
To conclude the section on A/ill + generating systems, one should mention
that besides the redox or hydrolytic (ATPase) H + pumps and bacteriorhodopsin,
one more mode of development of the proton potential difference has been
revealed. In some bacteria, peculiar types of fermentation have been found. The
fermentation occurs in such a way that H + ions are taken up and the end product
appears to have less negative charge than the substrate (e.g. conversion of oxalate
to formate by Oxalobacter formigenes). As a result, fermentation and the
downhill output of the product in exchange for input of the extracellular substrate
can produce ApH and A ~ , respectively (Anantharam et al., 1989). The
contribution of such processes to cell energetics remains to be elucidated.
AfiH+-CONSUMING SYSTEMS
The H+-Driven ATP Synthase
The H+-driven ATP synthase is the enzyme catalyzing the A/lH+-driven
ADP phosphorylation by inorganic phosphate in mitochondria, chloroplasts and
respiring or photosynthetic bacteria. The enzyme complex can be dissociated into
two subcomplexes, factors F0 and F1. Eubacterial factors Fo and F1 are composed
of three (a-c) and five (ol-e) types of polypeptides, respectively. The subunit
stoichiometry is shown to be 3o/: 3/3 : c : 6 : c : a:2b : (6-15)c. Mitochondrial and
chloroplast FoF1 complexes contain a larger number of subunits (13 and 9,
respectively). (For reviews, see Senior, 1990; Skulachev, 1988).
404
Skulachev
The sequence of major subunits of the archaebacterial H+-driven ATP
synthase has been shown to resemble those of the tonoplast H+-motive ATPase
rather than of eubacterial H+-driven ATP synthase (Inatomi et al., 1989; Denda
et al., 1988a, b). The number of subunits remains uncertain, for H. halobium, H.
saccharovorum, Methanosarcina barkeri and Sulfolobus acidocaldarius, different
subunit compositions have been reported (see, e.g. Nanba and Mukohata, 1987,
and Ltibben et al., 1988).
The most important observations concerning the mechanism of the H §
driven ATP synthase action may be formulated as follows.
(i) ADP and Pi bound to the FoF1 complex or isolated F1 in solution can produce
bound ATP in an energy independent fashion.
(ii) Energy in the form of A/~H + is required to transport the bound ATP from the
F1 catalytic site to water and/or to transport ADP and P~ from water to the
catalytic site.
(iii) ApH or AW produced by a A/~H + generator (or artificially imposed across
an FoFa-eontaining membrane) are equally effective in supporting the bound
ATP release.
(iv) It is F0 that is plugged through the membrane and is responsible for H +
transfer, whereas F1 protrudes from the membrane and is involved in reversible
ATP hydrolysis.
(v) In certain cases, Na + ions effectively substitute for H + ions in the H+-ATP
synthase of the FoF1 type (Skulachev, 1988; Boyer 1989; Laubinger and Dimroth,
1989).
Observations (iv) and especially (v), as well as indications that the H +/ATP
ratio may be higher than 2, seem to me to be very difficult to reconcile with a
model assuming that the H + ions transported by the H+-driven ATP synthase are
directly used to form H20 when ATP is synthesized from ADP and Pi (see above,
Fig. 2B). Rather, translocation of nH+ ions through F0 energizes the FoF1
complex in such a way that bound ATP is released from the F1 catalytic site. As
to the 2H + required to form H 2 0 from ADP and phosphate anions, they are not
involved in the transmembrane H + flux via F0 (Fig. 10). Most probably, two
processes i.e. (a) the proton translocation by F0 and (b) the bound ATP release
from F1 are conformationally coupled. (For reviews, see Senior, 1990; Boyer,
1989). This may be an example of indirect chemiosmotic energy coupling similar
to that in the transhydrogenase (see below) and distinct from direct coupling in
the RC complex, discussed above, where the energy released in the course of
chemical action (electron transfer) is immediately transduced to A/~H + without
being intermediately invested into an energized conformation of the enzyme.
The mechanism of H + translocation via F0 remains obscure, mainly due to
the absence of information on the three-dimensional structure of this complex.
This might be H + transfer relayed through protolytic groups (or of a chain of
bound water molecules) as in the outer H + pathway of bacteriorhodopsin, or the
reversible formation of a cleft in the hydrophobic F0 region similar to that
suggested for the inward bacteriorhodopsin H + pathway.
Chemiosmotic Bioenergetics
405
Fig. 10. A tentative scheme of the H+-driven
H~
ADPOH
+ HOP
ADPO'-'P
ATP synthase. It is assumed that translocation
of H § (in some bacterial ATP synthases, Na §
via F0 is conformationally coupled to the release of the bound ATP. The scheme takes into
account that (i) bound ATP is equilibrated with
bound ADP and P~ in the F1 catalytic site in an
energy independent manner so that bound
ATP should be regarded as a low energy
compound (ADPO-P) in contrast to high
energy ATP in the solution (ADPO - P). Protons, required to form H20 when ADP (in the
scheme, ADPOH) is phosphorylated by inorganic phosphate (HOP), are assumed to be not
identical to the protons translocated across the
membrane via F0.
H+-Driven Pyrophosphate Synthase
This enzyme is found in bacterial c h r o m a t o p h o r e s (Baltscheffsky et al., 1966;
Isaev et al., 1970; Kondrashin et al., 1980) and plant tonoplasts ( R e a Pa and
Poole, 1985; Dupaix et al., 1989; Sarafian and Poole, 1989). In c h r o m a t o p h o r e s ,
the enzyme can catalyze PPi formation from 2P i at the expense of A/~H §
produced by the p h o t o r e d o x chain or, alternatively by the FoF1 complex splitting
ATP. In the tonoplast, A T P hydrolysis by the H+-motive A T P a s e seems to be
the only way to form A/~H + which can be used to support the H+-driven PPi
synthase action. Nyren and Baltscheffsky (1983) succeeded in demonstrating a
protonophore-sensitive A T P synthesis from A D P and Pi, coupled to PP~
hydrolysis by proteoliposomes containing FoF1 and bacterial H+-driven pyrophosphatase. I have suggested that a function of the H§
PPi synthase m a y be to
buffer A ~ H + at a level sufficient for A T P formation (Skulachev, 1978).
The molecular properties of the H+-driven PPi synthase remain obscure.
T h e r e are some indications that 52 and 54 k D a subunits are present in the
c h r o m a t o p h o r e enzyme (Baltscheffsky and Nyren, 1987), whereas in the tonoplast a 67 k D a polypeptide has been identified (Sarafian and Poole, 1989).
Skulachev
H+-Driven Transhydrogenation
As proposed by Mitchell (1966), the energy-linked transhydrogenase, discovered by Danielson and Ernster (1963), is an additional respiratory chain
energy transducer, catalyzing the following process:
NADH + NADP + + nHout
+ ~ NAD + + NADPH + nHi+
(1)
where Uou
+ t and Hi,+ represent extra- and intra-mitochondrial H + ions, respectively. Since the redox potentials of the N A D H / N A D + and N A D P H / N A D P +
couples differ by no more than 5 mV, the AGo of the transhydrogenase action is
close to zero. To generate A/2H + in the proper direction, the transhydrogenase
must oxidize NADPH by NAD + under conditions when [NAD +] x [NADPH] >
[NADH] x [NADP+]. Simple calculation shows that the transhydrogenase could
effectively compete with the respiratory chain or ATPase for A/~H + formation
only if the [NAD +] x [NADPH] were several orders of magnitude higher than
[NADH] x [NADP+], a most unlikely situation for the living cell. However,
under anaerobic conditions and at very low [ATP], the transhydrogenase was
assumed to be competent in the generation of A/~H + in hepatocyte mitochondria
oxidizing NADPH by NAD + (Hoek and Rydstr6m, 1988).
It seems obvious that the usual direction of this process in respiring
mitochondria is opposite, i.e. NADH oxidation by NADP +. Maintaining a high
[NADPH]/[NADP +] ratio at the expense of the A/~H+ produced by the
redox-chain, the H+-driven transhydrogenase creates an additional driving force
for reductive biosynthesis which, as a rule, includes NADPH-oxidizing steps. A
high NADPH level is also favourable for cytochrome P-450-mediated detoxication of xenobiotics and SH-glutathion-mediated reduction of SS-bonds in
proteins.
The H+-driven transhydrogenase is present in mitochondria and some
bacteria which do not have a non-cyclic photoredox chain. On the other hand, it
is absent from chloroplasts and cyanobacteria where the problem of the high
[NADPH]/[NADP +] ratio is solved by reduction of NADP + as the final electron
acceptor of the redox chain.
The H+-driven transhydrogenase has been found to catalyze a direct
stereospecific transfer of hydride ion (H-) between the A position of the
nicotinamide ring in NAD and the B position of that in NADP. The H - transfer
occurs with no proton exchange with water (Lee and Ernster, 1964). This means
that the proton translocated through the membrane by the transhydrogenase is
not identical to the proton transferred from one nicotinamide nucleotide to the
other in the course of the redox reaction. When deuterated reduced nicotinamide
adenine dinucleotide phosphate (NADOD) is used as a reaction substrate, the
process is described by equation (2):
NADP +
NADPD + NAD + + nHout----~
+
NADD
+ nUin+
(2)
where Hout
+ and Hin
+ are hydrogen ions outside and inside the everted submitochondrial vesicles. Thus the H+-driven transhydrogenase exemplifies an
indirect chemiosmotic system since the A/2H § generation results from an event
ChemiosmoticBioenergetics
407
other than the energy-releasing chemical reaction per se (Mitchell, 1966). This
is in contrast to, e.g., the RC complex, where AfiH + is formed as a direct
consequence of the energy-releasing redox reaction (CoQH2, when being
oxidized, releases 2H § on one side of the membrane, whereas CoQ, when being
reduced, combines with 2H § on the other side).
The mechanism of generation and consumption of A#H § by the H+-driven
transhydrogenase remains obscure. (For details of this system, see Rydst6m et
al., 1984; Hoek and Rydstr6m, 1988). The transhydrogenase action in its usual
(NADP+-reducing) direction (see equation (1)) can be regarded as an example of
reverse transfer of reducing equivalents, which are transported from a reductant
of a higher redox potential to an oxidant of a lower redox potential.
