Springer-VerlagTokyo102650918-94401618-086030669031Journal
of Plant Research J
Plant Res010810.1007/s10265-003-0108-4
J Plant Res (2003) 116:401–418
Digital Object Identifier (DOI) 10.1007/s10265-003-0108-4
© The Botanical Society of Japan and Springer-Verlag Tokyo 2003
JPR SYMPOSIUM
Hiroshi Kitasato
Membrane potential genesis in Nitella cells, mitochondria, and thylakoids
Received: December 31, 2002 / Accepted: April 4, 2003 / Published online: August 13, 2003
Abstract The resting membrane potential of Nitella cells
shifts in parallel with the change in H+ equilibrium potential, but is not equal to the H+ equilibrium potential. The
deviation of the membrane potential from the H+ equilibrium potential depends on the extrusion rate of H+ by the
electrogenic H+-pump. The activity of the electrogenic H+pump was formulated in terms of the change in the free
energy of ATP hydrolysis. The deviation of membrane
potential from the H+ equilibrium potential induces a passive H+ flow. The passive inward H+ current may be coupled
with Cl- uptake. The coupling rate of H+,Cl- co-transport
was discussed. The membrane potential of mitochondria
was electrochemically formulated in terms of oxidation–
reduction H2/H+ half-cells spontaneously formed at the inner
and outer boundaries of each trans-membrane electronconducting pathway. The membrane potential formed by a
pair of H2/H+ redox cells is pH-sensitive in its nature, but
deviates from the H+ equilibrium potential to an extent that
depends on the logarithm of the ratio of H2 concentrations
at the inner and outer boundaries. The membrane potential
of thylakoids is considered to be primarily due to the electromotive force of photocells embedded in the thylakoid
membrane, as far as the anode and cathode of each photocell are in contact with the inner and outer solutions, respectively. The light-induced electronic current yields oxygen at
the inner boundary and causes an increase in the H2 pool
at the outer boundary of the electron-conducting pathway,
which has no shunting plastoquinone chain between these
two boundaries.
Key words Cells · Mitochondria · Thylakoids · H+-pump ·
Electron-conducting pathway · Membrane potential
H. Kitasato (*)
Department of Physiology, Shiga University of Medical Science,
Ohtsu, Shiga 520-2192, Japan
Tel. +81-75-9211368; Fax +81-75-9211368
e-mail: hkitasat@mbox.kyoto-inet.or.jp
Introduction
A lipid bi-molecular layer membrane plays a key role in
converting and storing energy, owing to its high hydrophobic property that does not allow electrochemical energy to
dissipate without performing any tasks valuable to the cell.
The resting membrane potential is inside-negative even in
plant cells. In animal cells where the high concentration of
cytoplasmic K+ ions and the relatively high permeability of
the plasma membrane to K+ play an essential role in maintaining the resting potential. It is generally accepted that in
plant cells Na+ and K+ are not the main cations subjected to
active transport, although K+ ions are accumulated in the
cytosol (Spanswick and Williams 1964; Gutknecht 1965;
Kishimoto and Tazawa 1965; Kotyk and Janacek 1970;
MacRobbie 1970; Hope and Walker 1975). The permeability
to K+ is not large enough to be able to account for the
membrane potential of Nitella cells. In 1968, the author
found that the resting membrane potential alters nearly
58 mV for a 10-fold increase in extracellular H+ concentration in the pH range between 5 and 7 (Kitasato 1968). This
finding was considered to be what indicates that the H+
conductance is much higher than the conductance for any
other ion. Usually, when any one species of ion has a very
high conductance compared with those for other ions, the
membrane potential may be very close to the equilibrium
potential of the ion species. However, experiments showed
that the membrane potential of Nitella cells was more negative (by as much as about -100 mV) than the H+ equilibrium potential. From this large discrepancy, it was deduced
that a large current carried by H+ ions must always flow
down across the plasma membrane from outside to inside.
Although the continuous H+ influx was expected, the H+
concentration of cytosol remained at a steady level. These
findings led him to propose the presence of an electrogenic
H+ pump in Nitella cells, besides the H+ channels (Fig. 1). At
the same time, two questions arose from the expected large
inward H+ flow: one is whether is it rational that the H+ ion
flows down across the plasma membrane without performing any work valuable for the cell; and the other is that, if
402
ture or equipment is called the H+-pump. The tendency of
ATP hydrolysis to ADP and Pi is expressed by the Gibbs’
free energy change. When dn moles of ATP are hydrolyzed,
the free energy change in this chemical system, DGATP, is
described as follows:
DG ATP = m ATP (- dn) + m ADP dn + mPi dn,
+
+
Fig. 1. Electrogenic H -pump and H channel in Nitella cells
the passive H+ flow were coupled to any work useful for the
cell, would the apparent conductance of the pathway for the
passive H+ flow be high enough to account for the total
membrane conductance. These questions have remained to
be solved for long time in his mind. Consideration on these
matters led to the following discussions concerning the
potential dependence of the electrogenic H+-pump and the
work done by the passive H+ flow.
where mATP, mADP, and mPi respectively represent the chemical potentials of ATP, ADP, and Pi. For each of these,
the chemical potential is expressed by the following
equation:
0
m ATP = m ATP
+ RT ln[ ATP ],
0
+ RT ln[ ADP ],
m ADP = m ADP
mPi = mPi0 + RT ln[ Pi ].
Before solving the question whether H+ flows down without
accomplishing any work valuable for the cell, we have to
understand the potential dependence of the electrogenic
H+-pump. In 1960s, a yellow dye (2,4-dinitrophenol; DNP)
was already known to suppress sodium efflux in the squid
giant axon (Hodgkin and Keynes 1955, 1956). It was widely
accepted that DNP uncouples oxidative phosphorylation
in mitochondria (Carafoli and Rossi 1967; Caswell and
Pressman 1968). In this background, the effect of the uncoupler of oxidative phosphorylation on the membrane potential was examined. The resting potential of Nitella was
diminished by DNP (Kitasato 1968), suggesting the contribution of ATP to the deviation of resting membrane potential from the H+ equilibrium potential. Evidence supporting
the hypothesis that ATP may contribute to the activation of
the electrogenic H+-pump was accumulated by many
researchers (Higinbothan et al. 1970). Slayman et al. (1973)
observed the close relationship between the membrane
potential and the internal ATP concentration in Neurospora
crossa. Soon after, Shimmen and Tazawa (1977) directly
demonstrated the ATP-driven electrogenic pump in Chara
cells, using the newly invented internal perfusion of
tonoplast-free cells with solutions containing a variety of
various phosphate compounds. They found that the negative resting potential is maintained by Mg2+-ATP but not by
adenylyl imidodiphosphate.
The energy released from ATP hydrolysis
ATP spontaneously breaks down to ADP and inorganic
phosphate, Pi. If this chemical tendency is coupled with the
translocation of H+ ions in a membrane structure, H+ can be
transported against its electrochemical potential. The struc-
(2)
0
ADP
R and T have their usual meanings, respectively. m , m0Pi,
and m0ATP are the standard free energies, namely the free
energies of 1 mol/l ADP, Pi, and ATP in a standard state.
Thus, DGATP is:
0
0
+ mPi0 - m ATP
+ RT ln
DG ATP = Ê m ADP
Ë
Fundamental thermodynamics of ATP hydrolysis and
ATP-driven H+-pump
(1)
[ ADP ][ Pi ]ˆ
dn.
[ ATP ] ¯
(3)
m0ADP + m0Pi - m0ATP is the standard free energy change.
When in quasi-equilibrium, DGATP is very close to zero and
the concentration ratio is nearly equal to the equilibrium
concentration ratio. Let us denote the equilibrium concentration ratio as:
Ê [ ADP ][ Pi ]ˆ .
Ë [ ATP ] ¯ eq
(4)
Since DGATP is zero when in equilibrium, the following
relation can be obtained:
[ ADP ][ Pi ]ˆ
0
0
.
m ADP
+ mPi0 - m ATP
= - RT lnÊ
Ë [ ATP ] ¯ eq
(5)
However, the equilibrium concentration ratio is defined
as the equilibrium constant of ATP hydrolysis, KATP:
K ATP ∫ Ê
Ë
[ ADP ][ Pi ]ˆ
.
[ ATP ] ¯ eq
(6)
Thus, the well known relation can be derived as:
m0ADP + m0Pi - m0ATP = -RT(ln KATP). Namely, the equilibrium
constant reflects the standard free energy change. By inserting this relation into Eq. 3, the following equation can be
obtained:
[ ADP ][ Pi ] ˆ
DG ATP = Ê RT ln
dn.
Ë
K ATP [ ATP ]¯
(7)
If DGATP is negative, this reaction proceeds spontaneously. Equation 7 shows that the released energy from this
chemical system depends on the concentrations of ATP,
ADP, and Pi. The value of RT is given as 2.48 kJ/mol (at
25°C). The standard free energy change of ATP hydrolysis
(the free energy change for the hydrolysis of 1 mol ATP
under standard conditions; i.e., all the concentrations of
ATP, ADP, and Pi are 1 mol/l, dn = 1 mol) is listed as
-30.5 kJ/mol (Harper et al 1979, Table 18-1). Using the following equation:
403
-30.5 kJ mol = 2.48 kJ mol ¥ ln
1 mol l
, or
K ATP
30.5 ˆ
mol l ,
K ATP = expÊ
Ë 2.48 ¯
(8)
we may readily calculate the value of KATP as 2.24 ¥ 105 mol/
l. Using this value of KATP, the energy released from ATP
hydrolysis can be calculated at any concentration of ATP,
ADP, and Pi.
