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In this paper, we provide an overview of mitochondrial bioenergetics and specific conditions that lead to the formation of non-bilayer structures in mitochondria. Secondly, we provide a brief overview on the structure/function of cytotoxins and how snake venom cytotoxins have contributed to increasing our understanding of ATP synthesis via oxidative phosphorylation in mitochondria, to reconcile some controversial aspects of the chemiosmotic theory. Specifically, we provide an emphasis on the biochemical contribution of delocalized and localized proton movement, involving direct transport of protons though the Fo unit of ATP synthase or via the hydrophobic environment at the center of the inner mitochondrial membrane (proton circuit) on oxidative phosphorylation, and how this influences the rate of ATP synthesis. Importantly, we provide new insights on the molecular mechanisms through which cobra venom cytotoxins affect mitochondrial ATP synthesis, mitochondrial structure, and dynamics. Finally, we provide a perspective for the use of cytotoxins as novel pharmacological tools to study membrane bioenergetics and mitochondrial biology, how they can be used in translational research, and their potential therapeutic applications.

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Significance: Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) act as signaling molecules, regulating gene expression, enzyme activity, and physiological responses. However, excessive amounts of these molecular species can lead to deleterious effects, causing cellular damage and death. This dual nature of ROS, RNS, and RSS presents an intriguing conundrum that calls for a new paradigm. Recent Advances: Recent advancements in the study of photosynthesis have offered significant insights at the molecular level and with high temporal resolution into how the photosystem II oxygen-evolving complex manages to prevent harmful ROS production during the water-splitting process. These findings suggest that a dynamic spatiotemporal arrangement of redox reactions, coupled with strict regulation of proton transfer, is crucial for minimizing unnecessary ROS formation. Critical Issues: To better understand the multifaceted nature of these reactive molecular species in biology, it is worth considering a more holistic view that combines ecological and evolutionary perspectives on ROS, RNS, and RSS. By integrating spatiotemporal perspectives into global, cellular, and biochemical events, we discuss local pH or proton availability as a critical determinant associated with the generation and action of ROS, RNS, and RSS in biological systems. Future Directions: The concept of localized proton availability will not only help explain the multifaceted nature of these ubiquitous simple molecules in diverse systems but also provide a basis for new therapeutic strategies to manage and manipulate these reactive species in neural disorders, pathogenic diseases, and antiaging efforts.

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Recently in this Journal, James Lee employed his transmembrane electrostatically localized proton (TELP) hypothesis and the notion of a transient protonic capacitor to explain the force holding protons at the surface of bacteriorhodopsin purple membrane fragments. Here we show that purple membrane fragments cannot maintain the requisite transient non-zero transmembrane potential, and even if they could, it would not support the surface proton current moving from the P side to the N side that was reported by Heberle et al. (Nature, 1994). Currently accepted models explain the force keeping protons at the membrane surface by invoking the unusual structure of water at the interface which serves to stabilize the proton (energy well) and/or raise the activation ∆G‡ (energy barrier) for release to the bulk phase. Any future invocations of TELP should be required to include experimental measurements carried out at the surfaces of lipid bilayer membranes and/or biological membranes.

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Traditionally, proposed molecular mechanisms of fundamental biological processes have been tested against experiment. However, owing to a plethora of reasons-difficulty in designing, carrying out, and interpreting key experiments, use of different experimental models and systems, conduct of studies under widely varying experimental conditions, fineness in distinctions between competing mechanisms, complexity of the scientific issues, and the resistance of some scientists to discoveries that are contrary to popularly held beliefs-this has not solved the problem despite decades of work in the field/s. The author would like to prescribe an alternative way: that of testing competing models/mechanisms for their adherence to scientific laws and principles, and checking for errors in logic. Such tests are fairly commonly carried out in the mathematics, physics, and engineering literature. Further, reported experimental measurements should not be smaller than minimum detectable values for the measurement technique employed and should truly reflect function of the actual system without inapplicable extrapolation. Progress in the biological fields would be greatly accelerated, and considerable scientific acrimony avoided by adopting this approach. Some examples from the fundamental field of ATP synthesis in oxidative phosphorylation (OXPHOS) have been reviewed that also serve to illustrate the approach. The approach has never let the author down in his 35-yr-long experience on biological mechanisms. This change in thinking should lead to a considerable saving of both time and resources, help channel research efforts toward solution of the right problems, and hopefully provide new vistas to a younger generation of open-minded biological scientists.

