5.1. Several Lines of Biochemical Evidence that Cannot be Accounted for by the Chemiosmotic Theory
but Are Logically Explained by the Torsional Mechanism of Energy Transduction and ATP Synthesis
Recently, we have shown that several lines of biochemical evidence, many from our own
laboratory, do not support the chemiosmotic theory, but are readily explained by the torsional
mechanism of energy transduction and ATP synthesis. These include: (i) the acid concentration
dependence of the rate of ATP synthesis [11], (ii) the isolation and characterization of several
uncoupler-resistant mutants of oxidative phosphorylation, (iii) the increase in oxidative
phosphorylation uncoupling efficacies with increase in lipid solubility of the uncoupler, other things
remaining the same, and (iv) experimental data on the inhibition of ATP synthesis by known specific
anion channel blockers such as the triorganotin compound, tributyltin chloride (TBTCl), and the
stilbene compound 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) [10]. The experiments on
inhibition of ATP synthesis at nanomolar concentrations of the potent, specific anion channel blocker,
DIDS were repeated with improved methods (Section 8) and similar results were obtained (Nath and
Agarwal, in preparation). It is very difficult to explain why specific anion channel blockers inhibit the
rate of ATP synthesis using theories such as chemiosmosis [6] that energetically link ATP synthesis to
the translocation of a single agent only, i.e. protons. However, if ~50% of the energy to synthesize
ATP comes from anion translocation (by a small inorganic anion such as chloride or by an organic
anion such as succinate which is singly charged under the experimental conditions in our plant
system), and the other ~50% is donated by proton movement through specific half-access channels in the FO portion of ATP synthase, and both Cl- (or succinate) and H+ move in a strongly coupled way
(with 1:1 stoichiometry being the favored and most likely case) through access pathways that form a
rigid intramembrane link, a structural union, as it were, in the membrane for the addition and joint
utilization of energy and for regulation of transport and metabolism, as predicted by the torsional
mechanism [1, 2, 10-13, 15], then such an inhibition in the concentration range of inhibitor that does
not saturate the membrane binding sites is in fact the expected outcome. Thus, ATP synthesis becomes
a multicomponent reaction involving anions and cations, and the FO portion of ATP synthase is then an
A-–C+ symsequencecotransporter. A difference between the molecular mechanisms in mitochondria,
chloroplasts, and bacteria is the nature of the anion (chloride, succinate) and cation (proton, sodium
ion) to which the energy-transducing membrane is permeable.
For ATP synthesis by mammalian mitochondria, a singly-charged dicarboxylic acid anion such as
succinate serves as the permeant anion in vitro and in vivo (distinct from its well-known role as a
respiratory substrate in mitochondria). In chloroplasts, both small inorganic anions such as chloride
[10] and organic acid anions such as succinate [11] support ATP synthesis at physiologically high rates
in vitro. However, the narrow range of HCl concentrations in which measurable rates of ATP synthesis
are found in vitro (Figure 4), the fact that chloride is the principal anion in plant chloroplasts present at
high concentrations of ~150 mM, necessitating an ultra-high intra-thylakoid concentration > ~150 mM
to generate a gradient that can support outward anion translocation and ATP synthesis in chloroplasts
that have not been observed experimentally, the strong acid nature of HCl that may interfere with
stability of ionic conditions in the chloroplast, the broader range of succinate concentrations that supports ATP synthesis (~1 to ~10 mM) at high rates [11], the clean exponential nature of the data
[11], the weak acid nature of succinic acid, and finally the known experimental fact that chloride is
essential only for those photosynthesis steps in which oxygen evolution occurs but is not necessary for
cyclic photophosphorylation in bacteria and isolated chloroplasts involving photosystem I and ATP
synthase (which we believe is operative under our acid-base/light-dark experimental conditions) all
suggest that chloride is not the permeant anion in vivo. Since organic acids are the major products of
photosynthetic activity, weak acid organic anion substances are the ubiquitous type of carbon
compounds expected to be present at moderately appreciable concentrations (~millimolar) within the
chloroplasts in vivo, and hence the type of mechanism involving such organic acid anions and protons
described above can be readily expected to occur in vivo. If this is so, then we also have a unity in the
nature of the permeant anion that is translocated through the a-subunit access channel in FO and
supports ATP synthesis. It should however be noted that whether the specific permeant cation is H+ or
Na+ and the permeant anion is Cl- or a low molecular weight weak acid anion does not directly impact
the torsional mechanism.
Inhibition studies [10] have helped to experimentally establish a role for anions in ATP synthesis.