The characteristics of the membrane potential of
Nitella cells
+
The reversal potential of the H -pump
We assume the translocation of H+ ions is rigidly coupled to
the decomposition of ATP. The coupling ratio of H+ translocation to ATP hydrolysis is denoted by m, i.e., the extrusion of mdn moles of H+ is coupled to the hydrolysis of dn
moles of ATP. The work done by the H+-pump for pumping
up the mdn moles of H+, DW, against its electrochemical
potential gradient is:
DW = - F (E - EH + )mdn,
(9)
where EH + conventionally represents the equilibrium potential of the H+ concentration electrochemical cell (or the H+
equilibrium potential). The energy consumed by the pump
is preserved in a form of electrochemical potential energy
of H+. Thus, the total change in the free energy with respect
to the whole cell, DGtotal, is:
(10)
DG total = DG ATP + DW .
+
If DGtotal is negative, then the pump extrudes H ions
from inside to outside at the expense of ATP hydrolysis. In
contrast, if it is positive, the pump allows H+ ions to flow
down through the pump and ATP is synthesized from
ADP and Pi. When H+ ions are neither pumped up nor
flow down, DGtotal is zero. In other words, the force acting
on H+ ions located at the transporting site of the pump
molecule is counter-balanced by the force provided by the
hydrolysis of ATP via a possible conformational change of
the transport molecule. The membrane potential where
the movement of H+ through the pump is zero is called the
reversal potential of the H+-pump. The reversal potential,
(Erev)pump, can be derived by inserting Eqs. 7, 9 into Eq. 10
as follows:
(E rev ) pump
RT [ ADP ][ Pi ]
= EH +
ln
.
mF K ATP [ ATP ]
the plasma membrane, and if such an apparatus were so
arranged as to shift the membrane potential more negative
than the reversal potential of the H+-pump, then the H+pump would synthesize ATP from ADP and Pi in the
reverse mode of active H+ extrusion. However, such a
membrane potential-generating apparatus has not been
reported so far to exist in the plasma membrane of the cell.
(11)
Spanswick (1966, 1974) and Felle and Bentrup (1976)
also obtained a similar expression for the reversal potential
of the H+-pump. On the assumption that the concentrations of ATP, ADP, and Pi are all 1 mM, the potential
difference between the reversal potential and the H+ equilibrium potential, (Erev)pump-EH + can be calculated at a variety of coupling ratios (m) as: -480 mV (m = 1), -240 mV
(m = 2), -160 mV (m = 3), -120 mV (m = 4), and -80 mV
(m = 6).
If there were any apparatus generating electromotive
force other than the above-described electrogenic H+pump, such as a redox electrochemical cell or a photocell in
The membrane potential and its pH dependence in
a steady state
The electric current carried by the actively transported H+
ions through the H+-pump can be written in the form of a
function of the difference between the existing membrane
potential and the reversal potential of the H+-pump as
follows:
( IH )pump + ( gH )pump [E - (E rev )pump ],
+
+
(12)
where ( gH + )pump is the conductance of the H+-pump and
(Erev)pump is its reversal potential. As far as the membrane
potential is not equal to the H+ equilibrium potential, H+
ions passively flow across the membrane through the H+
pathways or channels. The passive H+ current is conventionally described as follows:
( IH )passive = ( gH )passive (E - EH ),
+
+
+
(13)
where ( gH + )passive is the conductance of the passive pathway
for H+. In a steady state with regard to the membrane
potential, ( IH +)passive + ( IH +)pump = 0. From these relations, the
following equation can be derived:
E = EH + +
( gH )pump
RT [ ADP ][ Pi ]
ln
.
( gH )passive + ( gH )pump mF K ATP [ ATP ]
+
+
(14)
+
This equation indicates that the resting membrane
potential behaves as if the cell limited by a plasma membrane were a H+ concentration electrochemical cell, except
the potential level is more negative than the EH + , by as
much as:
( gH )pump
RT [ ADP ][ Pi ]
ln
.
( gH )passive + ( gH )pump mF K ATP [ ATP ]
+
+
(15)
+
A similar equation was derived by Kishimoto et al.
(1984) from the kinetic analysis of membrane currents.
Equation 14 indicates that the membrane potential
shifts in parallel to the change in the H+ equilibrium potential independently of the conductance of the passive pathway for H+. This implies that the membrane potential shift
of 58 mV for a 10-fold increase in the external H+ concentration does not necessarily show a high conductance of the
passive pathway for H+. In other words, the high sensitivity
of the membrane potential to pH does not always deny the
possibility that the passive H+ current may accomplish work
useful for the cell. The force acting on the H+ ions can be
given from Eq. 15 in the following form:
404
(force)H + = E - EH +
( gH + )pump RT [ ADP ][ Pi ]
=
ln
.
gH + + ( gH + ) pump mF K ATP [ ATP ]
(16)
It should be noted that the H+ driving force depends both
on the [ADP][Pi]/[ATP] concentration ratio and on the conductance fraction for ( gH + )pump, but does not depend on the
H+ equilibrium potential, against the presupposition evoked
from a form of E - EH + . From the above discussions, the
experimental observation that the resting membrane potential shifts by 58 mV per unit pH change within the pH range
from 5 to 7 may be said to indicate that the force pushing
forward the H+-pump remains constant within this pH
range.
Accumulation of Cl- and cations
An increase in Cl- efflux was reported during the action
potential in Nitella cells (Mullins 1962). This finding implies
that Cl- ions are accumulated in the intracellular space.
Soon after this report, Spanswick and Williams (1964)
demonstrated, using gentle centrifugation, that the cytosolic
Cl- concentration is 65 mM, whereas the vacuolar Cl- is
160 mM in N. translucens. In N. flexilis, the cytosolic Clconcentration is 35.9 mM and the vacuolar Cl- is 136 mm
(Kishimoto and Tazawa 1965). The latter values were
obtained using a sophisticated perfusion technique to separate the flowing cytoplasm from the chloroplast layer
(Tazawa 1964). This perfusion technique newly invented by
Tazawa greatly contributed to the development of research
in the field of the H+-pump in plant cells.
Even though most cations other than H+ can be accumulated in the cytoplasm in a manner to equilibrate with both
their external concentration and the existing membrane
potential, the uptake of anions requires energy under the
conditions of negative membrane potentials. Since Cl- is
one of the major anions in the internal solution, it is important to consider how Cl- is accumulated. The energy to
accumulate Cl- may come from the passive H+ flow (Fig. 2).
Sanders (1980) observed a depolarization in Chara cells
when the Cl--starved cell was exposed to Cl- and postulated
2H+,1Cl- co-transport. If the Cl-,H+ co-transporter has a
fixed coupling ratio (H+:Cl- = r:1) in the plasma membrane
of Nitella cells, the force to drive this co-transport is
described as follows:
(force)Cl - , rH + = r (E - EH + ) - (E - ECl - ).
(17)
When the coupling ratio, r, is more than one, the cotransport carries positive charges in the direction of the Clflux. Namely, such a co-transporter is rheogenic. When
(force)Cl - , rH + is negative, H+ ions flow inward through this cotransporter, and Cl- ions are obliged to translocate from
outside to inside. The membrane potential that makes
(force)Cl - , rH + zero is called the reversal potential of Cl-,rH+
co-transport. The reversal potential of Cl-,rH+ co-transport,
(E rev )Cl - , rH + , is derived from Eq. 17:
(E rev )Cl - , rH + =
r
1
EH + E -.
r -1
r - 1 Cl
(18)
Fig. 2. Schematic drawing of the electrogenic H+-pump, the Cl-,H+ cotransporter, the gated Cl- channel, and the cation channel in Nitella
cells. There may be gated Cl- channels responsible to the regenerative
depolarization at action potential generation. Other ion channels and
transporters are omitted
The cytoplasmic pH of plant cells is within the range 7.0–
7.5 (Walker and Smith 1975; Mimura and Kirino 1984;
Guern et al. 1991). The external Cl- concentration is 3 mM
(Hope and Walker 1975; Kotyk and Janacek 1970). Taking
the value of the cytoplasmic Cl- concentration as 36 mM
(Kishimoto and Tazawa 1965), the ECl - is given as 62 mV.
Assuming that EH + is 0 mV, the value of (E rev )Cl - , rH + is
calculated to be -62 mV. Provided the existing membrane
potential is more negative than (E rev )Cl - , rH + , Cl- ions are
accumulated. If there are no routes to allow the leak-out
of Cl- ions, the cell continues to accumulate Cl- ions
while the membrane potential remains more negative than
(E rev )Cl - , rH + . However, the (E rev )Cl - , rH + eventually becomes
equal to the membrane potential and the membrane potential finally reaches the (Erev)pump, because the inward current
gradually decreases to zero. Under these conditions, the
final value of ECl - is described as follows:
ECl - @ rEH + - (r - 1)(E rev ) pump .
(19)
Taking values of EH + = 0 mV, (Erev)pump = -120 mV, and
r = 2, the final value of ECl - is calculated to be 120 mV, which
gives a cytosolic Cl- concentration of 365 mM. This value is
much higher than the experimentally obtained figure,
implying the presence of Cl- leakage. The function of the
Cl- leak flow is unknown.