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Following structural determination by recent advances in electron cryomicroscopy, it is now well established that the respiratory Complexes I-IV in oxidative phosphorylation (OXPHOS) are organized into supercomplexes in the respirasome. Nonetheless, the reason for the existence of the OXPHOS supercomplexes and their functional role remains an enigma. Several hypotheses have been proposed for the existence of these supercomplex supercomplexes. A commonly-held view asserts that they enhance catalysis by substrate channeling. However, this - and other views - has been challenged based on structural and biophysical information. Hence, new ideas, concepts, and frameworks are needed. Here, a new model of energy transfer in OXPHOS is developed on the basis of biochemical data on the pure competitive inhibition of anionic substrates like succinate by the classical anionic uncouplers of OXPHOS (2,4-dinitrophenol, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and dicoumarol), and pharmacological data on the unique site-selective, energy-linked inhibition of energy conservation pathways in mitochondria induced by the guanidine derivatives. It is further found that uncouplers themselves are site-specific and exhibit differential selectivity and efficacy in reversing the inhibition caused by the Site 1/Complex I or Site 2/Complexes II-III-selective guanidine derivatives. These results lead to new vistas and sufficient complexity in the network of energy conservation pathways in the mitochondrial respiratory chain that necessitate discrete points of interaction with two classes of guanidine derivatives and uncoupling agents and thereby separate and distinct energy transfer pathways between Site 1 and Site 2 and the intermediate that energizes adenosine triphosphate (ATP) synthesis by Complex V. Interpretation based on Mitchell's single-ion chemiosmotic theory that postulates only a single energy pool is inadequate to rationalize the data and account for the required complexity. The above results and available information are shown to be explained by Nath's two-ion theory of energy coupling and ATP synthesis, involving coupled movement of succinate anions and protons, along with the requirement postulated by the theory for maintenance of homeostasis and ion translocation across the energy-transducing membrane of both succinate monoanions and succinate dianions by Complexes I-V in the OXPHOS supercomplexes. The new model of energy transfer in mitochondria is mapped onto the solved structures of the supercomplexes and integrated into a consistent model with the three-dimensional electron microscope computer tomography visualization of the internal structure of the cristae membranes in mammalian mitochondria. The model also offers valuable insights into diseased states induced in type 2 diabetes and especially in Alzheimer's and other neurodegenerative diseases that involve mitochondrial dysfunction.

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Mechanisms coupling the chemical reactions of oxidation and ATP synthesis in cellular metabolism by the fundamental biological process of oxidative phosphorylation (OX PHOS) in mitochondria provide > 90% of the energy requirements in living organisms. Mathematical graph theory methods have been extensively used to characterize various metabolic, regulatory, and disease networks in biology. However, networks of energy coupling mechanisms in OX PHOS have not been represented and analyzed previously by these approaches. Here, the problem of biological energy coupling is translated into a graph-theoretical framework, and all possible coupling schemes between oxidation and ATP synthesis are represented as graphs connecting these processes by various intermediates or states. The problem is shown to be transformed into the hard problem of finding a Hamiltonian tour in the networks of possible constituent mechanisms, given the constraints of a cyclical nature of operation of enzymes and biological molecular machines. Accessible mathematical proofs of three theorems that guarantee sufficient conditions for the existence of a Hamiltonian cycle in simple graphs are provided. The results of the general theorems are applied to the set of possible coupling mechanisms in OX PHOS and shown to (1) unequivocally differentiate between the major theories and mechanisms of energy coupling, (2) greatly reduce the possibilities for detailed consideration, and (3) deduce the biologically selected mechanism using additional constraints from the cumulative experimental record. Finally, an algorithm is constructed to implement the graph-theoretical procedure. In summary, the enormous power and generality of mathematical theorems and approaches in graph theory are shown to help solve a fundamental problem in biology.

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In his Transmembrane Electrostatically Localized Proton hypothesis (TELP), James W. Lee has modeled the bioenergetic membrane as a simple capacitor. According to this model, the surface concentration of protons is completely independent of proton concentration in the bulk phase, and is linearly proportional to the transmembrane potential. Such a proportionality runs counter to the results of experimental measurements, molecular dynamics simulations, and electrostatics calculations. We show that the TELP model dramatically overestimates the surface concentration of protons, and we discuss the electrostatic reasons why a simple capacitor is not an appropriate model for the bioenergetic membrane.