They have revealed the requirement of permeant anions (in addition to protons), and have indicated an
energy provision role of the anion in bioenergetic coupling of ATP synthesis by translocation through
the membrane-bound FO portion and binding to the protein-in-the-membrane (i.e. trapping of chloride
in its binding pocket in a lipophilic region of the membrane). The properties of these lipophilic regions
have been considered and detailed molecular mechanisms developed within FO [1, 2, 10-13, 15, 19];
such detailed explanations of the role of membrane elements have led to a deeper understanding and
offer a more realistic and complete picture of biological energy transduction than a theory such as
chemiosmosis [6] that strictly permits only “energized bulk aqueous phases” and considers the bilayer
as “mere insulation”.
5.2. Establishment of the Type of Inhibition Found with Potent, Specific Anion Channel Blockers Such
as DIDS and its Logical Explanation
It now becomes important to determine, classify and explain the type of inhibition found with the
inhibitor (DIDS) at various HCl concentrations within the framework of the torsional mechanism.
Inhibitor concentrations in the nanomolar range between 1 nM and 8 nM and HCl concentrations
between 0.5 mM to 1.4 mM at which we could obtain measurable rates of ATP synthesis were chosen.
Inhibitor and HCl at concentrations mentioned above were used to define the type of inhibition, and
the result was inferred from a plot made between the inverse of the rates at a particular HCl
concentration and different inhibitor concentrations. The results are shown in Figure 4.
Based on the cumulative observations depicted in Figure 4, the results were inferred as showing
inhibition of a mixed type, which we suggest to be predominantly competitive with respect to chloride
and predominantly uncompetitive with respect to chloride and proton, depending on the HCl
concentration. Such a mixed type of inhibition has generally been encountered in multi-substrate
reactions. The binary enzyme-inhibitor complex or ternary enzyme-substrate-inhibitor complex thus
formed does not lead to any rates or results in decreased rates of enzyme activity. It is a virtue of
competitive inhibition that once the inhibitor has bound to its site on the enzyme to form the enzyme-inhibitor complex, the ion/substrate for which the inhibitor is competitive has to remain in an unbound
state irrespective of whether they share the same binding site or have different binding sites. On the
other hand, a prerequisite condition for uncompetitive inhibition is that bound inhibitor cannot unbind
and leave as long as the ion/substrate for which the inhibitor is uncompetitive remains bound to its site
on the enzyme.
In our system, the inhibition can be competitive with respect to the primary ion, i.e. the one that
binds/unbinds before the binding/unbinding of the second/secondary ion takes place, while it can be
uncompetitive with respect to the primary ion, or with respect to both ions, i.e. both chloride and
proton, which remain bound to their respective sites (e.g. if unbinding of the primary ion is required
before the secondary ion can unbind, as in an ordered and sequential mechanism). Any mechanistic
explanation has to be also consistent with the known role of DIDS as a potent anion channel blocker.
In our mode of study, the DIDS inhibitor was added to the external aqueous medium on the stromal
side of the membranes at the time of phosphorylation i.e. in the base stage. Hence we logically expect
the DIDS to bind to its site on the membrane surface on the stromal side and block ion exit from the
access channel/pathway and not interfere with ion entry from the intra-thylakoid space (lumen side).
Moreover, since the inhibitor used in our experiments is a potent anion channel inhibitor, we infer that
DIDS inhibition is competitive with respect to the chloride ion, and in fact all our results could be
successfully explained by considering the ability of the chloride ion (i) to remain bound to its site or
(ii) to unbind from its site but not exit from the access channel due to the presence of bound inhibitor
at its own site. The inhibition can be classified as uncompetitive or competitive for the cases (i) and (ii)
respectively. Thus, after both chloride and proton have bound to their binding sites on the a- and csubunits
respectively at the a-c interface of the FO portion of the F1FO-ATP synthase, the inhibition
would be competitive with respect to chloride if the Cl- unbinds from its site but cannot come out of
the a-subunit anion exit access channel due to the block caused by binding of DIDS to its site at the
surface of the a-subunit anion access channel. The inhibition would be uncompetitive with respect to
chloride if the chloride is unable to unbind from its site at the HCl concentrations employed, and
uncompetitive with respect to both Cl- and H+ if the chloride ion cannot unbind from its binding site,
and if the two ions translocate in a coupled way and the H+ can only unbind following unbinding of the
Cl-, in which case both Cl- and H+ will remain bound to their binding sites on the a- and c-subunits
respectively. One would expect the tendency of the anion to remain bound to its site in the a-subunit
access channel to be readily subject to modulation by the stromal side concentrations of the substrate
for the FO portion of the F1FO (e.g. HCl or succinic acid).