Sometimes, the H+-driven transhydrogenase is employed by the cell jointly
with other systems of reverse electron transfer along the redox chain. This is
apparently the case in Rh. rubrum chromatophores oxidizing substrates of about
zero potential, such as sulfur. In the light, AfiH § (produced by the cyclic
photoredox chain) can be utilized: (i) to reverse NADH-CoQ reductase and,
hence, to reduce NAD § by sulfur; and (ii) to oxidize the resulting NADP + via
the H+-driven transhydrogenase (Skulachev, 1972).
Thiobacillus ferroxidans oxidizing Fe 2+ to Fe 3+ (midpoint potential +0.77 V)
by oxygen at acidic pH, has been shown to build all the components of its cell
from CO2, H20 and NH3. To do this, it is necessary to reverse not only
NADH-CoQ reductase and transhydrogenase but also CoQH2-cytochrome reductase (Skulachev, 1969; 1988).
ApiH+-Driven Osmotic Work
The term "osmotic work" refers to all the work done when a solute is
transported across a membrane from its lower to higher concentration. Such an
uphill transport is accompanied by the appearance of a transmembrane osmotic
imbalance due to changes in the concentration of the solute molecules (and in
their hydration) in at least one of the membrane-separated compartments.
In the great majority of cases, such processes are driven by A#H + (or
AfiNa +, see below), ATP or phosphoenol pyruvate energy. In the inner
mitochondrial membrane, plant or fungal plasmalemma, tonoplast, as well as in
the membrane of the secretory granules of animal cells, A#H + is used as the
driving force for the uphill transport of metabolites. On the other hand, the
animal plasmalemma utilizes, for the same purpose, AfiNa + or ATP. As to
bacteria, here one can find all the above driving forces (Skulachev, 1988).
For a solute S, accumulated by a AfiH+-driven system in a negatively
charged compartment, the accumulation ratio [S]in/[S]out obeys the following
equation:
RTln iS]out
[S]n = - ( n +z)FA
-nRTln[ [H+]io
]oo
H~
(3)
408
Skulachev
when n is the number of H § ions symported with one S molecule, and z is the
number of positive charges on S (Kotyk, 1983).
For a monovalent cation C § which is translocated electrophoretically by a
uniporter, the accumulation ratio is described by equation (4):
[C+]in
RT In [C+]out
=
-FAW
(4)
If ApH is the only driving force, equation (3) is simplified as follows:
[S]in
~ [n§
=
~n
(5)
There are several types of porters or ion channels, usually protein or, in
rather rare cases, short peptides, which can be used to carry out A/~H +dependent osmotic work. The simplest natural systems of this kind have been
found among hydrophobic peptide antibiotics playing the role of K § uniporter
(valinomycin), K § + antiporter (nigericin) or N a t / H § antiporter (monensin).
Valinomycin increases the K § permeability of the membrane since it reversibly
combines with K § and the resulting K§
complex diffuses through
the hydrophobic membrane barrier. Thus, K § moves to the negatively charged
compartment, ApK being formed at the expense of AW.
Antiporter antibiotics can combine with either K§
+) or H § (more
probably n30+). In this case, ApH appears to be the driving force. All the
biological porters other than antibiotics are found to be membrane proteins which
are much larger, for example, than valinomycin. The involvement of a protein to
carry out a biological function makes it possible to organize a system that not only
facilitates the process but also allows it to be regulated (the latter facility is absent
when we deal with antibiotics, "weapons" used by a microorganism to fight the
others).
The A/~H+-driven transport proteins include ion uniporters, H§
symporters and H§
antiporters (for reviews, see West, 1983; Skulachev,
1988; Henderson, 1990, 1991). The three-dimensional structure at atomic
resolution has not been reported for any of them. As to the transport mechanism,
a lot of hypothetical schemes have been proposed which are still awaiting direct
verification. This is the case even for the most intensely studied porters, such as
the E. coli H§
symporter (for discussion, see Kaback et al., 1990; Kaback,
1990).
An unsolved problem of the A/~H +- (or A/~Na§
transport consists in
the following. Transported solutes are often rather large hydrophilic molecules.
Their translocation into the hydrophobic membrane core seems to require a large
amount of energy. Formation of a stable hydrophilic channel is improbable since
it must increase the H § ( O H - ) permeability of the A/~H§
membrane
and, hence, reduce the driving force of the transport. For example, it is hardly
possible to imagine a channel able to transport a large hydrophilic protein, such
as the precursor of a major subunit of factor F1, without a dramatic increase in
the H § permeability of the mitochondrial membrane.
Chemiosmotic Bioenergetics
409
In the 'fifties, Mitchell suggested the concept of the mobile m e m b r a n e barrier
to explain the transport of hydrophilic compounds across the hydrophobic
membrane core (Mitchell, 1957, 1959). According to his scheme, a hydrophilic
solute comes to the m e m b r a n e core, moving into a cleft formed by the porter.
Then the porter changes its conformation in such a manner that the first cleft
closes and the solute passenger becomes accessible to another cleft that opens so
as to connect the m e m b r a n e core with the solution on the opposite side of the
membrane. The scheme in question suggests that the hydrophobic membrane
barrier is much more narrow in the place occupied by the porter because of two
oppositely oriented clefts. This may create, however, some difficulties in
maintaining a high A # H + level due to the increased probability of electric
breakdown and H § leakage through the porter.
Another variation of the mobile membrane barrier theme is shown in Fig.
11. The scheme illustrates a possible mechanism of an electrogenetic export of a
hydrophilic anion (in the scheme, shown as a black body). It is assumed that this
anion is bound to the specific binding site of the porter protein, which faces the
vesicle interior (Step 1). Then the anion is translocated to a hydrophilic, reverse
micelle-like microvolume localized in the middle part of the protein (Step 2). The
binding and translocation of the anion is facilitated by a cationic group localized
in the lower part of the protein, between the binding site and internal
microvolume. At the next stage (Step 3), a conformation change occurs,
increasing the affinity of the anion to one more cationic group, which is localized
closer to the upper protein part. In the scheme, this rearrangement of the protein
Fig. U A tentative scheme of electrophoretic uniport of a hydrophilic anion through a membrane. It
is assumed that there is a AW across the membrane (the interior negative). This AW serves as the
driving force for the anion efllux. For other explanations, see the text.
410
Skulachev
is shown as the change in position of two transmembrane cr helical columns. At
Steps 4 and 5, the anion is translocated from the internal microvolume to the
outer binding site and is released to the outer water phase. It is essential that (i)
the hydrophilic anion is always in a hydrophilic environment and (ii) the
probability of H § leakage via the porter is low since the protein domains below
and above the hydrophilic microvolume are of high hydrophobicity.
An interesting consequence of the scheme is that the same porter might also
transport hydrophobic anions provided that they could reach the cationic groups
of the porter without participation of the specific binding site for hydrophilic
anions. This appears to be the case for the transport of hydrophobic anions by the
A T P / A D P - antiporter (see the next section). Presumably, the porter might
easily be converted to a non-specific channel for small hydrophilic solutes, if the
microvolume-shielding domains were somehow damaged. In agreement with this
assumption, Kr/imer's group reported that the mitochondrial aspartate/glutamate
and ATP/ADP antiporters in proteoliposomes acquire pore-like properties after
modification by SH-reagents (Dierks et al., 1990a, b).
In Fig. 12, the above scheme is adapted for the transport of large molecules
such as proteins. In this figure, it is taken into account that the transport of
(1)
'I',; .......
~,7;',$',i,,,,~
'l . . . . . .
x~|ltlll
i I
t
(2)
4)
(3)
XX~IIII,
~1111111111l
'/ht,
dO
Fig. 12 The "sluice hypothesis" of protein transport into mitochondria.
The upper and lower bilayers, outer and inner mitochondrial membranes,
respectively. The transported protein is assumed to contain two (N- and
C-terminal) hydrophilic domains linked with a hydrophobic 0c-helix. For
details, see the text.
ChemiosmoticBioenergetics
411
proteins, for example, into mitochondria occurs at the contact sites of the outer
and inner mitochondrial membranes. Let us assume that in the contact sites, (i)
the two membranes form a sluice, a small water space separated from both the
cytosol and the mitochondrial matrix, and (ii) pore-forming proteins are inlaid
into both membranes in the sluice region. The pores are postulated to be
potential-dependent being closed when AW is high. In the ground state, the pore
in the outer membrane is open whereas that in the inner membrane is closed.
Thus, it is the inner membrane that forms the H § impermeable barrier in the
contact site.
The process starts with the recognition of the protein precursor by a
cytosol-facing receptor in the outer membrane. Then a N-terminal part of the
protein crosses the outer membrane via the open pore in such a fashion that the
positively charged signal sequence and the first hydrophilic domain comes to the
sluice, whereas the hydrophobic or-helical region localized after the hydrophilic
domain occupies the pore like a plug, decreasing its permeability. Then the signal
sequence enters the inner membrane pore (Step 1). At this stage, it is the outer
membrane that bears AqJ. At Step 2, the first hydrophilic domain is translocated
to the matrix via the inner membrane pore, the or-helix occupies this pore and
then the second hydrophilic domain crosses the outer membrane. At Step 3, the
second hydrophilic domain leaves the sluice so that the entire precursor protein
molecule appears in the matrix. Here the signal peptide is removed by a specific
peptidase. The process is completed with the closing of the inner membrane pore
and opening of the outer membrane pore, so that the inner membrane becomes
again the AqJ bearing one (Step 4).
The above scheme is supported by several observations.
(i) Protein transport is localized in the contact sites (Schleyer and Neupert, 1985;
Pain et al., 1990).
(ii) There is an intermediate stage of the transport when the N-terminal part of
the protein precursor is already in contact with the matrix, whereas the
C-terminal part is still outside the mitochondrion (Hartl et al., 1989).
(iii) Porin permeability is potential-dependent (it decreases with the Aq t rise)
(Roos et al., 1982). This property is difficult to reconcile with the common point
of view assuming that the outer membrane is never exposed to AW.
(iv) It is Art/, not ApH, that is necessary to transport the positively charged signal
sequence (Pfanner and Neupert, 1985).
(v) To be transported, a protein precursor must be structurally rearranged in
some way (partial or complete unfolding) (Pfanner et al., 1990; Rassow et al.,
1990).
The scheme shown in Fig. 12 may also explain a phenomenon discovered in
our group by Bakeeva et al., (1978, 1983, 1985) and called the mitochondrial
junction. It was found that two adjacent skeletal or heart muscle mitochondria
can form a specific four-membrane contact. In the contact region, two outer
membranes of mitochondria are very close to each other (resembling the tight
junction of the outer cell membranes) whereas the inner membranes remain at
some distance from the outer ones, the intermembrane space being filled with
412
Skulachev
..~/,'///,,,,,,, , , , , , , , , ~
outer
membranes_.
