The physiological role of complex V in ATP synthesis: Murzyme functioning is viable whereas rotary conformation change model is untenable

Abstract Complex V or FoF1-ATPase is a multimeric protein found in bioenergetic membranes of cells and organelles like mitochondria/chloroplasts. The popular perception on Complex V deems it as a reversible molecular motor, working bi-directionally (breaking or making ATP) via a conformation-change based chemiosmotic rotary ATP synthesis (CRAS) mechanism, driven by proton-gradients or trans-membrane potential (TMP). In continuance of our pursuits against the CRAS model of cellular bioenergetics, herein we demonstrate the validity of the murburn model based in diffusible reactive (oxygen) species (DRS/DROS). Supported by new in silico derived data (that there are ∼12 adenosine nucleotide binding sites on the F1 bulb and not merely 3 sites, as perceived earlier), available structural information, known experimental observations, and thermodynamic/kinetic considerations (that de-solvation of protons from hydronium ions is facile), we deduce that Complex V serves as a physiological chemostat and a murzyme (enzyme working via murburn scheme, employing DRS). That is- Complex V uses ATP (via consumption at ε or proteins of F1 module) as a Michaelis-Menten substrate to serve as a pH-stat by inletting protons via the c-ring of Fo module. Physiologically, Complex V also functions as a murzyme by presenting ADP/Pi (or their reaction intermediates) on the αβ bulb, thereby enabling greater opportunities for DRS/proton-assisted ATP formation. Thus, the murburn paradigm succeeds the CRAS hypothesis for explaining the role of oxygen in mitochondrial physiologies of oxidative phosphorylation, thermogenesis, TMP and homeostasis. Communicated by Ramaswamy H. Sarma


Introduction
While some important problems in biology/medicine continue to remain unsolved conundrums for long (e.g. what are the roles of unique alkaloids produced in plants, how do anesthetics work on animal nervous system, etc.), some other issues are hotly debated. How is ATP, the energy currency of life, synthesized in a living cell? This is a concern of fundamental significance and most basic biochemistry textbooks do address this topic. Since the last five years, the issue has once again reached flammability points, reminiscent of the "oxphos wars" on the subject prevalent in the mid-parts of 20 th century. In this context, we investigate the currently prevailing structure-function correlations on Complex V (the 'molecular motor enzyme' that is purported to turn over the high amounts of ATPs required for maintaining cellular function) and inspect its validity, fundamentality, even its historical background, and then delineate our new perspective based in murburn concept. This article is contributed keeping with the core philosophy that-"When a proposal is demonstrated to be invalid or a hypothesis is nullified, the outcome is definitive and enables the search for alternative viable solutions. Thus, the pursuit of science advances by negation." Therefore, scientific pursuits have witnessed that some highly popular perceptions did undergo gradual or dramatic paradigm shifts. We envisage such a perception-change imminent with a critical inspection of the popular ideas on Complex V. In recent times, we have comprehensively addressed the various aspects of this subject, pointed out the errors in history, and provided new murburn conceptbased explanations for bioenergetic oxidative-/photo-phosphorylations in diverse life forms (e.g. Manoj, 2018a;Manoj, 2018b;Manoj et al., 2019aManoj et al., , 2019bManoj et al., 2021aManoj et al., , 2021bManoj et al., , 2021c; and the relevant citations mentioned therein). Murburn concept is an anagram of 'mured burning' (closed oxidation) and espouses a ubiquitous 'molecule-unbound ion-radical' interaction scheme (Manoj, 2018c). Contrary to the currently prevailing mainstream perceptions that consider one-electron active diffusible reactive (oxygen) species (DRS/DROS) as toxic and wasteful intermediates, murburn concept advocates that reactions involving such DRS are the mainstay of routine life. The new murburn proposal considers respiratory Complexes I through IV as the major ATP-synthases and attributes secondary roles for Complex V towards ATP-synthesis. Herein, we provide an analytical/theoretical/critical investigative write-up focusing on the activity of Complex V alias F o F 1 ATPase. A demonstrable ATPase in vitro, this protein complex is also considered to work reversibly as the ATP-synthase of mitochondria/chloroplasts of eukaryotes and periplasmic membrane of eubacteria/archaea. Citing the case of lactate dehydrogenase, we had also shown why the perception of "reversibility" is not facile for physiological reactions with significant thermodynamic barriers (Manoj et al., 2021d). Herein, we project how/why the multi-phasic and multi-component protein of Complex V cannot be the primary physiological ATP-synthase of diverse living organisms, although it may enable some ATP-synthesis (both in vitro and in situ) and enhance ATP-yields in steady-state. We also present details of the much simpler murburn model for the structure-function correlations of Complex V and delineate agenda for further research.