The above analysis has summarized the inhibition of ATP synthesis in the presence of anion
channel blocker as competitive or uncompetitive depending on the tendency of the primary ion bound
to its site to unbind and leave or to remain bound to its site. This tendency itself will be a strong
function of the concentration gradient of the substrate for which measurable rates can be obtained in
thylakoid membranes. At lower HCl concentrations (0.5 mM to 0.75 mM), mixed inhibition that is
predominantly competitive has been found (Figure 4), and the second, minor component of this mixed
inhibition has been characterized in the above discussion as uncompetitive in nature. At higher
concentrations of HCl (1 mM to ~1.5 mM), the inhibition was also interpreted to be mixed inhibition
but predominantly uncompetitive in nature (Figure 4) while the minor component of this inhibition
was characterized as competitive.
In our experiment, we have a large population of thylakoids (approximately 1012 per mg
chlorophyll) and further there would be a large number of sites in each thylakoid. Taking only FO sites
that contain bound inhibitor, there will be heterogeneity of the ionic microenvironment around such
sites and in particular, we shall only have a statistical distribution of chloride ions. Each of the ~1012
thylakoids per mg chlorophyll in the isotonic medium cannot be expected to be perfectly swollen if the
ion permeation process in both acid and base stages is intrinsically statistical in nature. This will lead
to heterogeneity in the number of chloride ions around each chloride binding site. Finally, the
microdistribution of ions among the FO sites in each thylakoid will not be perfectly uniform leading to
further spatial heterogeneity in ionic distribution. Hence, even in experiments tailored to unbind
chloride from its binding site in each FO, there will always be a small population in which chloride
does not unbind due to the above mentioned heterogeneity. In other words, only the average bulk
concentration (but not the local number of Cl- ions around each binding site) can be controlled by
the experimentalist.
Hence, at low HCl (~0.5 mM), Cl- will unbind from a majority of the sites and lead to competitive
inhibition in the presence of inhibitor; however, due to fluctuations in the stromal chloride
concentration around the exit access channel, Cl- will be unable to unbind and this will cause
uncompetitive inhibition in a minority of sites containing bound DIDS.
At higher average chloride stromal concentration the situation is the reverse. Chloride remains
bound in the majority of the sites because the driving force for unbinding of chloride is low. Hence, we
have the presence of an ESI bound form and a predominantly uncompetitive inhibition. In a minority
of sites, due to special heterogeneity for the reasons discussed above, the Cl- ion can unbind from its
binding site but cannot exit from the access channel due to the presence of bound inhibitor, and
therefore we have a competitive inhibition with respect to chloride in such sites. It should however be
emphasized that despite the presence of heterogeneity, the data (Figure 4) show that predominantly we
do obtain the type of inhibition that we expect, implying the distribution of Cl-/H+ is not too nonuniform.
Due to the reasons discussed above, it would be difficult to obtain pure
competitive/uncompetitive behavior even in a single molecule experiment, i.e. to reproducibly
simulate an all-or-none situation when chloride will unbind or remain bound to its site each and
every time.
5.3. Explanation of Rate Enhancement of ATP Synthesis at Very High Inhibitor Concentrations
At very high concentrations, [I], of tributyltin chloride (TBTCl) ([I] ≥ 100 nM) and 4,4′-
diisothiocyanostilbene-2,2′-disulfonate (DIDS) (100 nM ≤ [I] ≤ 2 μM), a rate enhancement of ATP
synthesis (~8-fold with TBTCl and ~2-fold with DIDS) was reproducibly observed [10]. An attempt
was made to explain the observation of rate enhancement but this proved to be a difficult exercise in
the absence of a proposal of a different mechanism or of a special phenomenon operating at these
artificially high TBTCl or DIDS concentrations [10]. Here we propose that after unbinding from their
respective binding sites on the a- and c-subunit in FO, because of lipid solubility at the high
concentrations of TBTCl or DIDS (≥ 100 nM), the Cl- and the H+ combine to form neutral HCl in the
microenvironment of the exit access half-channel. The neutral HCl can relieve the block caused by
bound [I], for example by diffusing out by molecular diffusion. We propose that such molecular diffusion occurs at enhanced rates compared to ion transport as individual H+ and Cl- charges and
thereby leads to more cycles of ATP synthesis per unit time. Moreover, neutralization by formation of
HCl is electrostatically akin to removing H+ and Cl- to infinity. Hence no inhibition is observed at
these conditions, and since the rate of transport of molecular HCl is faster than that of H+ and Clindividually,
a rate enhancement of ATP synthesis occurs, as observed [10]. It should also be
understood that energy transduction is not hampered if H+ and Cl- recombine after unbinding from
their respective binding sites at the a-c lipid-water interface.