~
......
II I I I I j l h l l l l l l * l l t l l l -
,~
I~1~ ' , ' , ' , ' , ~
-IIiItllHlltllll
II
Ill/
~ ' , " ' , ' :,h ' , ' ~
,nn~r
m~mhrnn~_r
Fig. 13 Possible structure of the conductive junction connecting two adjacent
mitochondria. For explanations, see the text.
electron-dense material as in the gap junction. We have found that mitochondrial
junctions are of low electrical resistance, uniting many mitochondria in an
equipotential cluster called Streptio mitochondriale. Such clusters may be involved
in A/~H § transmission in large heart muscle cells (Amchenkova et al., 1988;
Skulachev, 1990).
One may assume that the mitochondrial junction arises in the place where
two mitochondria make contact in such a fashion that two "sluices" oppose each
other and two outer membranes stick together. As a result, two matrix spaces
appear to be connected via four pores and two "sluices" (Fig. 13). It is important
that all four pores should be open since there is no electric potential difference
between matrix spaces of two energized mitochondria. However, when one of the
mitochondria is damaged and de-energized, a trans-junctional potential difference
may arise, which should result in closing the potential-inhibited pores and, hence,
in isolating the damaged mitochondrion from its partners in the cluster.
AI~H + as an
Energy S o u r c e
for H e a t P r o d u c t i o n
Dissipation of the respiratory chain-produced A/~H + seems to be a major
mechanism of urgent heat production in warm-blooded animals. Such an effect
called thermoregulatory uncoupling was discovered by our group in 1960 when we
studied the effect of sudden decrease in the ambient temperature on pigeon
breast muscle mitochondria (Skulachev and Maslov, 1960; Skulachev, 1963).
Later, the phenomenon of thermoregulatory uncoupling was confirmed in
experiments on mitochondria of mouse skeletal muscle (Skulachev et al., 1963)
and fur seal (Grav and Blix, 1979), of rodent brown fat (Smith and Horwitz,
1969; Nicholls, 1976) and of ground squirrel liver (Brustovetsky et al., 1990a).
Several pieces of evidence have been obtained indicating that free fatty acids
are responsible for thermoregulatory uncoupling.
(i) Addition of delipidized serum albumin, which is known to bind fatty acids,
abolishes this uncoupling (Skulachev, 1963; Grav and Blix, 1979).
(ii) Fatty acids isolated from mitochondria of cold-treated animals and added to
mitochondria from the non-treated ones induce uncoupling (Levachev et al.,
1965).
(iii) Uncoupling caused by adding low concentrations of (1 x 10 -5 M - 3 x 10 -5 M)
palmitate to muscle or liver mitochondria from non-treated animals is abolished
Chemiosmotic Bioenergetics
413
by very low (5 x 10 -7 M) carboxyatracylate (CAtr), a specific inhibitor of the
ATP/ADP antiporter (Andreyev et al., 1988, 1989: Sch6nfeld, 1990). Thermoregulatory uncoupling in these mitochondria is also CAtr-sensitive
(Brustovetsky et al., 1990a).
(iv) Thermogenin, the brown fat mitochondrion protein responsible for thermoregulatory uncoupling in this tissue, requires free fatty acids to increase the H +
permeability of the mitochondrial membrane (Nicholls, 1976). This effect was
recently reproduced on thermogenin proteoliposomes (Garlid, 1990b).
It is not yet clear how fatty acids activate thermogenin as well as how they
uncouple in tissues other than brown fat where there is no thermogenin. Fatty
acids operate in a fashion distinct from classical protonophorous uncouplers such
as trifluoromethoxy-p-carbonylcyanide phenylhydrazone (FCCP). Fatty acids fail
to increase the conductance of planar phospholipid bilayers or cytochrome
oxidase proteoliposomes (Andreyev et al., 1989). This observation is in agreement with the fact that fatty acids can traverse the phospholipid membrane only
in their protonated form (RCOOH), whereas the anion (RCOO-) always
occupies a position in the lipid/water interface with the carboxylate group facing
the water and the hydrocarbon chain penetrating into the membrane core
(Skulachev, 1988; 1991).
In 1988 I presented a hypothesis that some mitochondrial proteins allow the
anionic form of fatty acids to traverse the mitochondrial membrane (Skulachev,
1988). The proteins in question were supposed to be thermogenin in brown fat
and the ATP/ADP antiporter in other tissues (Fig. 14). It is assumed that H §
nucleotirt~
Fig. 14 The futile fatty acid circuit mediated by the ATP/ADP antiporter or thermogenin. (From
Skulachev, 1991).
414
Skulachev
ions, which are pumped from the mitochondrial matrix by the respiratory chain
(Step 1), protonate fatty acid anions on the outer membrane surface (Step 2).
Protonated fatty acid, being a penetrant for the phospholipid bilayer (see
Andreyev et al., 1989), diffuses to the inner membrane surface (Step 3). Here it is
deprotonated (Step 4). The fatty acid anion that is produced scans the inner
membrane surface until it reaches the anion-binding site of the ATP/ADP
antiporter (or thermogenin) which may be localized in the internal hydrophilic
microvolume postulated for the anion porters (see above, Fig. 11). Due to the
hydrophobicity, the fatty acid anion needs no interaction with the specific
binding site involved in the transport of adenine nucleotide anions by the ATP/
ADP antiporter. At Step 5, flip-flop of the fatty acid anion inside the porter
takes place, so that the anion appears again on the outer membrane surface.
The above scheme explains why CAtr, the specific ATP/ADP antiporter
inhibitor, blocks fatty acid-induced uncoupling. C A t r has been found to decrease
the State 4 respiration and H § conductance stimulated by fatty acids. Such a
decrease was accompanied by an increase in the electric membrane potential
(Andreyev et al., 1988, 1989; Brustovetsky et al., 1990b; Sch/Snfeld, 1990).
Another inhibitor of the ATP/ADP antiporter, pyridoxal 5-phosphate was found
to substitute for CAtr (Brustovetsky et al., 1990b). An effect qualitatively similar
to that of CAtr and pyridoxal 5-phosphate, but not so potent, was found to be
given by ADP, an ATP/ADP antiporter substrate (Andreyev et al., 1989). Under
the same conditions, FCCP-induced uncoupling proved to be resistant to CAtr,
pyridoxal 5-phosphate and ADP (Andreyev et al., 1989; Brustovetsky et al.,
1990b).
The efficiency of fatty acids as CAtr-sensitive uncouplers proved to be
proportional to A T P / A D P antiporter content in mitochondria of different tissues
(heart muscle > kidney > liver) (Sch/Snfeld, 1990). Several pieces of evidence
indicate that the ATP/ADP antiporter and thermogenin are related proteins.
They are of similar molecular mass, sequence, secondary structure, arrangement
and domain composition. Both of them are formed without the stage of a larger
precursor, a situation unusual for mitochondrial inner membrane proteins
encoded by the nuclear genes. Both of them are shown to bind purine nucleotides
and fatty acids. Binding results in uncoupling (fatty acids) or recoupling (purine
nucleotides). Not only the A T P / A D P antiporter but also thermogenin can be
regarded as an anion carrier since thermogenin increases the membrane permeability for CI- (for refs., see Skulachev, 1991). Garlid, (1990a) reconstituted
CI- transport in thermogenin proteoliposomes and showed that fatty acids are not
necessary for this process as they are for H + transport. Rather, H + seems to
compete with C1- for thermogenin. Transport of CI- occurred at high CIconcentration and was slow. Other anions were also transported by brown fat
mitochondria and thermogenin proteolipsomes. The rate of transport was found
to increase with the increase of hydrophobicity of anions. The process proved to
be very unspecific when monovalent, monopolar anions were tested. The list of
transportable anions included alkylsulfates, alkylsulfonates, benzenesulfonate,
oxohalogenoids, hypophosphate, hexafluorophosphate and pyruvate. In all cases,
transport was sensitive to purine nucleotides.
Chemiosmotic Bioenergetics
415
Similar non-specificity seems to be inherent in CAtr-sensitive uncoupling in
liver and heart mitochondria. Here it was found that dodecyl sulfate and cholate
could substitute for fatty acid but at much higher concentrations. Uncoupling by
cationic detergent (cetyltrimethyl ammonium) or non-ionic detergent (Triton
X-100) was CAtr-resistant (Brustovetsky et al., 1990b). To explain the dodecyl
sulfate-induced uncoupling within the framework of the above scheme, we
should postulate that this compound exists at neutral pH not only in anionic but
also in protonated form. This seems possible if we consider the membrane/
water interface rather than bulk water phase. According to Sankaram et al (1990),
the pKa of stearate, for example, increases from 5.0 in water up to 8.0 in liposomes and up to 9.6 in some proteolipsomes.
It seems interesting to compare thermogenin or the A T P / A D P antiporter
with the plasma membrane multi-drug resistance protein (Endicott and Ling,
1989; West, 1990). Both systems are rather non-specific to the transported
substances but the drug resistance protein pumps hydrophobic cations and
non-charged molecules rather than anions. At the same time, it should be
stressed that thermogenin and the A T P / A D P antiporter non-specificity for the
uncoupling anion is, most probably, not manifested under physiological conditions. The millimolar concentration of purine nucleotides, always present in the
cell, is quite sufficient to block the translocation of anions which are less
hydrophobic than fatty acids (Garlid, 1990a).
As to evolutionary relationships between the A T P / A D P antiporter and
thermogenin, we may speculate that the latter is a derivative of the former.
Apparently, fatty acid-induced uncoupling, a supplementary function of the
A T P / A D P antiporter, became the only function in thermogenin which still binds
purine nucleotides but cannot transport them.
A~H§
Mechanical Work: The Bacterial H + Motor
Mechanical work, like regulatory heat production, may be regarded as a
rather rare case among the processes of A/~H + utilization. Nevertheless we shall
pay attention to this kind of energy transduction since it represents one of the
most striking inventions of biological evolution. To date, three examples of
A/~H+-driven mechanical work have been described, namely swimming of
fiagellar bacteria (Skulachev, 1975; Belyakova et al., 1976; Manson et al., 1980;
Glagolev and Skulachev, 1978), gliding of cyanobacteria (Glagoleva et al., 1980;
Skulachev, 1980) and rotation of chloroplasts (Skulachev, 1980). Among them,
only the first phenomenon has been studied in detail.