In silico explorations in this study
Identification of probable binding sites of both ADP and ATP was explored by molecular docking of the flexible small molecule ligand with rigid Complex V of different species. The substrates, ADP and ATP, were retrieved in their 3 D format from PubChem and the crystal structures of Complex V from various species were retrieved from Protein Data Bank (PDB; rcsb.org). Eukaryota -Mammals (Bos), Plants (Spinacia) and Yeast (Saccharomyces); Bacteria -Gram positive (Caldalkalibacillus) and Gram negative (Thermusthermophilus) and Archaea (Nanoarchaeum) were selected for this study. Docking was mainly focused around the reported Walker Motifs on both a and b subunits and on the ATP binding site at e subunit. The PDB IDs used in this study are as follows: 1H8E and 2WSS (Bos), 1FX0 and 6VON (Spinacia), 2HLD (Saccharomyces), 5HKK (Caldalkalibacillus), 3W3A (Thermusthermophilus) and 5BN4 (Nanoarchaeum). Docking was done by creating grid boxes (as shown in Figure 1B) through the AutoGrid module followed by running the AutoDock 4.2 program (Morris et al., 2009). Auto Dock Tools, a module in the MGL Tools 1.5.6 was used to prepare the proteins in their 'pdbqt' format (Sanner, 1999). The ligands were prepared in the 'pdbqt' format with the help of OpenBabel (O'Boyle et al., 2011). After selecting the grid with spacing of 0.375 Å, Lamarckian genetic algorithm with a run of 20 was used to execute the docking. The results were visualized and analyzed for interactions using Chimera 1.12 (Pettersen et al., 2004). Data pertaining to binding energies and corresponding dissociation constants were obtained from the log files.

Solvation
We will consider the process of solvation of a proton in the form of two reactions: the addition of a proton to a water molecule in the gas phase and the solvation of the formed ion H 3 O þ gas : In the case of enthalpy we have Carrying out similar calculations for the Gibbs function, Therefore, we find For G function we have   Alberty (2003) and in our recent papers Manoj et al., 2021d).

Analyses, results & discussion
3.1. The rationale and criteria discrediting the CRAS model of complex V For a brief awareness on structure-function aspects of Complex V and how Boyer's CRAS (chemiosmotic rotary ATP synthesis) mechanism came to become the acclaimed explanation in bioenergetics, please refer the respective Items 1 & 2 of Supplementary Information (SI) file. In a critique published a few years back, one of us had systematically dissected most of the purported evidence/arguments available till date for the ETC (electron transport chain)-CRAS proposal, demonstrating the specific problems in each one of the components, and also showed how/why the various aspects of the explanations are unreal and just don't add up (Manoj, 2018b). Such crucial insight was enabled by the wealth of structural information and experimental observations reported by diverse researchers over the past half a century. Based on the critical insights availed in redox biochemistry spanning over two decades, we also put up a murburn-based model for explaining the bioenergetic phosphorylations Manoj et al., 2021aManoj et al., , 2021b and citations from Manoj's group therein). Herein, we focus first only on salient/greater issues pertaining to the activity of Complex V (particularly in the physiological context) and subsequently we shall detail how murburn model explicates things better. The critical arguments presented below are made due to four deductive logical standpoints: 1. There exists no direct and/or conclusive evidence till date to support Boyer's original hypothesis that Complex V is a rotary or motor like molecular machine, under physiological conditions. The wholesome/pure A. An overview of important motifs and chains is presented using the crystal structure of F-ATPase from Bos taurus (PDB ID: 2WSS) (Rees et al., 2009). The position of DELSEED (black) is on the b-monomer close to c-shaft. Walker motifs are shown in blue and green on a-monomer (sky blue) and b-monomer (pale yellow). The extension of c-shaft is presented in red and also with a distinct ribbon structure. The other two associated chains with the head of c-shaft are shown in pale tan (for d) and magenta (for e, another known site of ATP binding). Both a and b-monomers are shown from the inner core side by hiding other four monomers to provide a clarity on the positioning of the above mentioned motifs. B. Displays the grid boxes taken to scan for potential ADP and ATP binding sites. The grid covers the Walker motif regions on both aand b-monomers. Four monomers (two of a and two of b) are faded to depict the grid prominently.