5.4. Precise Explanation of Uncoupling Action by Weak Acid Anion Uncouplers of Oxidative
Phosphorylation
In the light of the discussion in Sections 5.1 – 5.3, the earlier explanation given for uncoupling
action by weak acid anion uncouplers (U-) of oxidative phosphorylation [see Figure 3 of Ref. 11 and
discussion below that Figure] can now be fine-tuned and made more precise. Since recombination of
U- and H+ after their unbinding does not dissipate ~50% of the energy (Section 5.3), an uncoupler
anion, U- is one that combines with H+ due to the lipid solubility of the uncoupler and forms neutral
UH in the microenvironment at or near the binding sites upon entry of U- and H+ sequentially through
their own access channels located in the a-subunit and c-subunit respectively at the a-c interface. The
formation of neutral UH precludes the conformational change in the α-helix containing the H+ binding
site (the Asp-61 residue in E. coli ATP synthase) from occurring. Therefore, entry of that helix into the
membrane [1, 12] is prevented from taking place. a-c electrostatic interactions cannot take place until
this helix buries its negatively charged Asp-61 and H+ charges inside the membrane, and without the
disequilibration and the electrostatic interactions [1, 12, 15, 19], rotation of the c-rotor cannot occur.
The neutral UH diffuses out, reaches the bulk aqueous phase, dissociates into U- and H+, and the Uand
H+ are pumped against their concentration gradients by the electron transport chain in
mitochondria, as explained earlier [11].
5.5. The Way Forward
Keeping the above discussion and the results of inhibition patterns of Figure 4 in mind, it was not
possible to explain the role of anions for ATP synthesis identified in this work using previous models,
such as the chemiosmotic theory. In a sense, this is clearly understandable, because chemiosmotic
dogma and other models of energy coupling only consider the proton as the coupling ion, and do not
invoke any role for other ions in energy coupling. Recent debate [29, 59, 60] has pointed out other
long-lasting difficulties, inconsistencies, and ambiguities in the earlier theories. However, no way
forward has been suggested [59, 60]. A mechanism that could explain all our experimental findings is
the torsional mechanism of energy transduction and ATP synthesis [1, 2, 10-29]. This mechanism
specifies the detailed events taking place in both the membrane-bound FO and the extra-membrane F1
portions of the enzyme and their temporal order during ATP synthesis. A unique tenet of this
mechanism is the significance and energetic role accorded to membrane-permeable anion translocation
(apart from proton/cation translocation) in providing the total energy needed for the synthesis of ATP.
The binding and unbinding of sequential anion and proton translocations in their respective half-access
channels which are physically located close to each other, the coupled nature of such transport, and the
local and direct energy transduction taking place has been described in consummate detail in the
mechanism [1, 2, 10-13, 15, 19]. The Δψ envisaged in the new theory is localized inside the halfaccess
channels and is created by each elementary act of anion and proton binding to and unbinding
from its binding site on the a- and c-subunit respectively at the a-c interface [10-13, 15]. This localized
Δψ (~35-45 mV due to each elementary act of anion/proton binding or unbinding to/from their
respective half-access channel) has no relationship with the large delocalized Δφ (~120-180 mV in
mitochondria) across bulk aqueous phases presumed to exist in chemiosmosis. (Since both Δψ and Δφ
are symbols for electrical potential differences, but have entirely different meanings in this context, it
is suggested that to avoid confusion, the symbol Δψ be used to represent the local electrical potentials
of the torsional mechanism of energy transduction and ATP synthesis which are created and destroyed
in aqueous access channels, while the symbol Δφ continue to be employed to refer to the delocalized
potential differences presumed to exist across bulk aqueous phases by the chemiosmotic theory). In the
new paradigm, violation of electrical neutrality of bulk aqueous phases to the extremely and
unrealistically large extent postulated in chemiosmosis by uncompensated, electrogenic proton
translocation is avoided [10, 11]. Numerous difficulties with the chemiosmotic theory, and how they
are readily overcome by the torsional mechanism, have been discussed earlier in considerable detail [1,
2, 10-13]. The major differences between the torsional mechanism and the chemiosmotic theory have
been summarized in tabular form on pp. 152-153 in Ref. [2]. In the present paper, the explanations for
the observed patterns of inhibition of ATP synthesis by potent, specific anion channel blockers (Figure
4) have been improved and ideally they serve to replace the previous explanation given in the second
paragraph on page 2221 of Ref. [10]. Revision of the previous theories along the lines described in
detail in the torsional mechanism and in the unified theory in this section provides a way out of the
present impasse, and resolves the problems by its new molecular systems biology ideas and
approaches once and for all [10], and allows us to go beyond the chemiosmotic theory, which is now
outdated, having first come into existence ~50 years ago, and can no longer provide a valid theoretical
basis for further experimental advances or for the progress of further theoretical research in the field in
the future.
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