Chronologically, Mitchell proved to be the first to indicate the possibility that
an ion gradient can be used as the driving force for motility of flagellar bacteria
(Mitchell, 1956). In 1974 Adler and coworkers (Larsen et al., 1974) showed that
an E. coli mutant deficient in oxidative phosphorylation required respiration to be
motile, although the ATP level was not affected by oxygen. A strong lowering of
the ATP level did not stop the motility, while the addition of an uncoupler did. In
1975 we found a good correlation between the Rhodospirillum rubrum motility
rate and the level of photoredox chain-produced membrane potential (Skulachev,
416
Skulachev
1975; Belyakova et al., 1976). Later it was shown that bacterial motility can be
supported by artificially-imposed AW or ApH (Skulachev, 1977; Manson et al.,
1980; Matsuura et al., 1977; Glagolev and Skulachev, 1978). Thus, it was
concluded that AfiH § powers ,the motility mechanism of flagellar bacteria.
A typical bacterial flagellum consists of a rigid filament several micrometers
long, a basal body inlaid in the cell wall, and a "hook", a curved structure of
larger diameter than the filament, attaching the filament to the basal body. The
basal body is composed of several discs, parallelling the membrane plane, and a
rod, axial to the discs and connected with the hook. In the isolated basal body,
one can usually see four discs called L, P, S and M, the last being the proximal
one. Apparently there are three more structures which are directly connected to
the basal body but are usually lost during its isolation. In the bacterial wall, one
can occasionally see a disc structure 50-140 nm in diameter, i.e. much larger than
the above-mentioned basal body discs (Remsen et al., 1968; Ferris et al., 1984;
B/ieuerlein, 1989; Kupper et al., 1989). Only once (when Wolinella succinogenes
was studied) was such a large disc isolated as part of the basal body (B/ieuerlein,
1989). The large disc is apparently localized between the S and P discs. It seems
to be in contact with the ring of 10-12 intramembrane particles surrounding the
M disc and protruding from the cytoplasmic membrane (Khan et al., 1988). At
the insertion site of the flagellum, a cap structure extending into the cytoplasm
was recently found (Swan, 1985; Metlina and Bakeeva, 1989; Driks and
DeRosier, 1990).
A putative scheme of the H § motor is given in Fig. 15. As we postulated in
I
1Onto
4
cwfosol
cytoplasmic
membrane
periplasm
pepfidoglycan
layer
oufer
membrane
oufer medium
Fig. 15 A scheme of the basal body of a bacterial flagellum. It is assumed that the rotor part of
the motor is formed by the large disc and rod whereas the stator is composed of L, P, S and M
discs, intramembrane particles (IP) and the cap (cytoplasm-facing particle, CFP).
ChemiosmoticBioenergetics
417
1978 (Glagolev and Skulachev, 1978; for detailed versions, see Mitchell, 1984,
and Kobayasi, 1988), the role of the rotor is performed by a basal body disc
firmly attached to t h e rod. Originally, we assumed that this was the M disc.
However, after discovery of the large disc, the latter seems to be a better
candidate for the role of rotor since it has the biggest diameter. The large disc is
an ideal cylinder of ca 50-140 nm in diameter and ca 5 nm in height. It is localized
in the periplasm just outside the cytoplasmic membrane. It is assumed that some
components of the stator are arranged in this membrane, namely the M and S
discs and the intramembrane particles (IP) surrounding the basal body. In the
particles, a proton well is assumed to be present, crossing the cytoplasmic
membrane and leading from the large disc to the cytosol. H § ions moving
downhill from the periplasm to the cytosol, pass along the large discintramembrane particle contact, come to the inward proton well and diffuse via
this well to the cytoplasmic membrane surface. The H § flux rotates, in some way,
the large disc.
To explain coupling of the downhill H + influx and the disc rotation, we
suggested (Glagolev and Skulachev, 1978) that protonation of a proton-acceptor
group (e.g., of the NH2-group of a lysine residue) on the rotor disc results in its
coulombic attraction to a fixed negatively charged group on the stator (this may
be - - C O O - of an aspartate or glutamate residue on the intramembrane particle).
Elegant calculations done by Kobayasi (1988) showed that such a system can
effectively operate as a diffusion motor.
According to Berg et al. (1982), ca 200 H + ions should be transported down
a protonic potential difference of 200 mV per rotational cycle of a single flagellum
if the radius of the bacterial cell is 1/~m and the rotation rate 10 Hz (other
calculations gave the value of 1240H +, see Meister et a L , 1987). In the same
group, it was also found that the torque of the motor energized by an
artificially-imposed AW or ApH is practically the same at 4~ and 38~ (Khan
and Berg, 1983). This fact indicates that the motor operates without conformational changes in its protein subunits. Substitution of D20 for HzO was also
without effect on the torque (Khan and Berg, 1983).
THE SODIUM W O R L D
The progress made in bioenergetic studies during the last few years has
stimulated interest in the role of Na § ions. The dogma, assuming H + as the
coupling ions in all the chemiosmotic systems, with the only exception being the
animal outer cell membrane, proved to be shaken.
In 1968 Mitchell pointed to the possible role of Na+/H § antiport as a
mechanism for increasing the pH buffer capacitance of the bacterial cytoplasm
(Mitchell, 1968). Later I suggested that, (i) it is electrogenic K § influx that
discharges the redox chain-produced AqJ, converting thereby AW to ApH and
ApK, and (ii) ApH is then converted to ApNa by the Na+/H+-antiporter. Such a
system was assumed to operate as a A g H + buffer (Skulachev, 1978). According
to this concept, the energy is invested in ApK and ApNa when the rate of
418
Skulachev
A/~H+-producing processes is higher than that of the AfiH+-consuming ones.
Under these conditions, K + is imported and Na t is exported. When A~H +consumption is faster than A~H+-production, AfiH § decreases and the direction
of K § and Na t fluxes reverses. Electrogenic efflux of K § generates AW, whereas
influx Na t in exchange for H § generates ApH. Thus, A~H + would be stabilized
until the K § and Na t gradients were dissipated. The idea of K+/Na § gradients as
a AgH + buffer was experimentally proved by our and other groups (Wagner et
al., 1978; Skulachev, 1980; Arschavsky et al., 1981; Brown et al., 1983; Michels
and Bakker, 1985).
Further studies have revealed that the bioenergetic role of Na § is not
restricted to A/~H § buffering. In some forms of life, Na § was shown to be directly
involved in energy transductions, performing the coupling-ion function previously
ascribed to H +. The great taxonomic variety of species employing Na + as the
primary coupling ion points to the ubiquitous distribution of this novel type of
membrane-linked energy transduction. An impression arises that besides the
world of living organisms using H § energetics there is a rather extensive area in
the biosphere which may be defined as a "sodium world".
A#Na+-Generating Systems
The Na+-motive Respiratory Chain
In 1981-1982 Tokuda and Unemoto demonstrated a respiration-dependent,
protonophore-resistant export of Na + from the cells of the marine alkalotolerant
Vibrio alginolyticus (Tokuda and Unemoto, 1981; 1982). Later the same group
succeeded in isolating from V. alginolyticus a Na +-motive NADHmena(ubi)quinone reductase, and they reconstituted proteoliposomes that import
Na + when oxidizing NADH by a quinone. The rate of oxidation was strongly
stimulated by Na + (Tokuda, 1984) and specifically inhibited by a very low
concentration of Ag + (Asano et al., 1985) or by 2-heptyl 4-hydroxyquinoline
N-oxide (HQNO) (Tokuda et al., 1985).
The molecular properties of H +- and Na+-motive NADH-quinone reductases
proved to be quite different. The H§
enzyme from mitochondria or
Paracoccus denitrificans is known to be composed of several subunits bearing
FMN and FeS clusters (reviewed by Skulachev, 1988). The Na+-motive reductase
contains only three subunits, FAD, FMN and no FeS clusters. The FADcontaining /3 subunit catalyzes oxidation of NADH and reduction of CoQ or
menaquinone to semiquinone anion (e.g. CoQ'-) whereas the ol and y subunits
are required for dismutation of two C o Q ' - to CoQH2 and CoQ. The latter (but
not the former) process is Na+-sensitive (Hayashi and Unemoto, 1986, 1987;
Unemoto and Hayashi, 1989).
The Na+-motive NADH-quinone reductase was later found in Vibrio
costicola (Udagawa et al., 1986), Vibrio parahaemolyticus (Tsuchiya and Shinoda,
1985; Tokuda and Kogure, 1989), halotolerant bacterium Bal (Ken-Dror et al.,
1986), Klebsiella pneumoniae (Dimroth and Thomer, 1989), alkalo- and halotole-
ChemiosmoticBioenergetics
419
rant Bacillus F T U (Kostyrko et al., 1991) and in eight out of the nine strains of
marine bacteria belonging to genera Vibrio, Alcaligenes, Alteromonas and
Flavobacterium, in which the possibility of the existence of Na + pumps was tested
(Tokuda and Kogure, 1989; Tokuda, 1989).
A Na+-motive terminal oxidase has recently been described in our group
(Verkhovskaya et al., 1988; Semeykina et al., 1989; Kostyrko et al., 1991). This
enzyme was found in a new alkalotolerant and halotolerant microorganism,
Bacillus FTU, which is, judging from the 5S RNA sequences, a close relative of
Bacillus subtilis and Bacillus licheniformis. In experiments on intact cells and
inside-out subcellular vesicles, oxidation of ascorbate via tetramethyl p-phenylene
diamine (TMPD) or diaminodurene was shown to be coupled to uphill Na +
transport. Pumping of Na § was: (i) stimulated by protonophorous uncouplers as
well as by valinomycin; (ii) inhibited by the Na+/H § antiporter monensin,
millimolar cyanide or Na § ionosphore ETH; and (iii) resistant to the Na+/H §
antiporter inhibitor amiloride, to the Na+-motive NADH quinone reductase
inhibitor HQNO, and to micromolar cyanide. The difference spectrum "millimolar cyanide minus micromolar cyanide" showed reduction of some b- and c-type
cytochromes.