The relationship between the ECl - and the membrane
potential indicates that the higher the ATP concentration
the more Cl- is accumulated in cytoplasm. Since only the
fraction (r - 1) of the H+ inflow mediated by the Cl-,rH+ co-
405
transporter is balanced by the H+ outflow driven by the
electrogenic H+-pump, H+ is also accumulated in the cytoplasm as the Cl- accumulation progresses. The accumulated
H+ should make the cytoplasm acidic. However, the cytoplasmic pH is always kept between 7.0 and 7.5 in characean
cells (Walker and Smith 1975; Mimura and Kirino 1984).
This fact implies the presence of some H+ extrusion mechanism other than the electrogenic H+-pump. A strict coupling of K+ uptake with H+ extrusion was reported in
Catharanthus reseus (Sakano et al. 1997). Although the H+/
K+ antiport is expected to play an important role in keeping
the cytoplasm slightly alkaline, there are very few reports
on this subject. This antiport also requires energy. Probably
the energy for driving the H+/K+ antiport is supplied by ATP.
In the steady state where the influx of H+ is balanced by the
efflux of H+ pumped up by the electrogenic H+ pump, the
cytosolic pH depends on the dissociation constant, Ka, and
the buffering capacity of organic acids in the cytosol.
Relating to the accumulation of cations, it seems obligatory
to note that the intracellular Na+ concentration is lower
than the K+ concentration (Spanswick and Williams 1964;
Kishimoto and Tazawa 1965). At present, we cannot completely exclude the possibility that some prototype of Na+/
K+-pump molecules (which are ubiquitous in the plasma
membrane of animal cells) is also functioning in plant cells.
This point will be discussed later.
Although some modification of cationic concentrations
in the cytosol occurs, the Cl-,rH+ co-transport seems to play
a key role in making the total solute concentration high in
the cytoplasm. Usually, the plasma membrane is permeable
to water to some extent. The accumulated Cl- and cations
draw water from the extracellular solution into the cytoplasm. The hydrostatic pressure resulting from absorbed
water or osmosis due to these solutes in the cytoplasm keeps
the form of the cell by generating turgor. Furthermore,
maintaining a high ionic concentration in cytoplasm is
important in terms of lowering the electrical resistance of
the chloroplast stroma and the thylakoid space, because the
high electrical conductivity of this solution is a prerequisite
condition for effectively progressing ATP synthesis and the
electrolysis of water for increasing the H2 pool.
The above discussions show the electrogenic H+-pump is
important not only for keeping the form of the cell but also
for providing materials indispensable for the photosynthesis
of carbohydrates.
Mitochondrial membrane potential
The fact that the Na+/K+-pump is fueled by ATP was beautifully demonstrated by Hodgkin and his collaborators
(Caldwell et al. 1960) in the squid giant axon, using the
technique of micro-injecting high-energy phosphate compounds into metabolically suppressed squid giant axons.
The investigation on ATP synthesis was stimulated by the
discovery of the reverse reaction of Na+/K+-transporting
ATPase artificially induced by the electrochemical potential
of K+ and Na+ in erythrocytes (Garrahan and Glynn 1966;
Glynn and Lew 1970). At present, it is widely accepted that
most ATP is oxidatively synthesized in mitochondria in the
reverse mode of the electrogenic H+-pump. The apparent
dehydration of reactants is accounted for in vague terms by
“chemiosmosis”, originally proposed by Mitchell (1966,
1979). Although the concept that ATP is synthesized from
ADP and Pi at the expense of the electrochemical potential
energy of H+ is supported by experiments (Thayer and
Hinkle 1975), the issue how the electrochemical potential
of H+ or the H+ driving force is formed still remains equivocal. Mitchell (1967) suggested that the H2/H+ oxidation–
reduction reaction contributes to the formation of the H+
driving force. Unfortunately, his proposal has not been fully
understood, partly due to the vagueness of his term, “chemiosmosis”, which is too attractively named. Some people
believe H+ ions are really pumped out by some means from
the internal solution to the external solution to form the H+
driving force (Wikström 1977; Nagel and Morowitz 1978).
Recently, many precise works were done on the structure
of cytochromes, using X-ray crystallography (Iwata et al.
1995, 1998; Tsukihara et al. 1995, 1996; Ostermeier et al.
1997; Xia et al. 1997; Yoshikawa et al. 1998), with the hope
of identifying the putative H+-pump. However, no clear
evidence has been obtained in the cytochromes of
mitochondria.
Boundary potentials between the end of the
electron-conducting pathway and the solution
Mitochondria have inner and outer membranes. The inner
membrane is the main barrier to ionic flows. Since the inner
membrane is not permeable to ions, it can store energy in
a form of ionic electrochemical potential. The most prominent difference in structure between mitochondria and
Nitella cells is that the mitochondrial inner membrane has
the trans-membrane respiratory complex, which is called
the electron transport system, while Nitella cell has no such
structure in the plasma membrane. Other cells also have no
such structure.
None-heme Fe, heme A, heme c, protoheme, Cucontaining protein, and cytochromes a, b, c, and c1 comprise
the electron transport system in respiratory complexes
(Palmer and Hall 1972). Within the molecules comprising the electron transport system, there is an electronconductive moiety. In the electron transport system, these
electron-conductive moieties of the component molecules
may form an electron-conductive chain. Let us now call this
chain in the electron transport system an “electronconducting pathway”. In mitochondria, membrane current
flows through two kinds of pathway: one is the transmembrane electron-conducting pathway and the other is
the ionic channel.
There seems to be some confusion in interpretation
when considering an electric current through the so-called
electron transport system. Metal wire, through which electrons flow, is a typical electron-conducting pathway. In
metal wire, an electric current flows along an electrical
potential gradient. When there is no potential gradient
406
within a metal wire, no current flows, i.e., when electrons do
not flow, the electrical potential is at the same level throughout the whole length of the metal wire. Like a metal wire,
an electric current flows through the mitochondrial electronconducting pathway along an electrical potential gradient
as far as the chain allows electrons to move. Namely, in a
system allowing the conduction of electrons, the electrical
potential gradient is the sole force driving an electric current. In an electron-conducting pathway, it is not important
how the molecules comprising the electron-conducting
pathway are arranged. The important thing is that the
electron-conducting moieties are tightly in contact with
each other, so as to give a low electrical resistance. Low
resistance is very important for accomplish tasks, because
otherwise the energy dissipates in a form of heat without
performing any valuable work.
A metal wire has another function. A chemically inert
metal, such as platinum, soaked in a solution can catalyze
the H2/H+ oxidation–reduction reaction. Depending on the
concentrations of H2 and H+, a potential evolves at the
metal/water boundary. The potential of the metal electrode
with respect to that of the bulk solution is called the electromotive force of the H2/H+ oxidation–reduction half-cell.
This is just the case in the events occurring at the boundary
between either the inner or the outer solution and the end
of the electron-conducting pathway. As far as the electronconducting pathway extends across the inner membrane, it
should be taken into consideration that a change in potential evolved at the boundary directly affects the membrane
potential or the potential difference between two solutions
separated by the inner membrane. For simplicity, let us
assume tentatively that there is only one electronconducting pathway across the membrane, that the outer
end of the electron-conducting pathway is connected via an
ubiquinone chain to the NADH dehydrogenase or complex
I, and that the inner end is associated with the cytochrome
oxidase or complex IV (Fig. 3).
At the boundary between an electron-conducting pathway and a solution, a potential difference always appears
between the metal and the aqueous solution. This potential
difference is a sort of boundary potential. The potential
difference at the boundary between metal and aqueous
solution is a H2/H+ redox potential. Usually, the end of the
electron-conducting pathway in contact with the solution is
thought to be chemically inert enough to catalyze the oxidation–reduction reaction of H2 molecules: H2 ´ 2H+ + 2e-.
This situation is exactly the same as that observed in the
H2/H+ half-cell composed of a platinum electrode and an
aqueous solution. In equilibrium, the potential of the
platinum electrode with respect to that of the solution is
described by the following equation:
2
(E redox )H 2 = (E 0 )H 2
(E )H
+
RT [ H ]
ln
.
+
2F
[ H2 ]
(20)
is the standard electromotive force of the H2/H
redox half-cell and represents the tendency of the H2 molecule towards ionization. Since the outer boundary of the
electron-conducting pathway is connected to the NADH
2
dehydrogenase via an ubiquinone chain, the H2 concentration is high (QH2 is high). At the inner boundary of the
electron-conducting pathway associated with the cytochrome oxidase, the concentration of H2 is kept very low,
because of the continuous oxidation by O2 which has diffused down from the extracellular solution through the
cytosol. When no current flows through the electronconducting pathway, there is no potential gradient within
the electron-conducting pathway. Namely, there is no
potential difference between the two ends of the electronconducting pathway. When no current flows, the redox
reactions of H2/H+ at both boundaries are in equilibrium
(Fig. 4A). The H+ concentration in the solution outside of
the inner membrane can be considered to be equal to that
in the cytoplasm, because the outer membrane is very leaky.
Under the conditions where there is no trans-membrane
electron current, the potential of the inner solution (matrix)
with respect to the outer solution (cytoplasm) is described
as follows:
E 0 = (Outer redox potential of H2 2H + )
- (Inner redox potential of H2 2H + ),
(21)
or:
+ 2
+
0
Fig. 3. Simplified drawing of circuit across the inner membrane of
mitochondria. The current is generated in a pair of H2/H+ redox halfcells. The thick line represents the electron-conducting pathway. Two
H2/H+ redox half-cells are formed at both the inner and outer boundaries between the electron-conducting pathway and the aqueous phase.