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The dynamics of ion translocation through membrane transporters is visualized from a comprehensive point of view by a Gibbs energy landscape approach. The ΔG calculations have been performed with the Kirkwood-Tanford-Warshel (KTW) electrostatic theory that properly takes into account the self-energies of the ions. The Gibbs energy landscapes for translocation of a single charge and an ion pair are calculated, compared, and contrasted as a function of the order parameter, and the characteristics of the frustrated system with bistability for the ion pair are described and quantified in considerable detail. These calculations have been compared with experimental data on the ΔG of ion pairs in proteins. It is shown that, under suitable conditions, the adverse Gibbs energy barrier can be almost completely compensated by the sum of the electrostatic energy of the charge-charge interactions and the solvation energy of the ion pair. The maxima in ΔGKTW with interionic distance in the bound H+ - A- charge pair on the enzyme is interpreted in thermodynamic and molecular mechanistic terms, and biological implications for molecular mechanisms of ATP synthesis are discussed. The timescale at which the order parameter moves between two stable states has been estimated by solving the dynamical equations of motion, and a wealth of novel insights into energy transduction during ATP synthesis by the membrane-bound FOF1-ATP synthase transporter is offered. In summary, a unifying analytical framework that integrates physics, chemistry, and biology has been developed for ion translocation by membrane transporters for the first time by means of a Gibbs energy landscape approach.

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Charge transfer across membranes is an important problem in a wide variety of fundamental physicochemical and biological processes. Since Mitchell's concept of the ion well advanced in 1968, several models of ion translocation across biomembranes, for instance through the membrane-bound FO portion of ATP synthase have been proposed. None of these models has considered the large desolvation free energy penalty of ~500 meV incurred in transferring a protonic charge from the aqueous phase into the membrane that hinders such charge transfer processes. The difficulty has been pointed out repeatedly. However, the problem of how the adverse ∆Gdesolvation barrier is overcome in order to enable rapid ion translocation in biomembranes has not been satisfactorily resolved. Hence the fact that the self-energy of the charges has been overlooked can be regarded as a main source of confusion in the field of bioenergetics. Further, in order to consider charges of a finite size (and not just point charges), the free energy of transferring the ions from water into a membrane phase of lower dielectric εm needs to be evaluated. Here a solution to the longstanding conundrum has been proposed by including the bound anion - the second ion in Nath's two-ion theory of energy coupling and ATP synthesis - in the free energy calculations. The mechanistic importance of the H+ - A- charge pair in causing rotation and ATP synthesis by ion-protein interactions is highlighted. The ∆G calculations have been performed by using the Kirkwood-Tanford-Warshel (KTW) theory that takes into account the self-energies of the ions. The results show that the adverse ∆Gdesolvation can be almost exactly compensated by the sum of the electrostatic free energy of the charge-charge interactions and the dipole solvation energy for long-range ion pairs. Results of free energy compensation using the KTW theory have been compared with experimental data on the ∆G of ion pairs and shown to be in reasonable agreement. A general thermodynamic cycle for coupled ion transfer has been constructed to further elucidate facilitated ion permeation between water and membrane phases. Molecular interpretations of the results and their implications for various mechanisms of energy transduction have been discussed. We firmly believe that use of electrostatic theories such as the KTW theory that properly include the desolvation free energy penalty arising from the self-energy of the relevant ions are crucial for quantifying charge transfer processes in bioenergetics. Finally, the clear-cut implication is that proton-only and single-ion theories of ATP synthesis, such as the chemiosmotic theory, are grossly inadequate to comprehend energy storage and transduction in biological processes.

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Recently, an exchange of views on key fundamental aspects of biological energy coupling and ATP synthesis in the vital process of oxidative phosphorylation appeared in the pages of this journal. The very difficult scientific problems are analyzed and clarified. Errors in the mathematical/thermodynamic equations of a previous analysis have been identified that invalidate previous assertions, and the correct equations are derived. The major differences between the two competing models - localized versus delocalized - for biological energy coupling and transduction are discussed from physical, chemical, and mathematical perspectives. The opposing views are summarized, so that the reader can assess for himself or herself the merits of the two coupling mechanisms. A fresh attempt has been made to go to the root of bioenergetics by calculating the desolvation free energy barrier, ∆Gdesolvation for ion transport across biomembranes. Several constructive suggestions are made that have the power to resolve the basic contradictions and the areas of fundamental conflict, and reach a consensus by catalyzing the progress of future research in this interdisciplinary field.