The stimulating effect of protonophore on Na + transport seems to be the
most demonstrative probe for electrogenic primary Na § pumps. Such a stimulation excluded involvement of the alternative mechanism, i.e. cooperation of an
H § pump and Na+/H § antiporter. Figs. 16A and 16B illustrate an approach
allowing discrimination between the primary Na + pump (A) and a combination of
the H + pump and Na+/H + antiporter (B). According to Fig. 16A, oxidation of
ascorbate via TMPD by inside-out subcellular vesicles of a bacterium is coupled
to Na + uniport which changes the membrane (the vesicular interior positive). AqJ
restricts large-scale Na § accumulation in the vesicles. In the presence of
protonophore (CCCP), A ~ is discharged due to H + efflux (reaction 2), Now
ApNa formation appears to be limited by operation of CCCP which is, in its turn,
restricted by ApH formation (the interior alkaline). Further stimulation can be
observed when ApH is discharged by influx of a penetrating weak acid, e.g. acetic
acid (reaction 3). In the presence of valinomycin + K § acetic acid should be
unnecessary for the maximal rate of Na + uptake since ApH is not formed
(reaction 4).
Exactly the above effects were observed in experiments on inside-out vesicles
from Bac. FTU. Maximal rates of Na § influx were obtained in the presence of
CCCP + diethylammonium (DEA) acetate or valinomycin (+K+). In the absence
of A~- and ApH-discharging agents the rate was very low. An intermediate rate
was found when only CCCP was added. DEA acetate without CCCP was
ineffective. All the above relationships were shown to be characteristic of the
vesicles from Bac. F T U growing under alkaline conditions (pH 8.6).
A quite different situation was revealed when the pH value of the growth
medium was decreased to 7.5. Now Na + uptake was completely inhibited by
CCCP. DEA acetate was also inhibitory whereas monensin was without effect.
Valinomycin ( + K +) was stimulatory. These results can be explained within the
framework of the scheme shown in Fig. 16B. The H+-motive oxidase pumps H +
420
Skulachev
A
K+
CISC
B
/f
1
~
K+
DEAW+
>N+-~DEA~
DEA
Ng. 16 The scheme illustrating mechanisms of the Na + uptakes by vesicles from Bacillus FTU
growing at pH 8.6 (A) or 7.4 (B). In the former case, the Na + uptake is due to activity of the
Na+-motive terminal oxidase. In the later case, it is mediated by cooperation of the H+-motive
oxidase and Na+/H + antiporter. For other explanations, see the text.
ions into the vesicle and forms Aqs (reaction 1). Valinomycin-mediated K + etitux
(reaction 2) converts Arts to ApH. The latter is utilized by an endogenous
Na+/H § antiporter to accumulate Na § (reaction 3). CCCP or D E A discharge
ApH (reaction 4 and 5). In agreement with the scheme, the Na+/H § antiporter
inhibitor amiloride abolished the Na § uptake by the vesicles from the bacteria
growing at neutral pH. If they were growing at pH 8.6, amiloride was without
Chemiosmotic Bioenergetics
421
effect. Direct measurement of ApH formation showed that the vesicles were
competent in valinomycin-stimulated, CCCP- or DEA-sensitive H § uptake
coupled to ascorbate ( + T M P D ) oxidation (Kostyrko et al., 1991). This oxidation
was found to be catalyzed by an aa3-type cytochrome oxidase, sensitive to
micromolar cyanide. As to the oxidase sensitive to millimolar and resistant to
micromolar cyanide, it proved to be specific to the cells growing at pH 8.6, being
absent from those growing at pH 7.5. Thus, the Na+-motive terminal oxidase is
an enzyme which is induced (or activated) when the concentration of H § ions in
the medium is lowered (for discussion, see Skulachev, 1988; 1989a; 1989b).
Na+-motive Decarboxylase
In 1980 Dimroth reported on the discovery of a prokaryotic, primary A/~Na +
generator creating a Na § potential difference with no A/)H + involved (Dimroth,
1980). It was found that the non-oxidative decarboxylation of oxaloacetate to
pyruvate by an enzyme from anaerobic Klebsiella pneumoniae, (i) requires Na §
for its activity, and (ii) carries out uphill export of Na § from the cytoplasm to the
external medium. The action sequence included (a) transfer of a carboxylic
residue to the biotin prostetic group and (b) release of CO2 from carboxylated
biotin in a Na+-dependent fashion (Dimroth, 1982; 1987). The enzyme is
composed of: (1) a peripheral, biotin-containing tr subunit; and (2) fl and y
subunits immersed into the membrane. The Na+-pump could be reconstituted in
proteoliposomes, using isolated subunits (Dimroth and Thomer, 1988). A similar
process was also described in Salmonella typhimurium (Buckel, 1986; Dimroth,
1987).
Two more Na+-motive decarboxylases were found when other bacterial
species were investigated. (i) In Acidaminococcus fermentans, Peptococcus
aerogenes, Clostridium symbiosum and Fusobacterium nucleatum, glutaconylCoA, an intermediate of the glutamate to acetate and butyrate fermentation was
shown to be decarboxylated to crotonyl-CoA, the process being coupled to Na +
efflux from the cell (Buckel, 1986). (ii) In Veilonella alcalensis, converting lactate
to acetate and propionate (Hilpert and Dimroth, 1983; 1984), and in
Propionigenium modestum, forming propionate from succinate (Hilpert et al.,
1984; Rohde et al., 1986; Hoffman et al., 1989), there is a Na§
enzyme
decarboxylating an intermediate of this process, methylmalonyl-CoA, to
propionyl-CoA. Two Na § ions were found to be transported per one molecule of
decarboxylated substrate. The Na § transport was stimulated by a protonophore,
an indication that the process was electrogenic.
Na+-motive ATPases
Chronologically, the first enzyme utilizing ATP to pump Na + was discovered
in the animal plasma membrane. This is the well-known Na+/K+-ATPase. The
enzyme is of the E1Ez type. It catalyzes ATP hydrolysis coupled to electrogenic
export of 3Na + in exchange for 2K + per ATP. (For reviews, see Schwartz and
Collins, 1982; Skulachev, 1988). This type of Na + pump proved to be specific for
animals. In bacteria, other Na+-ATPases have been found.
422
Skulachev
Heefner and Harold (1982) have reported on the ATP-dependent accumulation of Na § in inside-out vesicles from Streptococcus faecalis. The process proved
to be resistant to vanadate, an inhibitor of E1E2 ATPases, and dicyclohexylcarbodiimide (DCCD), an inhibitor of FoF~ ATPases, but sensitive to Nethylmaleimide and nitrate which inhibit the tonoplast H+-ATPase. Like the FoF~
and tonoplast ATPases, the Na§
ATPase of S. faecalis is composed of
membranous and detachable sectors, the latter mainly consisting of 73 kDa and
52 kDa subunits. The enzyme seems to catalyze an electroneutral Nain/I~ut+
+
exchange (Kakinuma and Iragashi, 1989; 1990). This ATPase was shown to be
induced under low A/~H § conditions: (i) at high outer pH; (ii) in the presence of
a protonophore in the growth medium; or (iii) in an H§
ATPase-deficient
mutant. Na § was necessary for the induction (Kinoshita et al., 1984; Kakinuma
and Harold, 1985; Kakinuma and Igarashi, 1989). Growth under alkaline
conditions was found to repress the H+-ATPase (Kobayashi et al., 1984).
A Na+-motive ATPase has been described in Mycoplasma (Benyoucef et al.,
1982; Shirvan et al., 1989a, b). The ATPase activity was shown to be stimulated
threefold by Na § ions at pH 8.5, but only very slightly at pH 5.5 (Shirvan et al.,
1989b).
A DCCD-sensitive Na+-driven ATP-synthase of the FoF1 type from P.
modestum is described below.
A~Na+-Consuming Systems
Na+-Solute Symporters
The performance of osmotic work appeared to be the first type of
A/~Na+-consuming process which was directly proved. It has been found that the
import of many metabolites by animal and bacterial cells is carried out by
Na+-metabotite symporters localized in the cytoplasmic membrane (reviewed by
West, 1983; Hoshi and Himukai, 1982; Krulwich, 1983; Skulachev, 1988). Among
bacteria, such a mechanism is typical of marine, halo- and/or alkalophilic species,
For example, in V. alginolyticus, 19 different amino acids and sucrose were found
to be imported together with Na + (Tokuda et al., 1982; Kakinuma and Unemoto,
1985). Transmembrane protein translocation seems also to be AfiNa+-dependent
(Tokuda et al., 1990).
In neutrophilic bacteria living at low or moderate [Na+], A/~H § rather than
A/~Na + is used to carry out osmotic work. However, some examples of
Na+-solute symporters have been reported also in this case. For instance, a
Na+-proline symporter is responsible for the uphill proline import in E. coli,
Salmonella typhimurium and Mycobacterium phlei (for refs., see Skulachev,
1988). The E. coli melibiose porter can operate with either H § or Na § (Bassilana
et al., 1985). In Klebsiella pneumoniae, citrate 3- is imported together with 2H §
and 2Na § so that ApH, ApNa and AW are the driving forces (Dimroth and
Thomer, 1986).
Chemiosmotic Bioenergetics
Na§
423
Motors
There is at least one example of a bacterium performing mechanical work at
the expense of A/~Na + generated by a primary Na + pump. This is V.
alginolyticus. As found in our group, motility of V. alginolyticus: (i) requires
Na§ (ii) can be supported by an artificially-imposed ApNa (ApH is ineffective);
(iii) is carried out at a lowered but measurable rate in the presence of
1 x 10 .4 M CCCP. A 100-fold lower CCCP concentration completely arrests the
motility if the medium is supplemented with monensin. Monensin without CCCP
lowers the motility rate only slightly (Chernyak et al., 1983; Bakeeva et al., 1986).
These data were recently confirmed in two other laboratories (Tokuda et aL,
1988; Liu et al., 1990).
We have isolated the basal body of the V. alginolyticus flagellum and found
that it is rather similar to the H§
motor of "protonic" bacteria (Sect. 3.6),
but some features (e.g. smaller diameter of the M-disc) proved to be specific for
this engine (Bakeeva et al., 1986). The structure of the V. alginolyticus flagellum
was identical to that of V. cholerae (Ferris et al., 1984).
A/lNa+-supported motility has also been found in alkalophilic Bacilli,
namely in Bac. firmus and Bac. YN-1 (Hirota et al., 1981; Kitada et al., 1982;
Hirota and Imae, 1983; Imae and Atsumi, 1989). ApNa + and AtIt proved to be
equivalent in supporting motility. The existence of primary Na § pumps in these
non-marine microorganisms is not yet established (for discussion, see the final
section).