The thin line represents the current carried by H+. NADH dehydrogenase (complex I) is connected to the outer end of the electronconducting pathway via a ubiquinone chain. Cytochrome oxidase is
attached to the inner end of electron-conducting pathway. F1 is the
coupling factor for ATP synthesis
E0 =
RT [ H2 ]inner
RT [ H ]outer [ H2 ]inner
ln
= EH + +
ln
.
2 F [ H2 ]outer [ H + ]2
2 F [ H2 ]outer
inner
(22)
The above equation describes the membrane potential
in a state where no electric current flows through the trans-
407
electrons simultaneously flows through the electronconducting pathway from inside to outside. The electric
current through the electron-conducting pathway is equal
to the sum of the ionic currents in magnitude but is opposite
in direction. As already discussed, when the membrane
potential is E0, no potential gradient exists within the
electron-conducting pathway and the electron current is
zero. Namely, only when the membrane potential is not
equal to E0 (Fig. 4B), can current flow through the electronconducting pathway. Thus, the electric current through the
electron-conducting pathway is described as follows:
I electron = gelectron (E - E 0 ),
(24)
where gelectron is the conductance of electron-conducting
pathway. If the total ionic current is carried only by H+ ions,
then:
(IH )passive = - I electron .
+
Inserting Eqs. 23, 24 into the above equation gives the
following equation:
Fig. 4A,B. Potential profile across the inner membrane of mitochondria. To the outer boundary of the electron-conducting pathway H2
molecules are supplied from NADH2+ via an ubiquinone chain in the
presence of NADH dehydrogenase. The H2 concentration at the inner
boundary is very low because of oxidation by O2 in the presence of
cytochrome oxidase. A Equilibrium potential profile. B Potential profile when an electric current is flowing through the electron-conducting
pathway. Boundary potentials are conventionally expressed by the
potential level of a metal electrode in reference to that of the solution
with which the metal electrode is in contact. The membrane potential
is defined by the internal potential in reference to the external
potential
membrane electron-conducting pathway. Namely, E0 represents the membrane potential in an equilibrium state. When
ATP synthesis does not proceed, no H+ ions flow down
through H+ channels attached to the F1 coupling H+-ATPase
and no electric current flows in the electron-conducting
pathways. Under this condition, the whole system is in equilibrium and the membrane potential is always expressed by
the above equation.
Membrane potential when ATP synthesis is in progress
The inward H+ ionic flow causes ATP synthesis (Mitchell
1966; Hinkle et al. 1991). The coupling ratio is reported to
be 1ATP/2H+ (Mitchell and Moyle 1965). The H+-pathway
directly contributing to ATP synthesis is believed to have
the coupling protein. The coupling factor is extensively
reviewed by Kagawa (1980). The current carried by H+ ions
passing through H+ channels attached to the F1 coupling
protein can be described as the product of the conductance
of passive pathways for H+, gH + , and the driving force causing H+ flow is described by:
( IH )passive = gH (E - EH ).
+
+
+
(23)
When the total ionic current flows down from outside to
inside through ionic channels, an electric current carried by
E=
gH +
gelectron
E0.
E + +
gH + + gelectron
gH + + gelectron H
(25)
Replacing E0 by Eq. 22 gives the relation between the
membrane potential in a steady state and the H2 concentrations at the inner and outer boundaries:
E = EH + +
gelectron
RT [ H2 ]matrix
ln
.
gH + + gelectron 2 F [ H2 ]cytosol
(26)
This equation shows again that the membrane potential
of mitochondria is sensitive to EH + , independently of H+
conductance.
Under conditions where the passive H+ flow is rigidly
coupled to the phosphorylation of ADP, the force required
to drive ATP synthesis is directly proportional to the driving force for H+ flow. Since the force for H+ flow is E - EH + ,
the relation between the force for H+ flow and the H2 concentration ratio is readily derived from Eq. 26 as follows:
force =
gelectron
RT [ H2 ]matrix
ln
gH + + gelectron 2 F [ H2 ]cytosol
(27)
This equation indicates that the driving force causing
ATP synthesis depends on the H2 concentrations at both
boundaries but not on the H+ concentration, notwithstanding the driving force for H+ flow is expressed in the form
E - EH + . Furthermore, the larger the electron conductance,
the stronger the force is. Namely, the energy generated in
a pair of H2/H+ redox half-cells is used for ATP synthesis,
in proportion to the conductance fraction of the electronconducting pathway. If the conductance of the electronconducting pathway is low, the energy generated in a pair
of H2/H+ redox half-cells dissipates as the Joule’s heat
from the electron-conducting pathway during a current
flow there. By the way, the suppression of the O2 supply to
the inner boundary directly results in an increase in the H2
concentration at the inner boundary, leading to a fall in the
driving force. An inhibition of the H2 supply to the outer
boundary causes a decrease in the H2 concentration at the
408
outer boundary; and a decrease in H2 concentration at the
outer boundary also brings about a fall in the driving force,
leading to the suppression of ATP synthesis.
Submitochondria are the inside-out vesicles of the mitochondrial inner membrane. Chance and his collaborators
(Azzi et al. 1969) showed in fluorescence experiments using
submitochondria that an injection of O2 into a suspension
of submitochondrial vesicles induces an increase in the fluorescence of 8-anilino-1-naphthalene-sulfonate (ANS) and
that an injection of NADH also causes an increase in the
fluorescence of the probe. At the time when these experiments were carried out, ANS was used as a hydrophobic
probe for the detection of structural changes in membrane
protein molecules. Later, this negatively charged substance
was confirmed to behave as a potential probe (Cohen et al.
1974) and many potential probes are now synthesized
(Ross et al. 1977; Fujii et al. 1980; Smith et al. 1980). Taking
account of the voltage-sensitivity of ANS fluorescence, their
findings can be re-interpreted as evidence indicating that
the injection of O2 or NADH induces a positive shift in the
submitochondrial membrane potential, or a negative shift
in the potential level of the suspending solution (corresponding to the inner solution of intact mitochondria) to
the level of the internal solution of the submitochondria
(corresponding to the external solution of intact mitochondria). This means that the injection of O2 or NADH brings
about a hyperpolarization in intact mitochondria. The
membrane potential of mitochondria measured by various
methods (Jasaitis et al. 1971; Nicholls 1974; Kamo et al.
1979; Demura et al. 1987) is reported to be about -180 mV
in the resting state.
already mentioned, coupled with ATP synthesis, H+ ions
flow down inwards through the H+ channel and an electric
current flows outwards through the electron-conducting
pathway. Thus, the O2 consumption and the dehydrogenation of NADH take place simultaneously when ATP
synthesis is in progress. The controlling effect of ADP on
the O2 consumption found in a mitochondrial suspension
(Chance and Williams 1956; Hall and Palmer 1969) reflects
this fact.
To briefly summarize this section, it may be said that, in
the case where there are both trans-membrane electronconducting pathways and H+ channels in a membrane limiting an electrically closed space from the bathing solution,
a membrane potential develops depending on two terms:
one is the H2 concentration ratio between the inner and
outer boundaries of the electron-conducting pathway; and
the other is the H+ equilibrium potential. In other words, a
pair of H2/H+ redox half-cells spontaneously formed at the
inner and outer boundaries of each electron-conducting
pathway plays an essential role in generating the membrane
potential. The ratio of H2 concentrations at the inner and
outer boundaries of the electron-conducting pathway determines the H+ driving force, but the H+ concentration gradient does not. It should be stressed that, in mitochondria, the
driving force for H+ is not generated by the H+-pump, in
contrast to the situation seen in Nitella cells, in which the
concentration ratio of [ATP]/[ADP][Pi] generates the H+driving force.
O2 consumption at the inner boundary and H+ dissociation
at the outer boundary
It is confirmed that three molecules of ATP are synthesized
for every dehydrogenation of NADH molecules (Mitchel
and Moyle 1965). The issue of how three ATP molecules
can be synthesized when only one NADH is dehydrogenated caught many researcher’s minds. In 1967, Mitchel
proposed a very beautiful hypothesis that there are three
trans-membrane electron transport systems in parallel
between the NADH dehydrogenase and the cytochrome
oxidase to account for the apparent transport of six H+ ions
against their electrochemical potential gradient for each
molecular dehydrogenation of NADH. Unfortunately, he
used the too-attractive term “chemiosmotic”, instead of
using the more clearly defined “electrochemical terms”. His
new term seems to have caused some confusion in understanding the mechanism for generating the driving force for
H+.
If there is only one trans-membrane electron-conducting
pathway between the outer boundary connected to NADH
dehydrogenase and the inner boundary associated with
cytochrome oxidase, one molecular dehydrogenation of
NADH is linked to both the evolution of two H+ ions at the
outer boundary and the disappearance of two H+ ions at the
inner boundary of the electron-conducting pathway, as discussed above. Let us consider another case, in which one
more electron-conducting pathway is inserted into the inner
membrane in parallel to the first electron-conducting pathway. Under physiological conditions, the membrane poten-
Electrochemical reaction takes place only when an electric
current flows across the boundary between an electronconducting pathway and a solution. When the membrane
potential is more positive than E0, an electric current flows
through the electron-conducting pathway from inside to
outside and, at the inner boundary, two H+ ions which have
carried positive electric charges from the matrix to the inner
end of the electron-conducting pathway receive two electrons from the end of the electron-conducting pathway,
yielding one H2 molecule: 2H+ + 2e- Æ H2.