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In a recent paper entitled "Chemiosmotic misunderstandings", it is claimed that "enough shortcomings in Mitchell's chemiosmotic theory have not been found and that a novel paradigm that offers at least as much explanatory power as chemiosmosis is not ready." This view is refuted by a wealth of molecular-level experimental data and strong new theoretical and computational evidence. It is shown that the chemiosmotic theory was beset with a large number of major shortcomings ever since the time when it was first proposed in the 1960s. These multiple shortcomings and flaws of chemiosmosis were repeatedly pointed out in incisive critiques by biochemical authorities of the late 20th century. All the shortcomings and flaws have been shown to be rectified by a quantitative, unified molecular-level theory that leads to a deeper and far more accurate understanding of biological energy coupling and ATP synthesis. The new theory is shown to be consistent with pioneering X-ray and cryo-EM structures and validated by state-of-the-art single-molecule techniques. Several new biochemical experimental tests are proposed and constructive ways for providing a revitalizing conceptual background and theory for integration of the available experimental information are suggested.

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Theoretical and experimental studies have revealed that that in the liver mitochondria an increase in the rate of free respiration in state 3 induced by protonophore uncouplers 2,4-dinitrophenol and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone is equal to or slightly greater than the increase in respiration rate in state 4 induced by these uncouplers. In contrast to these protonophore uncouplers, the decoupler α,ω-tetradecanedioic acid, increasing the rate of respiration in state 4, does not significantly affect the rate of free respiration in state 3. We have proposed quantitative indicators that allow determining the constituent part of the rate of respiration in state 4, associated with the decoupling effect of the uncoupler. Using the example of palmitic acid, we have found out the fundamental possibility of the simultaneous functioning of uncouplers by two mechanisms: as protonophores and as decouplers. The data obtained contradict the delocalized version of Mitchell's chemiosmotic theory, but are in complete agreement with its local version. It can be assumed that the F0F1-ATP synthase and nearby respiratory chain complexes form a local zone of coupled respiration and oxidative ATP synthesis (zones of oxidative phosphorylation). The uncoupler-induced stimulation of mitochondrial free respiration of mitochondria in state 3 is mainly due to the return of protons to the matrix in local zones, where the generation of a proton motive force (Δр) by respiratory chain complexes is associated with various transport processes, but not with ATP synthesis (zones of protonophore uncoupling). In contrast, respiratory stimulation in state 4 by decouplers is realized in local zones of oxidative phosphorylation by switching the respiratory chain complexes to the idle mode of operation in the absence of ATP synthesis.

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In a recent publication, Manoj raises criticisms against consensus views on the ATP synthase. The radical statements and assertions are shown to contradict a vast body of available knowledge that includes i) pioneering single-molecule biochemical and biophysical studies from the respected experimental groups of Kinosita, Yoshida, Noji, Börsch, Dunn, Gräber, Frasch, and Dimroth etc., ii) state-of-the-art X-ray and EM/cryo-EM structural information garnered over the decades by the expert groups of Leslie-Walker, Kühlbrandt, Mueller, Meier, Rubinstein, Sazanov, Duncan, and Pedersen on ATP synthase, iii) the pioneering energy-based computer simulations of Warshel, and iv) the novel theoretical and experimental works of Nath. Valid objections against Mitchell's chemiosmotic theory and Boyer's binding change mechanism put forth by Manoj have been addressed satisfactorily by Nath's torsional mechanism of ATP synthesis and two-ion theory of energy coupling and published 10 to 20 years ago, but these papers are not cited by him. This communication shows conclusively and in great detail that none of his objections apply to Nath's mechanism/theory. Nath's theory is further consolidated based on its previous predictive record, its consistency with biochemical evidence, its unified nature, its application to other related energy transductions and to disease, and finally its ability to guide the design of new experiments. Some constructive suggestions for high-resolution structural experiments that have the power to delve into the heart of the matter and throw unprecedented light on the nature of coupled ion translocation in the membrane-bound FO portion of F1FO-ATP synthase are made.