Na+-Driven ATP Synthases
In 1984, Dimroth and his colleagues (Hilpert et al., 1984) published some
evidence that A/~Na+--*ATP energy transduction occurs in P. rnodestum, a
strictly anaerobic marine bacterium which uses conversion of succinate to
propionate as the only energy source. Decarboxylation of an intermediate of this
process is coupled to Na + extrusion (see above). The resulting AONa + was
assumed to support ATP synthesis by a Na+-driven ATP synthase. In agreement
with this assumption, it was found that a Na+-stimulated FoFl-type ATPase is
present in P. modestum. The subunit composition and amino acid sequence of the
enzyme revealed homology with the E. coli FoF1 (Dimroth, 1987; Amann et al.,
1988; Ludwig et al., 1990). The enzyme incorporated into liposomes proved to
operate as an electrogenic Na + pump. When F1 was partially removed from P.
modestum ATP-synthase proteolipsomes, ATP hydrolysis was ineffective in
sustaining the Na + gradient, but the addition of F~ from E. coli made it possible
to sustain the gradient (Hoffmann et al., 1990). In everted subcellular vesicles of
P. modesturn, decarboxylation was shown to support some ATP synthesis.
Monesin dissipated the Na + gradient and blocked the synthesis. Unfortunately,
the rate of phosphorylation was very low, in fact 10 4 times lower than that of
decarboxylation and 103 times lower than the rate of hydrolysis of the added ATP
(Hilpert et al., 1984; Dimroth, 1987).
The synthesis of ATP supported by artificially-imposed A/~Na + has been
424
Skulachev
shown in intact cells of some methanogenic bacteria. The physiological role of the
putative Na+-driven ATP synthase or Na+-motive ATPase in this case remains
obscure. (For reviews, see Skulachev, 1988).
When studying cells of the same microorganisms, indications have been
obtained that some energy-releasing step(s) of the methanogenesis-linked electron transfer are coupled to A/~Na + formation (Kaesler and Sch/Snheit, 1989;
Gottschalk and Blaut, 1990) whereas the energy-consuming ones, to A/~Na §
consumption (Miiller et al., 1988).
In our group, it was found that ADP phosphorylation, coupled to lactate
oxidation by V. alginolyticus cells is resistant to CCCP. An artificially-imposed
ApNa § supported ATP formation in the presence of an inhibitor of Na§
NADH-quinone reductase, the effect being monensin- and DCCD-sensitive and
CCCP-resistant (Skulachev, 1985; Dibrov et al., 1986; 1989). Recently ApNa §
induced phosphorylation was reproduced by Guffanti and Krulwich (1988) in V.
alginolyticus and by Sakai et al. (1989) in V. parahaemolyticus. It was also found
that ATP hydrolysis by inside-out V. alginolyticus vesicles was coupled to Na §
accumulation, which was strongly stimulated by CCCP or valinomycin (+K§
completely inhibited by DCCD and diethylstylbestrol (Dibrov et al., 1988).
In our group, Dmitriev et al. (1989) succeeded in isolating an ATP-synthase
from V. alginolyticus. Its sensitivity to DCCD and venturicidin, subunit composition and N-terminal sequence of major F1 subunits proved to be similar to those
of the FoF1 H§
ATP synthase from E. coli and Na§
ATP synthase
from P. modestum. Further indications of similarities of ATP synthases from
these three bacterial species were revealed when the complete sequence of the V.
alginolyticus unc operon (Krumholz et al., 1989), as well as that of fl-subunit of F1
(Amann et al., 1988) and c-subunit of F0 (Ludwig et al., 1990) of P. modestum,
were obtained.
The carboxy-terminal sequences of the c subunits of V. alginolyticus and P.
modestum proved to be quite similar. The identity of 8 of the 9 carboxy-terminal
residues was shown. Among the next 9 residues, 4 were identical and 4 were of
similar size and hydrophobicity. Two facts should be stressed in this context.
(i) It is the c-subunit glutamic (or aspartic) acid carboxylate group that reacts with
DCCD (in both P. modestum and V. alginolyticus, the DCCD-sensitive residue is
localized in the 25th position from the C-end).
(ii) The carboxylate of the C-terminal glycine is the only anionic group facing the
extracellular water phase in the second transmembrane a-helical column of the
c-subunit. This carboxylate may, in principle, be involved in binding H § and/or
Na § .
A very important observation has recently been made by Laubinger and
Dimroth (1989). They found that the P. modestum Na§
ATP synthase
transports either Na § or H § depending on the [Na § level in the incubation
medium. It is the H § ion that is transported when [Na § is -<0.2 mM. At higher
[Na§ no H § pumping occurs. A similar situation was observed when the animal
Na§ + ATPase was studied. This enzyme, which is known to be very specific for
Na § was shown to catalyze H§ +, instead of Na+/K § antiport at slightly acidic
pH and low [Na § (Hara et al., 1986; Polvani and Blostein, 1988).
ChemiosmoticBioenergetics
425
A few examples have been described in which an ion-metabolite symporter
recognizes both H § and Na § (for refs., see Skulachev, 1989a). It is an H § flux
that is responsible for ion conductance through the Na § channel in nerve fibers in
the absence of Na § (Mozhayeva and Naumov, 1983). Two possible mechanisms
can be considered to explain such N a + / H § substitutions: (i) H § and Na § combine
with one and the same anionic group (e.g. carboxylate) of the enzyme, porter or
channel protein, located in the water/membrane interface; (ii) It is H3 O+, not
H § that combines with the protein in the interface. It is well known that in water
solution, most H § ions combine with H20 to form H30 +. As was recently
pointed out by Boyer (1988), crown ethers of Na § and H3 O+ are structurally very
similar. Boyer proposed that interchangeability of Na § and H § in biological
systems is accounted for by involvement of crown-like structures. Such structures
might apparently be formed by peptide bond carbonyls and a~-helical columns
which are typical of membrane proteins.
At neutral pH, the concentration of H3 O+ is 1 • 10-7M i.e. 5 • 106 times
lower than that of Na § in the sea water (5 • 10 -1 M). This means that the gate of
the proton channel in factor F0 must be of extremely high specificity to interact
with H3 O+ rather than with Na § Thus, it seems probable that any change, even
a small one, in the gate structure will result in a decrease of specificity and,
hence, it will facilitate the transport of Na § instead of H r. In agreement with the
above reasoning, Junge and co-workers showed that the rearrangement or
fragmentation of the chloroplast Fo under certain conditions causes Na § or K +
permeability which, like that of H § is venturicidin-sensitive (Sch6nknecht et al.,
1989). One may speculate that the FoFl-type Na § driven ATP synthase of P.
modestum has appeared as a result of a mutation in the H+-motive ATP synthase
gene, which causes some decrease in the ion specificity of F0.
At physiological [H +] and [Na+], P. modestum ATP-synthase operates with
Na § not H § (Laubinger and Dimroth, 1989). This is not surprising since A/~Na §
is the only driving force for ATP synthesis in P. modestum, and the Na § cycle,
not H § cycle, operates as the chemiosmotic system. As to the V. alginolyticus, it
seems to be equipped with both H § and Na + cycles. The initial and terminal
segments of the V. alginolyticus respiratory chain are Na § and H+-motive,
respectively (Skulachev, 1989a, b). According to Krumholz et al. (1989), there is
only one typical ATPase gene in V. alginolyticus. This conclusion is in agreement
with the sequence data of Dmitriev et al. (1989). Thus, it seems reasonable to
assume that, under physiological conditions, V. alginolyticus FoF1 ATP-synthase
can operate either with H § or with Na § (Skulachev, 1989b). Switching from H §
to Na + may be a result of a decrease in [Houri,
+
an event regularly occurring in
vitro. In fact, the V. alginolyticus cells live in mats of algae where strong pH
fluctuations take place due to the photosynthetic activity. The pH, which is
neutral in the morning, shifts to high values in the evening. Such a shift may
cause the energetic switch-over from the H + to the Na § cycle. Thus, we may say
that bacteria like V. alginolyticus "have H § for breakfast and Na § for dinner".
Thus, the F0F 1 ATP synthase exemplifies the case in which one and the same
enzyme is involved in Na § and H § pumping. This is in contrast to the situation in
the respiratory chain where the H+-motive enzymes strongly differ from the
Na+-motive ones (see above).
426
Skulachev
INTERRELATIONS OF THE H +- A N D Na+-CYCLES
The lowering of [Hout]
§ seems to be an obvious reason for the cell to
substitute Na § for H + as the coupling ion. Alkaline pH of the medium creates at
least two difficulties in the use of the H § cycle. One of them is kinetic. H + ions,
extruded from the cell by, e.g., the H+-motive respiratory chain, are immediately
neutralized by the external alkaline solution so that the proton-acceptor devices
of AgH + consumers must have a very high affinity for H + to be operative. This
should create a problem in releasing the H § ions, translocated by the consumer,
to the cytoplasm where pH is lower than outside.
An even more dramatic problem arises when we take into account the
thermodynamics of the process. As long as pHin is lower than pHout, the
formation of Atp (inside negative) by a H+-extruding A~H+-generator is
counterbalanced by ApH in the opposite direction (Fig. 17). Alkalinization of the
cytoplasm is impossible due to inactivation of intracellular enzymes. Further
increase in AtP (to compensate the opposing ApH) is also impossible because of
the electric break-down of the cytoplasmic membrane. Reorientation of A~H +generators in the membrane can hardly solve the problems since in this case, the
direction of the transmembrane electric field would be opposite to the usual one
(inside positive). This in its turn, would make it impossible to retain the normal
arrangement of all the membrane proteins which are electrophoretically oriented
inside the membrane because negatively and positively charged groups are
localized mainly on the outer and inner membrane sides, respectively (Skulachev,
1988).
Substitution of Na + for H + solves all these problems. The Na + extrusion
forms AW in the right direction. Downhill Na + influx actuates A/~Na+-consumers
which perform chemical, osmotic or mechanical work in spite of the unusual
direction of the pH gradient (Skulachev, 1984b, 1985). This is why all the
alkalophilic and alkalotolerant species require Na t to survive. The use of the
sodium cycle seems especially convenient for marine bacteria since here [Na+]out
is always high.
In non-marine alkalophilic Bacilli, osmotic and mechanical work were shown
to be also A/~Na+-dependent. However, the mechanisms of A/~Na+-generation
and ATP synthesis remain obscure. Attempts to identify primary Na § pumps in
these microorganisms are as yet unsuccessful (Krulwich and Guffanti, 1989).
