In this last article of the trilogy, the unified biothermokinetic theory of ATP synthesis developed in the previous two papers is applied to a major problem in comparative physiology, biochemistry, and ecology-that of metabolic scaling as a function of body mass across species. A clear distinction is made between intraspecific and interspecific relationships in energy metabolism, clearing up confusion that had existed from the very beginning since Kleiber first proposed his mouse-to-elephant rule almost a century ago. It is shown that the overall mass exponent of basal/standard metabolic rate in the allometric relationship [Formula: see text] is composed of two parts, one emerging from the relative intraspecific constancy of the slope (b), and the other (b\') arising from the interspecific variation of the mass coefficient, a(M) with body size. Quantitative analysis is shown to reveal the hidden underlying relationship followed by the interspecific mass coefficient, a(M)=P0M0.10, and a universal value of P0=3.23 watts, W is derived from empirical data on mammals from mouse to cattle. The above relationship is shown to be understood only within an evolutionary biological context, and provides a physiological explanation for Cope\'s rule. The analysis also helps in fundamentally understanding how variability and a diversity of scaling exponents arises in allometric relations in biology and ecology. Next, a molecular-level understanding of the scaling of metabolism across mammalian species is shown to be obtained by consideration of the thermodynamic efficiency of ATP synthesis η, taking mitochondrial proton leak as a major determinant of basal metabolic rate in biosystems. An iterative solution is obtained by solving the mathematical equations of the biothermokinetic ATP theory, and the key thermodynamic parameters, e.g. the degree of coupling q, the operative P/O ratio, and the metabolic efficiency of ATP synthesis η are quantitatively evaluated for mammals from rat to cattle. Increases in η (by ∼15%) over a 2000-fold body size range from rat to cattle, primarily arising from an ∼3-fold decrease in the mitochondrial H+ leak rate are quantified by the unified ATP theory. Biochemical and mechanistic consequences for the interpretation of basal metabolism, and the various molecular implications arising are discussed in detail. The results are extended to maximum metabolic rate, and interpreted mathematically as a limiting case of the general ATP theory. The limitations of the analysis are pointed out. In sum, a comprehensive quantitative analysis based on the unified biothermokinetic theory of ATP synthesis is shown to solve a central problem in biology, physiology, and ecology on the scaling of energy metabolism with body size.

The nonequilibrium coupled processes of oxidation and ATP synthesis in the fundamental process of oxidative phosphorylation (OXPHOS) are of vital importance in biosystems. These coupled chemical reaction and transport bioenergetic processes using the OXPHOS pathway meet >90% of the ATP demand in aerobic systems. On the basis of experimentally determined thermodynamic OXPHOS flux-force relationships and biochemical data for the ternary system of oxidation, ion transport, and ATP synthesis, the Onsager phenomenological coefficients have been computed, including an estimate of error. A new biothermokinetic theory of energy coupling has been formulated and on its basis the thermodynamic parameters, such as the overall degree of coupling, q and the phenomenological stoichiometry, Z of the coupled system have been evaluated. The amount of ATP produced per oxygen consumed, i.e. the actual, operating P/O ratio in the biosystem, the thermodynamic efficiency of the coupled reactions, η, and the Gibbs free energy dissipation, Φ have been calculated and shown to be in agreement with experimental data. At the concentration gradients of ADP and ATP prevailing under state 3 physiological conditions of OXPHOS that yield Vmax rates of ATP synthesis, a maximum in Φ of ∼0.5J(hmgprotein)-1, corresponding to a thermodynamic efficiency of ∼60% for oxidation on succinate, has been obtained. Novel mechanistic insights arising from the above have been discussed. This is the first report of a 3 × 3 system of coupled chemical reactions with transport in a biological context in which the phenomenological coefficients have been evaluated from experimental data.

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.

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.

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.