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Adenosine triphosphate (ATP) is the universal biological energy fuel, or nature's gasoline. The vast quantities of ATP required for sustenance of living processes in cells are synthesized by oxidative phosphorylation and photosynthesis. The chemiosmotic theory of energy coupling was proposed by Mitchell more than 50 years ago but has a contentious history. Part of the accumulated body of experimental evidence supports Mitchell's theory, and part of the evidence conflicts with the theory. Although Mitchell's theory was strongly criticized by several prominent scientists, the controversy was never resolved. Certain theoretical arguments and electrostatic calculations were originally made to justify the central tenet of the chemiosmotic theory of electrogenic proton transfer and violation of electrical neutrality in bulk aqueous phases by creation of a delocalized field. However, these calculations have not been scientifically scrutinized previously. Here it is proved from first principles that the original physical arguments and calculations made in support of steady state electrogenic ion transfer and chemiosmosis violate Gauss's law. Nath's two-ion theory of energy coupling in which the field is local, and ion translocation is dynamically electrogenic but overall electroneutral is shown to satisfactorily resolve the difficulties. Characterization of length scales in mitochondrial systems is shown to impose strong constraints on possible mechanisms of energy transduction. Some biological implications for energy coupling, transduction and ATP synthesis arising as a result of the above analysis are discussed. Examples of several other biological processes where the new theory is useful such as apoptosis, muscle contraction, the joint multisite regulation of oxidative phosphorylation and the Krebs cycle, and hindered protein aggregation arising from ATP's hydrotropic properties are outlined.

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Understanding how biological systems convert and store energy is a primary purpose of basic research. However, despite Mitchell's chemiosmotic theory, we are far from the complete description of basic processes such as oxidative phosphorylation (OXPHOS) and photosynthesis. After more than half a century, the chemiosmotic theory may need updating, thanks to the latest structural data on respiratory chain complexes. In particular, up-to date technologies, such as those using fluorescence indicators following proton displacements, have shown that proton translocation is lateral rather than transversal with respect to the coupling membrane. Furthermore, the definition of the physical species involved in the transfer (proton, hydroxonium ion or proton currents) is still an unresolved issue, even though the latest acquisitions support the idea that protonic currents, difficult to measure, are involved. Moreover, FoF1-ATP synthase ubiquitous motor enzyme has the peculiarity (unlike most enzymes) of affecting the thermodynamic equilibrium of ATP synthesis. It seems that the concept of diffusion of the proton charge expressed more than two centuries ago by Theodor von Grotthuss is to be taken into consideration to resolve these issues. All these uncertainties remind us that also in biology it is necessary to consider the Heisenberg indeterminacy principle, which sets limits to analytical questions.

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Starting from the universal concept of entropy production, a large number of new results are obtained and a wealth of novel thermodynamic, kinetic, and molecular mechanistic insights are provided into the coupling of oxidation and ATP synthesis in the vital process of oxidative phosphorylation (OX PHOS). The total dissipation, Φ , in OX PHOS with succinate as respiratory substrate is quantified from measurements, and the partitioning of Φ into the elementary components of ATP synthesis, leak, slip, and other losses is evaluated for the first time. The thermodynamic efficiency, η , of the coupled process is calculated from the data on Φ and shown to agree well with linear nonequilibrium thermodynamic calculations. Equations for the P/O ratio based on total oxygen consumed and extra oxygen consumed are derived from first principles and the source of basal (state 4) mitochondrial respiration is postulated from molecular mechanistic considerations based on Nath's two-ion theory of energy coupling within the torsional mechanism of energy transduction and ATP synthesis. The degree of coupling, q , between oxidation and ATP synthesis is determined from the experimental data and the irreversible thermodynamics analysis. The optimality of biological free energy converters is explored in considerable detail based on (i) the standard biothermodynamic approach, and (ii) a new biothermokinetic approach developed in this work, and an effective solution that is shown to arise from consideration of the molecular aspects in Nath's theory is formulated. New experimental data in state 4 with uncouplers and redox inhibitors of OX PHOS and on respiratory control in the physiological state 3 with ADP and uncouplers are presented. These experimental observations are shown to be incompatible with Mitchell's chemiosmotic theory. A novel scheme of coupling based on Nath's two-ion theory of energy coupling within the torsional mechanism is proposed and shown to explain the data and also pass the test of consistency with the thermodynamics, taking us beyond the chemiosmotic theory. It is concluded that, twenty years since its first proposal, Nath's torsional mechanism of energy transduction and ATP synthesis is now well poised to catalyze the progress of experimental and theoretical research in this interdisciplinary field.