According to Krulwich and Guffanti, A/~Na § is formed by a Na+/nH § antiporter
where n is a large number (to produce high A/~Na + at the expense of very low
A~H + created by the usual respiratory chain A/lH+-generators found in these
Bacilli). As to respiratory ATP formation, it is speculated that it may be
catalyzed by a "microchemiosmotic" mechanism in which H § ions, pumped by
the respiratory chain are directly channelled to an H§
ATP synthase
without being neutralized by the external alkaline medium (Krulwich and
Guffanti, 1989). A general microchemiosmotic coupling principle, applicable to
any coupling ion or ligand species, was described by Mitchell and Moyle (1958).
Also, a direct transfer of unhydrated protons between a respiratory enzyme and a
non-translocational ATP-forming enzyme, different in principle from the FoF1
Chemiosmotic Bioenergetics
427
|
.
~RGY
ACIDOPWILES:
[,+]o
A ~!-t = RT In +---~
[ij ;
~"~_.-~"~WOR K
|
NEUTROPI41LES"
11+
A ~t4 = FAy + RT In [14 +]o
[,+]~
K
|
~~/~N~ERGu
E
ALKALOPNILES"
A ~H'--o
Fig. 17 AtIt and ApH constituents of A/~H+ in acido-, neutro- and alkalophilic bacteria.
ATPase, was postulated by Williams (1961), but has never been experimentally
proved in spite of many attempts repeatedly undertaken by numerous bioenergeticists (for reviews, see Skulachev, 1984a, 1988).
It seems possible, however, that in non-marine atkalophiles, oxidative
phosphorylation is carried out by H+-motive respiratory chain enzymes and
H+-driven ATP synthases localized in "sluices" formed by outer and cytoplasmic
membranes, which we postulated to explain transmembrane transport (see above,
Fig. 12). In this specific case, one should assume that: (i) the porin channels are
428
Skulachev
outer medium
,S
X
cytoplasm
Fig. 18 Hypothetical scheme explaining operation of the H +
cycle in the non-marine alkalophilic Bacilli. It is assumed that a
lens in the cytoplasmic membrane allows respiratory enzymes
and the H+-driven ATP synthase to operate without direct
contact with the outer medium.
always closed; and (ii) the respiratory enzymes and H+-motive ATP synthase are
plugged through the cytoplasmic, not outer, membrane. Involvement of lenses
formed by the cytoplasmic membrane may be another version of this idea (Fig.
18). In both cases, one should postulate a "third" water phase separated from the
alkaline outer medium by an H+-impermeable membrane. Mechanisms of this
kind may be specific for such functions as ATP synthesis which require high
A/~Na + levels to be operative. In non-marine bacteria, [Nao+t] is apparently too
low to maintain high ApNa and, hence, high A~Na § On the other hand, failure
of attempts to find Na+-motive respiration and Na+-dependent ATP synthesis in
alkalophilic Bacilli may be due to inadequate in vivo or in vitro conditions. This
seems quite possible for bacteria exploiting both H § and Na + cycles, like V.
alginolyticus, Bac. FTU or S. faecalis. In this context, it should be noted that a
precedent when a non-marine aerobic bacterium possesses Na+-motive respiration is already described by Efiok and Webster (1990a, b), who studied
alkalotolerant VitreosciUa found in hypoxic habitats such as benthic regions of
fresh water sources. An indication was obtained that it is an o-type cytochrome
that operates as the Na+-motive terminal oxidase in Vitreoscilla.
Experiments on S. faecalis, Bac. FTU and recently on E. coli clearly showed
that in these bacteria, substitution of Na § for H § as the coupling ion is of more
general importance than adaptation to an alkaline niche. Rather, it is a response
to failure of the H § cycle to support the membrane energetic system of the cell.
In S. faecalis, the Na+-motive ATPase was induced when: (i) pHout increased; (ii)
a protonophorous uncoupler was present in the growth medium; or (iii) the
H+-ATPase was inactivated due to mutation (see above). In Bac. FTU,
activation of Na+-motive respiration was found to be induced by growth at
Chemiosmotic Bioenergetics
429
high pH or, alternatively, at neutral pH, but in the presence of uncoupler.
Recently similar relationships were shown in our group when E. coli growing on
succinate at pH 8.6 was studied. It was revealed that under these conditions,
besides the usual H+-motive N A D H - C o Q reductase and the H§
CoQH2
oxidase, E. coli possesses a Na§
N A D H - C o Q reductase and a Na §
motive CoQH2 oxidase (Avetisyan et al., 1989; 1991) (Fig. 19C). At pH 7.5, only
the H§
respiratory chain was operative in E. coli, and AfiNa + was formed
in a A/~H+-dependent fashion by the Na§247
The antiporter was
inactive in the cells growing at pH 8.6.
In V. alginolyticus, the Na§
NADH-quinone reductase was active
even when the cells were growing at neutral pH. At the same time, the terminal
oxidase was always H§
(Fig. 19B). Thus V. alginolyticus is always well
A
Propionyl
CoA+CO~
ka,Uk4
Fig. 19 Interrelations of Na +- and H-~-linked energy transducers. Heavy and light arrows,
systems specific for H + or for Na +, respectively. Dotted arrows, systems equally effective
with H t and Na t . A, the Na § cycle in P. modesturn. Purely sodium energetics (no A # H t
generators and consumers are present). 1, Decarboxylase converts methylmalonyl CoA to
propionyl CoA and CO 2 in a Na§
fashion. 2, Downhill Na t influx via Na+-driven
ATP synthase is coupled to ATP formation. (From Dimroth, 1987). B, V. alginolyticus
energetics. One and the same respiratory chain includes A/~Na +- and A#Ht-generators in
its initial (1) and terminal (2) segments, respectively. One and the same ATP-synthase (3) is
postulated to consume A/2Na + or A/~H +. C, Energetics of Bac. FTU and E. coli growing at
alkaline pH. In addition to the Hi-motive respiratory chain (1, 2) there is the Na§
one (la, 2a). Each chain includes at least two energy-coupling sites, one before quinone and
another after quinone. D, Energetics of the animal cell. The Na +- and Ht-cycles are
localized in two different membranes i.e. inner mitochondrial membrane and outer cell
membrane. 1. In mitochondria, respiration generates AfiH + which is utilized by the
Ht-driven ATP synthase (2). Formed ATP is exchanged for extramitochondrial ADP and
Pi via the A T P / A D P antiporter (3) and phosphate-H + symporter (to simplify the scheme,
H t influx via the latter system is not shown). 4, ATP is hydrolyzed by the N a t / K t ATPase
in the outer cell membrane and Na § is pumped out of the cell. 5, Downhill influx of Na +
via Nat-metabolite symporters results in accumulation of these metabolytes in the cytosol.
4.~
Skulachev
B
Nff
No,+ H +
H§
a
~
1
H+
'
c
~; ~.
No+ H +
d
D
~"~'1
NAD~I~ H ~ ~
AbP.~
+
" No'H+
~~02
outer cell \ ~
membrane~ \
+
~I
metobotytes\ \ me~abo,ytes
cytosol
Fig. 19
(Continued)
Chemiosmotic Bioenergetics
431
equipped to live under either neutral or alkaline conditions. This can easily be
explained by adaptation to a niche where the pH is fluctuating between low and
high values (see above).
For P. modestum (Fig. 19A), always living at neutral pH, we need to find
another explanation why Na + and not H + is used. The simplest idea is that
Na+-motive decarboxylases were discovered by evolution whereas H+-motive
ones were not. Such an explanation seems especially attractive if we assume, after
Rosen (1986), that the Na + cycle evolved prior to the H + energetics, which arose
as an adaptation to fresh water when marine microorganisms spread from sea to
rivers and lakes. It seems, however, more probable that the H + cycle was the first
one. In the case of the direct (redox loop) chemiosmotic mechanism shown above
in Fig. 2, the electrochemical potential difference of hydrogen ions appears to be
the inevitable consequence of the chemistry of the energy-supplying oxidative
reaction. The simplicity of such a system may point to its evolutionary priority.
On the other hand indirect chemiosmotic energy transducers, like the H +transhydrogenase and, most probably, the H+-driven ATP synthase look like
later inventions of biological evolution. Here, A#H + formation is not a direct
consequence of the energy-releasing process, and hence, the energy coupling is
organized in a more sophisticated manner. The systems of this type are not
inevitably connected with the transport of a hydrogen ion. Therefore it is not
surprising that one and the same enzyme can transport either H + or Na + as in the
case of the P. modestum ATP-synthase.
The A/~Na+-generators clearly fall into a class of indirect energy transducers
and, hence, may be regarded as the latest in the evolution of membrane
energetics.
It is remarkable, considering its relatively recent evolution, that the animal
outer cell membrane, employs Na + as the coupling ion. The only type of work
performed by this membrane is osmotic i.e. uphill transport of metabolites. Blood
and lymph are assumed to be little pools of the sea in our organism. Therefore, it
is not surprising that the osmotic work of the outer membrane of our cells, facing
the Na+-rich intercellular liquid apply Na + as the coupling ion like marine
bacteria when they perform the same type of work. As to the H+-cycle, it is
localized in quite another place in the cell, namely in the inner mitochondrial
membrane (Fig. 19D).
H+-linked and Na+-linked chemiosmotic systems are listed in Table 1.
This table shows the great diversity of chemiosmotic systems (the A/~H + and
A/INa + generators as well as the A/~H + and AfiNa + consumers) that are
employed by living cells. It also illustrates the general bioenergetic principle that
it does not matter how the cell produces convertible energy currencies, i.e. ATP,
AfiH + and AfiNa +, as long as the rate of their production is sufficiently high to
support all the energy-consuming processes in the cell. P. modestum on the one
hand, and plant and animal cells on the other hand, seem to be the most
demonstrative extreme examples. In the former case, there is only one type of
generator of convertible energy currency, i.e. the Na+-motive methylmalonyl
CoA decarboxylase, and only two such currencies, AfiNa + and ATP. In the plant
cell, there are several AfiH + generators (the mitochondrial respiratory chain and
Skulachev
432
Table 1.
Chemiosmotic systems based on circulation of H § or Na +
Type of system
A. A/~H+-generators
A1. Reaction centre complex in the cyclic bacterial
photoredox chain
A2. Reaction centre complex in non-cyclic bacterial
photoredox chain
A3. Reaction centre complex of Photosystem I
A4. Reaction centre complex of Photosystem II
A5. NADH-Q reductase
A6.
QH2-cytochrome c reductase
A7.
Cytochrome c oxidase
A8.
Fumarate-reducing complex
(may be involved in Na § pumping)
Cytochrome o complex
(may be involved in Na + pumping)
Cytochrome d complex
Methanogenesis-linked electron transfer
Bacteriorhodopsin
H+-ATPase of F0F 1 type
H+-ATPase of vacuolar type
A9.