Under physiological conditions, the H2 molecules
evolved are promptly oxidized by O2 to water in the presence of the cytochrome oxidase: H2 + 1/2O2 Æ H2O. The
oxygen is supplied through the cytoplasm from the extracellular solution.
In contrast, at the outer end of the electron-conducting
pathway, H2 molecules dissociate to two H+ and two
electrons: H2 Æ 2H+ + 2e-. The dissociated H+ carries a
positive electric charge from the outer end of the electronconducting system to the outer solution.
H2 is continuously supplied from NADH2+ via the
ubiquinone chain. It may be worthwhile to mention that the
O2 consumption at the inner boundary is always associated
with the dissociation of H2 to H+ at the outer boundary. As
Two parallel electron-conducting pathways
409
tial of the mitochondria is inside-negative. The newly
inserted second trans-membrane electron-conducting pathway forms a shunt, electrically short-circuiting the internal
solution to the outer solution. A current flows from the
external solution to the matrix through the second electronconducting pathway, because the membrane potential is
kept negative by a pair of redox half-cells of H2/H+ formed
at the inner and outer boundaries of the first electronconducting pathway. The inward current across the outer
boundary of the second electron-conducting pathway
causes an increase in the H2 concentration there. The
increase in H2 concentration makes the outer end of the
second electron-conducting pathway more negative with
respect to the potential level of the outer solution. In contrast, across the inner boundary, a current flows out from
the inner end of the second electron-conducting pathway to
the internal solution, leading to a decrease in H2 concentration at the inner boundary. The decrease in H2 concentration around the inner end of the second electron-conducting
pathway makes the inner end of the second electronconducting pathway more positive with respect to the internal solution. As these changes take place at the outer and
inner boundaries of the second electron-conducting pathway, the electrical potential gradient within the second electron-conducting pathway progressively declines, and finally
reaches zero. When the electrical potential gradient reaches
zero, the second electron-conducting pathway no longer
functions as a short circuit and the membrane potential
returns to the initial level determined by E0 described in
Eq. 22. In other words, the system concerning the second
electron-conducting pathway automatically attains a state
in equilibrium with the existing membrane potential, which
is generated by a pair of H2/H+ redox half-cells formed at
both ends of the first electron-conducting pathway. Therefore, such an electron-conducting pathway as stated above
does not contribute to the multiplication of the H+ current.
In contrast to the case mentioned above, when the outer
end of the first electron-conducting pathway is connected
to the inner end of the second electron-conducting pathway
by an ubiquinone chain, as shown in Fig. 5A, the H2 concentration at the outer boundary of the first electronconducting pathway is always equal to that at the inner
boundary of the second electron-conducting pathway. In this
arrangement, a decrease in H2 concentration at the outer
boundary of the first electron-conducting pathway simultaneously results in a decrease in H2 concentration at the inner
boundary of the second electron-conducting pathway, while
the H2 concentration at the inner boundary of the first
electron-conducting pathway is fixed to the level determined
by PO2 around the site and the H2 concentration at the outer
boundary of the second electron-conducting pathway is
fixed to the level determined by the NADH concentration
in the matrix. Let us assume that all the ionic channels
are closed. Because the electromotive force of the pair of
H2/H+ redox half-cells with respect to the first electronconducting pathway is not equal to that formed with respect
to the second electron-conducting pathway, electric currents
flow through both electron-conducting pathways in opposite
directions to each other. At the initial state, if the E0 of the
first electron-conducting pathway is more negative than the
E0 of the second electron-conducting pathway, a current
flows out of the outer end of the first electron-conducting
pathway and flows into the outer end of the second electronconducting pathway through the external solution; and it
then flows inwards through the second electron-conducting
pathway. This current brings about a decrease in H2 concentration, both at the outer boundary of the first electronconducting pathway and also at the inner boundary of the
second electron-conducting pathway. The decrease in H2
concentration at the outer boundary of the first electronconducting pathway makes the outward-down potential
gradient less steep within the first electron-conducting pathway, owing to the upward shift in the potential of the outer
end of the first electron-conducting pathway with respect
to the potential level of the external solution; and in the
same way the decrease in H2 concentration at the inner
boundary of the second electron-conducting pathway also
makes the inward-down potential gradient less steep within
the second electron-conducting pathway, owing to the
upward shift in the potential of the inner end of the second
electron-conducting pathway with respect to the potential
level of the internal solution. Finally, the potential gradient
within both electron-conducting pathways becomes zero.
Namely, the whole system reaches an equilibrium state. In
the equilibrium state, the E0 with respect to the first
electron-conducting pathway is equal to that with respect
to the second electron-conducting system. Namely, the
[H2]inner/[H2]outer ratio of the first electron-conducting pathway automatically becomes equal to the [H2]inner/[H2]outer
ratio of the second electron-conducting pathway, in addition
to the relation that ([H2]outer)1st is equal to ([H2]inner)2nd. The
finally attained potential profile is shown in the lower
drawing in Fig. 5A.
Let us consider one example, in which the H2 concentration is 1 mM at the inner end of the first electronconducting pathway associated with cytochrome oxidase
and is 100 mM at the outer end of the second electronconducting pathway connected to NADH dehydrogenase
via an ubiquinone chain. On reaching equilibrium, the
[H2]inner/[H2]outer of the first electron-conducting pathway
becomes 1 mM/10 mM; and the ratio of the second
electron-conducting pathway becomes 10 mM/100 mM. As
a result, the driving force for H+ becomes just half the
magnitude that would be generated when only one transmembrane electron-conducting pathway exists between the
cytochrome oxidase-associated inner site and the outer site
is connected to NADH dehydrogenase via an ubiquinone
chain. When the H+ channel opens, H+ flows down into the
internal solution. In the case described above, two electrons
flow through two parallel electron-conducting pathways
from outside to inside each time four H+ ions flow down
through the H+ channels. As two electrons flow down
through the electron-conducting pathway, one H2 molecule
dissociates into two H+ ions and two electrons at each outer
end of the two electron-conducting pathways while, at the
each inner boundary of the two electron-conducting pathways, two H+ ions are reduced to one H2 molecule by receiving two electrons from the electron-conducting pathway,
410
Fig. 5A,B. Schematic drawings of parallel electron-conducting pathways. A Schematic drawing of two parallel electron-conducting
pathways inserted between NADH dehydrogenase and cytochrome
oxidase. The outer end of the first electron-conducting pathway is
connected to the inner end of the second electron-conducting pathway
via an ubiquinone chain. The lower drawing shows the equilibrium
potential profile across the inner membrane. a The inner boundary
potential of the first electron-conducting pathway. b The outer boundary potential of the first electron-conducting pathway. The outer
boundary potential of the first electron-conducting pathway is equal to
the inner boundary potential of the second electron-conducting pathway, because both boundaries are connected by an ubiquinone chain.
c The outer boundary potential of the second electron-conducting
pathway. The magnitude of the outer boundary potential of the second
electron-conducting pathway is not influenced by the number of parallel trans-membrane electron-conducting pathways, because the H2
concentration at this outer boundary is fixed by the NADH2+ concentration in the matrix. The inner boundary potential of the first electronconducting pathway is also not affected by the number of parallel
trans-membrane electron-conducting pathways, because the H2
concentration at this inner boundary depends solely on PO2 (see text
for details). The membrane potential attained when two electronconducting pathways are inserted between cytochrome oxidase and
NADH dehydrogenase, (E0)1/2, is exactly half the magnitude that would
be attained when only one electron-conducting pathway exists, E0.
(E0)1/2 = a - b and E0 - (E0)1/2 = a - b. Thus, E0 = 2(a - b). For simplicity,
EH+ is assumed to be zero. The whole system is in equilibrium.
B Schematic drawing of three parallel electron-conducting pathways.
The outer end of the firstelectron-conducting pathway is connected
to the inner end of the second electron-conducting pathway via an
ubiquinone chain. The outer end of the second electron-conducting
pathway is connected to the inner end of the third electron-conducting
pathway via another ubiquinone chain. No current flows through the
electron-conducting pathways. The whole system is in equilibrium. The
lower drawing shows the potential profile between internal and external solutions. a The inner boundary potential of the first electronconducting pathway, b the outer boundary potential of the first
electron-conducting pathway. The outer boundary potential of the first
electron-conducting pathway is equal to the inner boundary potential
of the second electron-conducting pathway. The outer boundary potential of the second electron-conducting pathway, c, is equal to the inner
boundary potential of the third electron-conducting pathway. d The
outer boundary potential of the third electron-conducting pathway.