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A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F1FO-ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications.

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A wealth of molecular mechanistic insights has been provided into the coupling of ion transport to ATP synthesis based on a two-ion theory of biological energy coupling. A kinetic scheme that considers the mode of functioning of a single F1FO-ATP synthase molecule with H+-A- cotransport and unidirectional rotation of the c-rotor in the membrane-bound FO-portion of the enzyme has been developed. Mathematical analysis leads to a detailed enzyme kinetic model applicable to a population of molecules which is compared with experimental data on the pH dependence of ATP synthesis. The model agrees well with the experimental data, and a single equation with a single set of standard enzymological kinetic parameters has been shown to explain the experimental data over the entire range of conditions for the chloroplast ATP synthase. The analysis gives novel insights into kinetic and mechanistic characteristics of ATP synthesis in FO. These include an order imposed on ion binding and unbinding events in FO, the essential role of the anion in direct activation of the ATP synthase (in addition to its role as a permeant ion), and the integration in a novel way of the functions of cooperativity and cotransport of dicarboxylic acid anions and protons during physiological ATP synthesis. Further, Wyman's pioneering classical work on the thermodynamics of linked functions has been shown to offer a new approach to distinguish between various models of energy coupling in ATP synthesis. All these results have been found to be inconsistent with Mitchell's chemiosmotic theory and are shown to be in agreement with Nath's torsional mechanism of energy transduction and ATP synthesis.

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We have recently criticised the natural pH gradient hypothesis which purports to explain how the difference in pH between fluid issuing from ancient alkali vents and the more acidic Hadean ocean could have driven molecular machines that catalyse reactions that are useful in prebiotic and autotrophic chemistry. In this article, we temporarily suspend our earlier criticism while we consider difficulties for primitive organisms to have managed their energy supply and to have left the vents and become free-living. We point out that it may have been impossible for organisms to have acquired membrane-located proton (or sodium ion) pumps to replace the natural pH gradient, and independently to have driven essential molecular machines such as the ATP synthase. The volumes of the ocean and of the vent fluids were too large for a membrane-located pump to have generated a significant ion concentration gradient. Our arguments apply to three of the four concurrent models employed by the proponents of the natural pH gradient hypothesis. A fourth model is exempt from these arguments but has other intrinsic difficulties that we briefly consider. We conclude that ancient organisms utilising a natural pH gradient would have been imprisoned in the vents, unable to escape and become free-living.

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The vital coupled processes of oxidative phosphorylation and photosynthetic phosphorylation synthesize molecules of adenosine-5'-triphosphate (ATP), the universal biological energy currency, and sustain all life on our planet. The chemiosmotic theory of energy coupling in oxidative and photophosphorylation was proposed by Mitchell >50years ago. It has had a contentious history, with part of the accumulated body of experimental evidence supporting it, and part of it in conflict with the theory. Although the theory was strongly criticized by many prominent scientists, the controversy has never been resolved. Here, the mathematical steps of Mitchell's original derivation leading to the principal equation of the chemiosmotic theory are scrutinized, and a fundamental flaw in them has been identified. Surprisingly, this flaw had not been detected earlier. Discovery of such a defect negates, or at least considerably weakens, the theoretical foundations on which the chemiosmotic theory is based. Ad hoc or simplistic ways to remedy this defect are shown to be scientifically unproductive and sterile. A novel two-ion theory of biological energy coupling salvages the situation by rectifying the fundamental flaw in the chemiosmotic theory, and the governing equations of the new theory have been shown to accurately quantify and predict extensive recent experimental data on ATP synthesis by F1FO-ATP synthase without using adjustable parameters. Some major biological implications arising from the new thinking are discussed. The principles of energy transduction and coupling proposed in the new paradigm are shown to be of a very general and universal nature. It is concluded that the timely availability after a 25-year research struggle of Nath's torsional mechanism of energy transduction and ATP synthesis is a rational alternative that has the power to solve the problems arising from the past, and also meet present and future challenges in this important interdisciplinary field of research.

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