Complex V is a demonstrable ATPase in vitro, not an ATPsynthase. Some indirect and suggestive in vitro demonstrations with highly constrained and truncated derivatives of Complex V cannot be considered physiologically relevant. 2. Even if a low probability outcome of a rotary activity can be assumed to be operative in physiology, it is highly unlikely that it can work both clockwise and counterclockwise. (Another major concern is the difficulty to have spontaneously assembled rotary modules with bidirectional fidelity.) To rephrase the example made famous by Stephen Hawking: while a hot cup of tea can fall and spontaneously break into pieces with the tea spilling out and losing its heat, it is unlikely that the reverse can also happen spontaneously in time. In this regard, it is easier to accept that rotary ATPase activity might be a low probability event, but we deem that rotary ATPsynthase is an impossible event for Complex V. That is: the rotary reaction "ATP þ Water ¼ ADP þ Pi" (ATPase) appears somewhat probable whereas "ADP þ Pi ¼ ATP þ Water" (ATPsynthase) appears to be impossible to achieve with Complex V. 3. Even if a bidirectional rotary activity exists in Complex V due to some highly targeted evolutionary outcome, it is inconceivable how such a rotary motion translates to an endergonic bond formation between two negatively charged species (ADP and Pi). Just by saying that the energy of oxidation is required to push out a tightly bound ATP as the crucial step does not sound fundamental or monumental. The CRAS postulate that "K eq ' for ATP synthesis on the enzyme surface is near zero whereas the K eq ' for the reaction in free solution is 10 À5 … . F o F 1 binds ATP with very high affinity (K d 10 À12 M) and ADP with much lower affinity (K d % 10 À5 M). The difference in K d corresponds to a difference of about 40 kJ/mol in binding energy, and this binding energy drives the equilibrium toward formation of the product ATP. (Lehninger et al., 2004)" is false. High binding energy of ATP can be used for ATP hydrolysis, not for the formation of ATP. How does an environment that has zilch free protons pump out protons and if such a thing occurred, how does the proton support the ATP-synthesis when it is not involved in the bond formation step (as per Boyer proposal)? Also, in steady state, there is much higher physiological concentration of ATP than ADP, and there are zilch protons in the matrix to drive the equilibriumdriven "ADP þ Pi þ proton ¼ ATP þ Water" reaction (which is probable in vitro, as per the murburn interpretation of Jagendorf's experiment). This reaction is feasible because proton contributes a value of 452 kJ/mol on the favorable side of the equation. But this proton gets consumed in the murburn explanation (quite unlike the CRAS model, where the proton entering inward between the c-ring and protein-a is not involved in the reaction, but merely serves to push the gamma-rotor) . 4. Given the complexity of the mitochondrial composition, it is difficult to attribute the credit of bulk ATP synthesis to Complex V, although it may possess some reversibility. When isolated mitochondria, or mitoplasts or sub-mitochondrial particles are studied and ATP synthesis is observed, it is difficult to discriminate whether the outcome is due to Complex V or other agencies (like the redox complexes, which could also serve as murzyme ATP-synthases). And if the outcome arises due to a collaborative participation, it is difficult to gauge what is the contribution of each element. Let us re-assert: just because Complex V is an ATPase and it was found to be essential for physiological ATP synthesis, it does not mean that Complex V is the physiological ATPsynthase. We point out an analogy: In the bi-enzymatic heme-flavin cytochrome P450-reductase system, it was earlier believed that it was cytochrome P450 that bound oxygen and activated it (in accordance with the other proteins like hemoglobins). This false belief was reversed with the murburn explanations (Manoj et al., 2016;. Now, we move on to the specific criteria. To anyone concerned, the most important aspect is: how is the endergonic bond formation of ATP (bringing together reactants with negatively charged phosphate moieties) coupled with oxidation of respiratory substrates? Surely, a simpler solution must be present. For, what the classical explanation presents is a highly deterministic and definitely 'irreducibly complex' proposal. We present our critical arguments under the following subheads-3.1.1. Structure-function correlations that translate into mechanical/mechanistic aspects i. Demonstration of rotation by sticking the alpha-beta bulb onto a slide is unsatisfactory. If the