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Biology Articles » Biophysics » Medical Biophysics » The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease » Beyond the Chemiosmotic Theory: Details of the Molecular Mechanism of EnergyTransduction in the Membrane-bound FO Portion of F1FO-ATP Synthase

Beyond the Chemiosmotic Theory: Details of the Molecular Mechanism of EnergyTransduction in the Membrane-bound FO Portion of F1FO-ATP Synthase
- The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease

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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