The outer boundary potential of the third electron-conducting pathway
is independent of the number of parallel trans-membrane electronconducting pathways as is the inner boundary potential of the first
electron-conducting pathway. The membrane potential is exactly onethird of the magnitude that would have been attained with only one
electron-conducting pathway inserted between the cytochrome oxidase and the NADH dehydrogenase: (E0)1/3 = b - c = a - b, then
a = 2b - c, E0 - (E0)1/3 = a - c = 2b - 2c, E0 = 3(b - c). For simplicity, the
H+ equilibrium potential is assumed to be zero. The whole system is in
equilibrium
411
evolving two H2 molecules in total. At the inner boundary
of the first electron-conducting pathway, the evolved H2
molecule is oxidized by 1/2O2 which has diffused down from
the extracellar solution, while the H2 molecule evolved at
the second electron-conducting pathway’s inner boundary
diffuses to the outer boundary of the first electronconducting pathway through the shunting ubiquinone
chain. The outer boundary of the second electronconducting pathway is supplied with H2 from NADH2+ in
matrix via an ubiquinone chain in the presence of NADH
dehydrogenase. In total at each time, four H+ ions flow
down through the H+ channels, one molecule of NADH2+ is
dehydrogenated, and 1/2O2 is consumed. Thus, four H+ ions
look apparently to be transported against their electrochemical potential gradient from the matrix to the external
solution in association with one molecular dehydrogenation
of NADH2+. However, as already mentioned, the apparent
transport of H+ ions is not due to any pumping activity, but
is merely a result of electrochemical reactions induced by
electric current at both boundaries.
Three parallel electron-conducting pathways
In the case where three trans-membrane electronconducting pathways are inserted in parallel between the
inner boundary attached to the cytochrome oxidase and the
outer boundary connected to NADH dehydrogenase via an
ubiquinone chain, the driving force for H+ can be calculated
as done in the case having two electron-conducting pathways. This model was originally proposed by Mitchell (1966)
but has not been discussed extensively from the viewpoint
of electrochemistry.
The inner boundary of the first electron-conducting pathway is closely connected to the cytochrome oxidase; and the
outer boundary of the first electron-conducting pathway is
connected via an ubiquinone chain to the inner boundary
of the second electron-conducting pathway. The outer
boundary of the second electron-conducting pathway is
connected via another ubiquinone chain to the inner boundary of the third electron-conducting pathway. The outer
boundary of the third electron-conducting pathway is connected to the NADH dehydrogenase via an ubiquinone
chain, as shown in Fig. 5B. In the case having three electronconducting pathways the following relations can also be
attained automatically:
([ H2 ]outer ) 1st = ([ H2 ]inner )2nd ,
([ H2 ]outer )2nd = ([ H2 ]inner ) 3rd ,
(28)
Ê
[ H2 ]inner ˆ
Ê [ H2 ]cyt- ox ˆ
Ê [ H2 ]inner ˆ
=Á
Á
˜ =
˜
Ë
¯
Ë [ H2 ]outer ¯ 1st
[ H2 ]outer 2nd Ë [ H2 ]NADH - dehydr ¯ 3rd
Ê [ H2 ]cyt- ox ˆ
=Á
˜
Ë [ H2 ]NADH - dehydr ¯
13
(29)
.
In general, the membrane potential is described by the
following equation:
E = EH + +
[ H2 ]cyt- ox
gelectron
RT
ln
,
gH + + gelectron 2LF [ H2 ]NADH - dehydr
(30)
where L is the number of parallel electron-conducting
pathways between the inner boundary site associated with
the cytochrome oxidase and the outer boundary site connected to the NADH dehydrogenase by an ubiquinone
chain. The force acting on H+ becomes 1/L of that generated in the case having one electron-conducting pathway.
When six H+ ions flow down into the internal solution of
mitochondria through H+ channels, two H+ ions receive two
electrons from each inner end of three electron-conducting
pathways, yielding three molecules of H2 in total; and, at
each outer boundary of the three electron-conducting pathways, one H2 dissociates to two H+ ions and two electrons,
giving a total of six H+ ions in the external solution. The one
molecule of H2 evolved at the inner boundary of the first
electron-conducting pathway is oxidized by 1/2O2 which
has diffused down from the extracellular solution, while
one molecule of H2 is supplied to the outer boundary of the
third electron conducting-pathway via an ubiquinone chain
from NADH2+ in the matrix in the presence of the NADH
dehydrogenase. Thus, as six H+ ions flow down through the
H+ channels, one molecule of NADH +2 is dehydrogenated
and half a molecule of O2 is consumed.
One important question is still left to be clarified. That
is whether the driving force for H+ is large enough to drive
ATP synthesis. In order to drive the ATP synthesis, a driving force of about 200 mV is required at a stoichiometry of
2H+ for 1ATP. The driving force depends on the ratio of H2
concentration, [H2]inner/[H2]outer, as seen in Eq. 30. To generate a driving force of -200 mV, the H2 concentration ratio
must be 1:108. The H2 concentration at the outer boundary
is determined by the NADH 2+ concentration. The H2 concentration at the cytochrome oxidase site depends on PO2
in the mitochondrion. The affinity of H2 to O2 is very high.
The standard free energy change for water formation is
-237.35 kJ/mol (Moore 1962, Table 6.2). It is well known
that, despite the large negative DG between reactants and
product, the reaction mixture can be kept for more than
10 years without any detectable formation of water. This
apparent stability is due to the high barrier between reactants and product. The cytochrome oxidase facilitates water
formation by effectively lowering the barrier (the activation
energy) without causing an explosion. Thus, the water formation reaction at the inner boundary of the first electronconducting pathway can be considered to be very close to
equilibrium. The equilibrium constant of the water formation reaction is defined as follows:
KH 2 O ∫
[ H2 O]
.
12
[ H2 ][ O2 ]
(31)
RT is 2.5 kJ/mol (at 25°C). Using this value, the equilibrium constant can be calculated from the standard free
energy change above-stated as 3.57 ¥ 1041 (mol/l)-1/2. When
PO2 is 5.3 kPa in the matrix and [H2O] is 55 mol/l, the PH2
at the inner boundary of the first electron-conducting pathway is calculated to be 7.2 ¥ 10-33 Pa, i.e. a H2 concentration
of 3.2 ¥ 10-39 mol/l. Assuming that the H2 concentration at
the outer boundary of the third electron-conducting pathway is around 1 mM, the driving force of H+ is calculated
412
as 1.02 V if there is only one electron-conducting pathway
between the cytochrome oxidase and NADH dehydrogenase. This value is large enough to push ATP synthesis forward, even divided into three. Since O2 plays a critical role
in keeping the H2 concentration very low at the inner
boundary of the first electron-conducting pathway in order
to generate a membrane potential sufficient to push the
ATP synthesis forward, the suppression of the O2 supply is
lethal to animal cells. There is no substituting substance that
can sufficiently oxidize H2 molecules at the inner boundary.
In addition, the H2 concentration at the outer boundary of
the third electron-conducting pathway connected to the
NADH dehydrogenase is in equilibrium with the NADH2+
concentration in the matrix, which depends on the supply
of acetyl Co-A to the tricarboxylic acid cycle.
The coupling mechanism of ATP synthesis with passive
H+ flow at the F1 ATPase attached to the inner surface of
the inner membrane may also have to be discussed shortly,
in connection with the generation of membrane potential.
Although it has so far been suggested that ATP is synthesized in association with the downhill H+ flow, it is not
necessary to consider that the reaction requires such H+
channels as schematically illustrated in Fig. 3. The H+ channel current has not been recorded by the patch-clamp technique in mitochondria until now. A pore structure has been
suggested only in the uncoupling protein in the mitochondria of brown adipose tissue (Arechaga et al. 2001; Klingenberg et al. 2001). There is some possibility for a type of
electron-conducting pathway other than the electronconducting pathway described above. If the outer boundary
of some electron-conducting pathway is connected to the
inner boundary by a ubiquinone chain, the H2 concentration
at the outer boundary is equal to that at the inner boundary
of the electron-conducting pathway. Equation 22 shows that
the equilibrium potential with regard to such an electronconducting pathway should be equal to the H+ equilibrium
potential. This means that such an electron-conducting
pathway behaves electrochemically as if it were a H+ channel. Namely, when the membrane potential is not equal
to the H+ equilibrium potential, an electric current flows
through such an electron-conducting pathway. Let us here
call the electron-conducting pathway whose outer and inner
ends are shunted by an ubiquinone chain as the shunt (S)type electron-conducting pathway. The inner end of the Stype electron-conducting pathway is electrically positive to
the inner solution under physiological conditions, because
the external solution is electrically positive to the inner
solution. Anions in the matrix are attracted to the positive
charge on the surface of the inner end of S-type electronconducting pathway and are densely packed there. The
accumulated ADP- and Pi- anions may hand off electrons
to the electron-conducting pathway. When the membrane
potential is not equal to the H+ equilibrium potential, the
following reactions in the presence of proper ATPase may
take place at the outer and inner boundaries of the S-type
electron-conducting pathway.
At the outer boundary of the S-type electron-conducting
pathway:
2H+ + 2e- Æ H2 and H2 + Q Æ QH2
At the inner boundary of the S-type electron-conducting
pathway: ADP- + P-i Æ ADP* + P*i + 2e- and: ADP*·P*i +
QH2 Æ ATP + H2O + Q,
where ADP*·P*i is a highly reactive radical, or ATP·O**.
At the outer boundary of the S-type electron-conducting
pathway, two H+ ions disappear, and one molecule of H2
evolves. The H2 molecule evolved at the outer boundary
diffuses down to the inner boundary through the shunting
ubiquinone chain. At the inner boundary, two anions
(ADP-, P-i ) disappear, while two H+ ions having dissociated
from ADP and Pi remain intact in the matrix. In total, it
looks as if two H+ ions flow down from outside to inside
each time ATP is synthesized. These reactions are not mysteriously “chemiosmotic” but are purely “electrochemical”
events.