AI0.
All.
A12.
A13.
A14.
A15. H+-ATPase of EIE 2 type
B. AfiH+-consumers
B1. H+-ATP-synthase of FoFl-type
B2.
B3.
B4.
B5.
H+-ATP-synthase similar to vacuolar H+-ATPase
H+-PPi-synthase
H+-transhydrogenase
Reversalof NADH-Q reductase
B6.
Reversal of QH2-cytochrome c reductase
B7.
H +, solute-symporters, H +/solute-antiporters
B8.
B9.
H+-motor
Proteins mediating the fatty acid-induced uncoupling
(thermogenin, ATP/ADP-antiporter)
C. A/~Na+-generators
C1. Na+-NADH-Q reductase
C2.
Na+-QHz-oxidase
C3. Na+-decarboxylases
C4. Methanogenesis-linked electron transfer
C5. Na+-ATPase similar to vacuolar H+-ATPase
C6. Na+-ATPase of methanogenic bacteri
C7. Na+/K+-ATPase
D. AgNa+-consumers
D1. Na+-ATP-synthase
D2. Methanogenesis-linked electron transfer
D3. Na +, solute-symporters, Na+/solute-antiporters
D4.
Na+-motor
Distribution
Purple bacteria
Green bacteria
Chloroplasts, cyanobacteria
Chloroplasts, cyanobacteria
Mitochondria, many aerobic and
photo-synthetic bacteria, some
anaerobic bacteria
Mitochondria, some aerobic
bacteria, chloroplasts
Mitochondria, some aerobic
bacteria
Some anaerobic bacteria
Some aerobic bacteria
Some aerobic bacteria
Methanobacteria
Halobacteria
Some anaerobic bacteria
Tonoplast of plant and fungal
vacuoles, secretory granules of
animal cells
Outer membrane of plant and
animal cells
Mitochondria, chloroplasts,
eubacteria
Archaebacteria
Purple bacteria, tonoplast
Mitochondria, some bacteria
Bacteria oxidizing substrates of
positive redox potential,
mitochondria
Bacteria oxidizing substrates of
positive redox potential
Mitochondria, chloroplasts, nonmarine bacteria
Many bacteria, some chloroplasts
Mitochondria
Marine bacteria, E. coli growing
at alkaline pH
Bacilus FTU, E. coli growing at
alkaline pH, Vitreoscilla
Some anaerobic bacteria
Methanobacteria
Streptococcus faecilis
Methanogenic bacteria
Animal outer cell membrane
Some marine bacteria
Methanobacteria
Marine bacteria, animal outer cell
membrane
Alkalophilic and alkalotolerant
bacteria
Chemiosmotic Bioenergetics
433
the chloroplast photoredox chain, and the H+-motive ATPases of the plasma
membrane and tonoplast) and two energy currencies (ATP and A#H§ The
animal cell, possessing both A/ill + generators (mitochondria, secretory granules
and lysosomes) and A/~Na + generators (plasma membrane), uses all three energy
currencies, i.e. ATP, A/ill + and A/~Na +.
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DISCUSSION
G. Scarborough: I v e r y m u c h like y o u r m o d e l for t h e m e c h a n i s m o f b a c t e r i o r h o d o p s i n . I p u b l i s h e d o n e q u i t e s im i la r to it in M i c r o b i o l o g i c a l R e v i e w s 5 y ear s
ago. B u t m y q u e s t i o n is, d o y o u k n o w t h e N a § s t o i c h i o m e t r y o f t h e F o F l - t y p e
Na § pump?
V. P. Skulachev: A c c o r d i n g to D i m r o t h , t h e N a + / A T P
FoFl-type N a §
ATPase from Propionigenium
stoichiometry of the
m o d e s t u m s h o u l d be h i g h e r
442
Skulachev
than 2. Such a conclusion is an inevitable consequence of the facts that (i) the
Na+-motive methylmalonyl CoA decarboxylase is shown to transport 2Na + per
one molecule of the decarboxylated substrate and (ii) the energy yield of this
decarboxylation is about 7 Kcal per mole. Since the ATP synthesis costs, under
physiological conditions, about 10 Kcal per mole, 3 or more Na § should be
transported through Na§
ATPase to form one ATP. Direct measurement
of this stoichiometry is not done yet.
E. Carafoli: Could you comment on the mechanism of sodium extrusion linked
to the operation of the respiratory chain?
V. P. Skulaehev: The mechanism of Na + extrusion by the Na§
respiratory chain remains obscure. It is, however, already clear that the Na§
NADH:quinone reductase and the terminal oxidase are not identical to the
H§
reductase and terminal oxidase. E.g. the Na§
reductase of
Vibrio alginolitucus is, according to Unemoto's group, a three subunit enzyme
containing FAD and FMN and no FeS clusters. It is sensitive to very low HQNO
and Ag +, by contrast with the multisubunit H§
reductase which contains
FMN and 5 types of FeS clusters, and is resistant to low HQNO and Ag +. The
Bacillus FTU Na§
oxidase is resistant to micromolar cyanide, requiring
1 mM KCN to be completely inhibited, whereas the H§
oxidase from the
same Bacillus is arrested by low [KCN].
W. N. Konings: In Na + translocation by the NADH : Q oxidoreductase, quinones
are involved. The Bacilli like Bacillus FTU contain menaquinones. Is there any
information about the role of these menaquinones in the Na§
process coupled to NADH oxidation? You also indicated that the cytochrome
oxidase in E. coli and in Bacillus FTU is directly involved in Na § translocation.
The cytochrome c oxidase activity leads in any case to the generation of a A q9 and
ApH. In these two organisms the activities of cytochrome oxidase could then lead
to the generation of a A/~Na + in addition to a A#H + and ApH. Is there any
information about the generation of the A~H + components?
V. P. Skulachev: There is not yet any information about participation of
menaquinone in the Na+-motive respiration. An intriguing idea was put forward
some years ago by Lanyi and coworkers that it is the Na + salt of the quinol (or
semiquinone) anion that is transported across the membrane when the Na §
motive NADH-quinone reductase is involved. As to the Na+-motive terminal
step of the respiratory chain, it is catalyzed, apparently, by a quinol oxidase
rather than by cytochrome c oxidase. Our preliminary data indicate that H § is not
transported by the Na§
oxidase, at least at alkaline pH.
G. Hauska: In your list of the "sodium world" you included formaldehyde
reduction by hydrogen as a sodium pump in methanogenic bacteria. However,
according to the work of Gottschalk/G6ttingen and others, methanogenesis
primarily creates a protonmotive force, and sodium gradients are more likely to
be used for driving energy-requiring redox reactions, like the reduction of formic
acid to formaldehyde with hydrogen in Methanobacter, or the oxidation of
methanol to formaldehyde by F420 in Methanosarcina.
ChemiosmoticBioenergetics
443
V. P. Skulachev: I agree that this system operates as a A/iNa + consumer rather
than generator. However, formally speaking, it can also generate AfiNa +
assuming reversibility of such energy transducers.
G. Scarborough" Following up on my earlier question, is it possible that protons
move through the FoFl-type Na + pump with Na+? And if so, is it not premature
to rule out a direct oxideion-motive type mechanism for ATP synthesis by this
enzyme?
V. P. Skulachev: As it was directly shown by Dimroth, H + is taken up by the
Na+-motive ATPase proteoliposomes only if Na + is absent. It does not occur
when millimolar Na + is present. This does not exclude that the "intra-protein"
H + is somehow involved in the Na + transport (e.g. an "internal" Na+/H +
antiporter which cannot carry out transmembrane Na+/H + exchange but can
catalyze Na+/H + exchange between F0 and F1). However, the subunit composition of the P. modestum Na+-motive ATPase proved to be identical to that of the
E. coli H+-motive ATPase. So, there is no room for a Na+/H + antiporter or any
additional protein(s) in the Na+-motive ATPase. Moreover, the sequences of the
subunits are also similar.
J. Ingledew: With respect to the indicated Na+-translocation by the E. coli
oxidase: E. coli can synthesize two (quinol) oxidases, cytochrome bo and
cytochrome bd. Interestingly, the former has extensive similarities including
sequence homology, with cytochrome aa3. Is it known which of the two E. coli
oxidases is Na+-translocating?
V. P. Skulachev- The resistance to low cyanide is today the only information
about the properties of the Na+-motive E. coli oxidase. In this respect, it
resembles the Na+-motive oxidase of Bacillus FTU. The E. Coli ba oxidase is also
of low cyanide sensitivity. However, it is not excluded that our enzyme is a
special type of bo oxidase rather resistant to cyanide. Webster insists that
Na§
oxidase of VitreosciUa is of bo type. Unfortunately he did not titrate
the activity by cyanide, simply indicating that 5 mM KCN kills the enzyme.
E. Padan: Do you suggest a complete Na + cycle in E. coli including the
Na+-motive ATPase?
V. P. Skulachev: It seems possible that E. coli uses the respiratory chainproduced AfiNa + to form ATP by its FoF1 ATPase as in V. alginolyticus. An
alternative possibility is that, under the low AfiH + conditions, AfiNa + supports
membrane-linked work other than ATP synthesis whereas ATP is produced by
glycolysis. This problem is now studied by our group.
F. Harold: We now have a variety of sodium-transport pathways, including
electron transport chains and ATPase. Is it possible to draw direct coupling
mechanisms for sodium-coupled energy transduction?
V. P. Skulachev: If Lanyi's idea on (semiquinone anion-Na) or (quinol bianion2Na) is right, decomposition of the complexes due to semiquinone (quinol)
oxidation may be regarded as an example of a (rather) direct coupling mechanism
of the Na + pump. Today, however, this is pure speculation.
444
Skulachev
T. A. Link: After the "first w o r l d " - - p r o t o n bioenergetics--, the "second
world"--sodium bioenergetics--was detected. Do you expect a "third world" to
be discovered, i.e., Bioenergetics using other ions like potassium or anions (e.g.
chloride)?
V. P. Skulaehev: There are no indications of the existence of such a "third
world" in bioenergetics. Rather, all the events already described seem to be
explained within the frameworth of H § or Na § cycles. In some membranes, the
primary pumps were found to translocate K § Ca 2§ CI- and some other ions or
non-ionized compounds. However, these membranes lack consumers of A/~K §
A/ICa 2+, etc. This means that, say, the Ca 2§ cycle cannot be organized.