The thylakoid membrane potential
Hill (1939) discovered that fresh leaves ground in water
containing suitable hydrogen acceptors give off oxygen
when exposed to light, even though the cells are crushed
and photosynthesis of carbohydrate has ceased. Ferricyanide is photochemically reduced and quinone is reduced to
hydroquinone. These findings indicate that a reduction pool
of hydrogen is produced when light is absorbed, even by the
fragmented chloroplasts.
Photosynthesis of carbohydrate in the chloroplast consists of a light reaction and a dark reaction. The light reaction is divided into two categories: the non-cyclic electron
transfer and the cyclic electron transfer. In the former
system, light absorption reduces NADP+ to NADPH2+ (or
NADPH), giving off O2, while in the latter system light
absorption yields ATP. In the dark reaction, carbon dioxide
is fixed to carbohydrate at the expense of ATP and reducing
power is provided from NADPH2+. In the production of ATP
and the reducing pool, the electrochemical potential of H+
is likewise considered to play a principal role in mitochondria. Since there is no significant difference in H+ concentration between internal (thylakoid space) and external
(chloroplast stroma) solutions (as in Nitella cells), the force
acting on H+ is almost completely determined by the membrane potential.
Electrochemical decomposition of water
Chlorophylls are arranged in the thylakoid membrane so
as to effectively capture photons. Roughly 300 chlorophylls
are supposed to form one photocell (Barber 1987). An
internal electron-conducting pathway connected to the
anode of the photocell may extend to the inner surface of
the thylakoid membrane; and another electron-conducting
pathway connected to the cathode extends to the outer
surface. Exposing the photocell to light makes the internal
electron-conducting pathway positive with respect to the
external electron-conducting pathway. At a boundary between the electron-conducting pathway and the solution,
413
there is a boundary potential which is characterized by the
oxidation–reduction reaction of H2/H+. It is also supposed
that there are H+ channels or passive routes of electric current across the thylakoid membrane, some of which serve
as apparatus for ATP synthesis (Fig. 6). Generally, an equation describing the membrane potential of a particle
enclosed by a lipid bi-layer membrane having an electronconducting pathway which is connected to a photocell can
be derived in a similar way to that for mitochondria. The
membrane potential of thylakoid with no current flow
through the electron-conducting pathway, E0, can be
described by the following equation:
RT [ H2 ]inner
ln
+ E photocell ,
2 F [ H2 ]outer
E 0 = EH + +
(32)
where Ephotocell is the electromotive force of the photocell
embedded in the thylakoid membrane. Potential profiles in
an equilibrium state are shown in Fig. 7A,B. The electromotive force of the photocell contributes directly to the
membrane potential in an equilibrium state.
When H+ ions flow down through the H+ channel, an
electric current of the same magnitude as that carried by
the H+ ions flows in the opposite direction through the
electron-conducting pathway and photocell. In a steady
state, the membrane potential is described as follows:
E = EH + +
gelectron
( gH )passive + gelectron
+
RT [ H2 ]inner ˆ
Ê
E
+
ln
.
Ë photocell 2 F [ H2 ]outer ¯
(33)
where (gH+)passive is the conductance of the H+ ionic current.
Thus, the driving force for H+, (force)H + is:
gelectron
(force)H + =
( gH )passive + gelectron
+
RT [ H2 ]inner ˆ
Ê
ln
E
+
Ë photocell 2 F [ H2 ]outer ¯
(34)
Again the driving force for H+ is independent of the H+
equilibrium potential. The larger the electronic conductance, the larger is the driving force for H+.
There may be two kinds of H+ channel: one associates
with the coupling factor that provides the catalytic site for
ATP synthesis and the other has no coupling protein. The
current carried by H+ ions is:
( IH )passive =
+
(gH )passive (gelectron )
(gH )passive + gelectron
+
+
RT [ H2 ]inner ˆ
Ê
ln
.
+
E
Ë photocell 2 F [ H2 ]outer ¯
(35)
A current equal in magnitude to (IH+)passive flows across
both the inner and outer boundaries between the electronconducting pathway and the aqueous phase; and electrochemical reactions take place at the boundaries. The higher
the H+ conductance, the more the energy provided from the
photocell is used for the electrochemical decomposition of
water.
Fig. 6. Hydrogen-store production and oxygen evolution. The internal
electron-conducting pathway is connected to the anode of a photocell
embedded in a thylakoid membrane. The external electron-conducting
pathway is connected to the cathode of the photocell. There are two
groups of H+ channels: one is short-circuiting the thylakoid membrane
and the other is equipped with the coupling factor, F. The chloroplast
has no H2-yielding chemical system, such as the tricarboxylic acid cycle.
Under conditions where there is no difference in H2 concentration
between the inner and outer boundaries and no difference in pH
between the thylakoid space and the chloroplast stroma, the inner
boundary potential is equal to the outer boundary potential. The electromotive force of the photocell induced by light is used for the electrolysis of water, when the short-circuiting H+ channels are open. At
the outer boundary, two H+ ions (or other cations) receive two electrons from the cathode of the photocell through the external electronconducting pathway. The evolved H2 reduces NADP+ to NADPH2+ in
the presence of an appropriate enzyme, such as NADPH dehydrogenase. At the inner boundary, two OH- (or Cl-) ions hand out two
electrons to the anode of the photocell through the internal electronconducting pathway, yielding water and oxygen at the inner boundary.
This system corresponds to noncyclic photophosphorylation. Under
these conditions, most of the H+ ions flow through the short-circuiting
channels
The H2 concentration ratio also has an influence on the
driving force for H+, in addition to the electromotive force
of photocell. When thylakoids are exposed to light, initially
a large amount of H+ flows out from the thylakoid space
to the external solution. The electric current flows from
the external solution into the outer end of the external
electron-conducting pathway connected to the cathode of
the photocell. Two H+ ions, which carried electric current
across the outer boundary, receive two electrons from the
external electron-conducting pathway, evolving H2 which
reduces NADP+ to NADPH2+. In contrast, at the inner
boundary the current may be carried by either OH- or Cl-
414
H2 at the outer boundary decreases gradually. The next flash
induces a large current again, reducing NADP+ to HADPH2+
at the outer boundary and yielding H2O and O2 at the inner
boundary. This situation may explain the gradual decrease
in O2 production during continuous illumination in relation
to the non-cyclic photophosphorylation proposed by
Clayton (1971).
ATP production
Arnon and his collaborators (1965) obtained direct evidence of the conversion of photic energy into ATP in
chloroplasts, beside the reduction of NADP+ to NADPH2+.
Bishop (1973) reported the presence of an ubiquinone-like
substance, plastoquinone, in chloroplasts. In the case where
there is a plastoquinone chain facilitating the diffusion of
H2 between the outer and inner boundaries of the electronconducting pathway (Fig. 8), the H2 concentration at both
boundaries remains always equal. In this case, the membrane potential is described as follows:
E = EH + +
gelectron
E
.
( gH + )passive + gelectron photocell
(36)
Then the force acting on H+ ions is:
Fig. 7A–C. Potential profiles across the thylakoid membrane in various states. A Without light, no electromotive force is generated in the
photocell. No potential difference between thylakoid space and chloroplast stroma. The whole system comprising the electric circuit is in
equilibrium. B A flash generates electromotive force in the photocell.
This electromotive force makes the electrical potential of the thylakoid
space positive with respect to the chloroplast stroma. C Continuous
illumination causes the accumulation of O2 at the inner surface and H2
at the outer surface, resulting in a decrease of the H+ driving force
ions, because the H2 concentration is extremely low at the
boundary unless H2 is supplied to the site. Two hydroxyl
ions hand out their electrons to the internal electronconducting pathway, yielding 1H2O and 1/2O2. A part of the
evolved water dissociates into H+ and OH- to some extent.
Alternatively, two Cl- ions hand out their electrons to the
internal electron-conducting pathway, yielding 2Cl which
promptly react with water, evolving 1/2O2 and 2HCl. However, the results of these two reactions are the same from
the viewpoint of O2 generation and the constancy of ionic
contents. The elevation of PO2 at the inner boundary causes
a positive shift in the potential of the internal electronconducting pathway with respect to the internal solution,
resulting in a decrease in membrane potential, as shown in
Fig. 7C. The accumulation of H2 at the outer boundary also
causes a decrease in membrane potential. As the membrane
potential decreases, the current becomes smaller. However,
while the membrane potential is more positive than EH + , H+
ions flow out of the thylakoid space through H+ channels
and both the reduction of NADP+ at the outer boundary
and the evolution of O2 at the inner boundary continue.
After shutting off the light, the once-elevated PO2 at the
inner boundary falls to its initial level and the accumulated
(force)H + =
gelectron
( gH )passive + gelectron
E photocell .
(37)
+
If all the short-circuiting channels are closed, the force
will be used only for driving the ATP synthesis. These situations correspond to the cyclic photophosphorylation proposed by Clayton (1971).
In conclusion of this section, it may be worth pointing
out that the membrane potential of thylakoids primarily
depends on the electromotive force of the photocell embedded in the thylakoid membrane and on the H+ equilibrium
potential. The dependence of the membrane potential on
the H+ equilibrium potential is due to the H2/H+ redox halfcells automatically formed at the boundaries between the
electron-conducting pathway and the aqueous solution. The
driving force for a passive H+ current is determined by
the sum of the electromotive force of the photocell and the
potential due to the concentration ratio of H2 at the inner
and outer boundaries. Furthermore, the larger the electron
conductance, the stronger is the driving force for H+. This
force is used either for the electrolysis of water or for ATP
synthesis. Under the condition where all the short-circuiting
H+ channels which are not associated with the catalytic site
of the coupling ATPase are open, all of the driving force is
used for the electrolysis of water. This situation corresponds
to “non-cyclic photophosphorylation”. In contrast, when
the shunting H+ channels are closed and the H+ channels
equipped with the coupling factor for ATP synthesis are
open, all of the driving force is used for ATP synthesis in
the presence of a short-circuiting plastoquinone chain which
connects the external and internal boundaries of the
electron-conducting pathway. This situation corresponds to
“cyclic photophosphorylation”. In the thylakoid also, the
generation of a membrane potential is considered to be
415
neither chemical nor osmotic, but electrochemical and photochemical. ATP may be synthesized through electrochemical reactions similar to those in mitochondria.
(Wingo and Smolka 1995). However, reports on H+/K+transporting ATPase in plant cells are very scarce. In contrast to the scarcity of reports on this transporter in plant
cells, there are many reports regarding ATP-dependent
Na+/H+-transporters. Clint and MacRobbie (1987) reported
that the Na+ efflux depends not only on the external pH but
also on the internal ATP in tonoplast-free Chara cells.
Cells of the marine alga Heterosigma akashiwo have
Na+-activated ATPase which has an immunologically identical epitope to Na+/K+-ATPase (Wada et al. 1992). Two
cDNA clones were isolated from that plant. The longer
cDNA has conserved regions of eukaryotic P-type ATPases
(Wada et al. 1994). More recently, Shono et al. (2001)
succeeded in cloning the full length of the cDNA which
encodes a 1,330-amino-acid protein. The deduced product
is estimated to have about 40% identity in amino acids
with Na+/K+-ATPase alpha-subunits. If the Na+/H+-pump
extrudes Na+ ions in exchange for external H+ ions across
the plasma membrane, H+ ions will gradually accumulate in
the cytoplasm. Only the situation where the inflow of H+ is
in balance with the extrusion of H+ keeps the level of the
cytosolic pH unchanged. The steady level of cytosolic pH
depends on the Ka and buffering capacity of the organic
acids produced within the cell. From the fact that the cytoplasmic pH remains within a range of 7.0–7.4, it may be
reasonable to suppose that the Na+/H+-pump works coupled with the H+/K+-pump so as to result in no accumulation
of H+.
Although the rate of transport of H+ ions by the H+pump is expected to be proportional to the sum of a
constant and the logarithm of cytosolic ADP/ATP concentration ratio (Eq. 16), the dependence of H+-pump activity
on the cytosolic ATP concentration sometimes agrees with
the saturation curve of a Michaelis–Menten type. This finding suggests that the H+-pump molecules have some regulatory sites sensitive to cytosolic ATP. The opening of
stomata is driven by the accumulation of K+ in guard cells.
The accumulation of K+ is induced by blue light, which
activates the extrusion of H+. With regard to the dependence of H+-pump activity on ATP, very important findings
were obtained by Kinoshita and Shimazaki (1999), who
showed that the H+-ATPase prepared from guard cells illuminated by blue light was phosphorylated and the phosphorylation level was parallel to the ATP hydrolytic activity.
This finding indicated that cytoplasmic ATP not only provides the H+-pump molecule with the energy required to
pump out H+ but also regulates the turning rate of the
pump. The issue how the affinity of the regulatory site is
modified by blue light is an interesting problem.
Discussion
Conclusion
There are many reports on gastric H+/K+-ATPase (Morii et
al. 1990, 1996; Reuben et al. 1990). The gastric type of H+/
K+-ATPase is expressed in various organs, such as the distal
colon (Asano et al. 1998; Rajendran et al. 2000), the inner
ear and choroids plexus (Lecain et al. 2000), and the kidney
The resting membrane potential of Nitella cells depends not
only on the H+ equilibrium potential but also on the activity
of the electrogenic H+-pump. The force pushing the H+pump forward comes solely from the concentration ratio of
ATP, ADP, and Pi, while the force counter-acting is propor-
Fig. 8A–C. ATP production in the thylakoid. A, B Potential profiles
across the thylakoid membrane in the dark and illuminated state,
respectively. C Schematic structure of components participating in
ATP synthesis. Coupling factor, F, is attached to the outer orifice of
the H+ channel. When ATP synthesis is in progress, H+ flows down
through the H+ channel from the thylakoid space to the chloroplast
stroma and an electric current carried by electrons flows in the opposite
direction through an electron-conducting pathway. Evolved H2 molecules at the outer boundary (connected to the anode of the photocell)
promptly diffuse down to the inner boundary through a plastoquinone
chain. Under these conditions, As shown in B, the inner boundary
potential, a, is always kept equal to the outer boundary potential, b,
and the membrane potential is equal to the electromotive force of
photocell, c, induced by light. For simplicity, the H+ equilibrium potential is assumed to be zero. Pi Inorganic phosphate
416
tional to the potential difference between an existing membrane potential and the H+ equilibrium potential. The total
force acting on the H+-pump is the difference between these
two forces. This relation gives a pH-sensitivity to the membrane potential. At the reversal potential of the H+-pump,
the pushing-forward and counter-acting forces are in balance. Since any deviation of the membrane potential from
the H+ equilibrium potential derives from the chemical tendency towards the hydrolysis of ATP, it may be said that the
force causing a passive H+ flow is determined by the concentration ratio of ATP, ADP and Pi. The passive H+ current
seems to be coupled with Cl- uptake. If the coupling ratio
of the H+,Cl- co-transporter is two, the value of E Cl
calculated using the reported values of the cytosolic pH and the
resting membrane potential is higher than the experimentally obtained value.
In considering the mitochondrial membrane potential
genesis, the potential gradient within the trans-membrane
electron-conducting pathway should be taken into account.
When no electronic current flows through the electronconducting pathways, the membrane potential of mitochondria is principally due to the electromotive force generated
in a pair of oxidation–reduction H2/H+ half-cells spontaneously formed at the inner and outer boundaries of each
trans-membrane electron-conducting pathway. This membrane potential is also pH-sensitive in its nature. The driving
force for passive H+ flow is determined by the ratio of
the H2 concentrations at the inner and outer boundaries.
The H2 concentration at the outer boundary depends on the
concentration of NADH in the matrix, while the H2 concentration at the inner boundary depends on the PO2 in the
matrix. In the presence of O2 at 5.3 kPa, the H2 concentration is sufficiently low to generate the force to drive ATP
synthesis, even though the force is divided by three.
The membrane potential of thylakoids is primarily due to
the electromotive force of photocells embedded in the thylakoid membrane. The internal electron-conducting pathway connected to the anode of the photocell is kept in
contact with the inner solution of the thylakoid, while the
external electron-conducting pathway which is connected to
the cathode of photocell is in contact with the outer solution.
Light absorbed into photocells generates the inside-positive
membrane potential, causing an outward flow of H+ mostly
passing through short-circuiting H+ channels; and an electric
current of the same magnitude flows through the electronconducting pathways in the opposite direction. This lightinduced current yields O2 at the inner boundary between the
electron-conducting pathway and the aqueous phase and
causes an increase in the H2 pool in the form of NADPH at
the outer boundary. When short-circuiting H+ channels are
closed, H+ flows through H+ channels equipped with the
coupling factor for phosphorylation. If the inner and outer
ends of the electron-conducting pathway are connected with
each other by a plastoquinone chain, the light-induced
current effectively drives the ATP synthesis without yielding
H2 and O2.
The ATP synthesized in chloroplasts is utilized to transport H+ ions from inside to outside of the cell. The hyperpolarization resulting from the activation of H+-pump
causes an increase in the accumulation of Cl- through the
rheogenic Cl-,rH+ co-transporter, leading to a rise in salt
concentrations in the cytoplasm. In a steady state, the
reported value of the cytosolic Cl- concentration is slightly
lower than the calculated value obtained using the values
of the cytosolic pH and the resting membrane potential,
under an assumption that the co-transporter has the fixed
coupling ratio of 2:1. This slight discrepancy indicates the
presence of either the electrically neutral Cl-,H+ symporter
together with the rheogenic Cl-,rH+ co-transporter in the
plasma membrane or a small leakage of Cl-. Furthermore,
from the fact that the cytosolic pH is kept around 7.0, it may
be considered that the Na+/H+-pump is associated with the
H+/K+-pump in activity.
Acknowledgments The author is greatly indebted to Prof. M. Tazawa
and the Committee of the JPR Symposium on “Plant plasma membrane H+ pumps: past and present” for the opportunity to write this
theoretical article rather than a review. The theory presented in this
article is based on thoughts that the author developed over some
30 years. After the publication of papers on the electrogenic H+ pump
in Nitella cells and on the affinity of the K+ channel to potassium ions
in Nitella, the author devoted himself to research on the relationship
between electrical activity and the metabolic state in pancreatic beta
cells at a newly founded Medical School. This situation made it difficult
for him to continue the study on plant cells. If this opportunity had not
been given to him, the results of his thinking would go into oblivion
without leaving any trace. Again, the author would like to express
special gratitude to Prof. M. Tazawa and the Committee of the JPR
Symposium.
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