gamma þ epsilon þ cring association is significant, introduction of torsion in cring would easily destabilize the Complex V structure. Also, since the rotation of the c-ring is the most crucial aspect in coupling chemiosmosis with rotation, the Noji experiment (Noji et al., 1997) does not translate to any discernible interpretation with physiological relevance. Further, the ratio of linear dimension of sigma stalk to the length of attached actin filament was 1:100. We find it difficult to accept that a purported torque exerted on the slender gamma stalk enables it to plough through the alpha-beta bulb and also account for the immense hydrodynamic drag that long actin filament would experience. (In the bacterial flagellar motors, for a radial rotation of the rotor with much lesser hydronamic drags, thousands of protons are needed. For an incredibly long and perpendicularly connected actin vane that would experience much higher drag, the Noji demonstration was made with ATP hydrolysis, and not with the 2-3 protons that is sought in Boyer's proposal for physiological ATP synthesis.) However, the simpler explanation for the original experiment could be that a mechanical twanging of the gamma filament (due to ATP hydrolysis) could send a rippling vibration, which could work finally as a torque due to a wobble at the gamma-(biotin-avidin)-actin junction. All that is needed to perpetuate the rotation of the actin filament is the continuous twanging (mechanical perturbation) at the base. Even otherwise, actin is also known to bind to ATP and show mechanized movements thereafter. So, it was important to demonstrate via a suitable negative control that the rotation seen was not within the junction of the actin filament with the gamma stalk. This could have been possible with a covalent linking of the gamma stalk with the alpha-beta bulb. In this regard, strong counter-evidence against gamma-rotation being a contributor to ATPase activity exists. It was shown by cross-linking of the gamma-rotor using maleimide functionality (specific for sulfhydryl groups) that any purported rotary effect had insignificant impact on the activity of Complex V (Musier & Hammes, 1987). This is not expected if the gamma module rotated within the alpha-beta bulb. Such experiments with truncated single molecules do not demonstrate a realistic rotation or the lack of rotation of the gamma-stalk of Complex V. It is neither a demonstration of ATP synthesis by movement of protons enabling a rotary movement in Complex V. In this regard, the proposal that "the secondary structure of the flexible DELSEED loop in the beta-monomers serves as a rigid hinge type of framework for the gamma stalk to rotate" is purely wishful thinking. If the structure was a well-defined helix of low thermal signature, there could have been some merit. It must be noted that the gamma stalk is slender at the distal base (into the bulb) and broader at the proximal side (towards the c-ring) ( Figure 1). If the movement of protons can exert a torque at c-ring (as strongly as the one demonstrated to rotate the actin filament), such a force should easily unscrew the gamma stalk from the alpha-beta bulb. ii. It was known that each of the beta monomer has two adenosine nucleotide binding sites (ANBS) (Khananshvili & Gromet-Elhanan, 1984). While it may be possible that one site per monomer can change conformations from high affinity for ATP to low affinity for ATP, it is quite unlikely that all six ANBS of Complex V can deterministically switch conformations and cooperate to serve the mandates of Boyer's proposal. Particularly, if one sees that the alpha monomers are intercalated between the beta-subunits and some subunits are connected to peripheral stalks also (rendering the structure asymmetric), Boyer's deterministic proposal looks stretched out. Such a predicament makes Complex V's structure even more incapable for the rotary reversibility of functions. iii. While it is possible that a given directional movement of gamma stalk may enable the transition of a beta monomer's binding site from high binding affinity for ATP (the native state) to low binding affinity for ATP, it does not make the converse to be true. That is, changing the direction of the movement of gamma stalk alone does not now make the Complex V to have the ANBS preferring ADP. This is because the Walker site retains its original high affinity for ATP, regardless of the direction of gamma's rotation. This is a mirage woven in Boyer's model, quite like the closed circulation of protons in Mitchell's proposal.
iv. The structure of the F o or F 1 module varies significantly across various species, particularly with respect to the number of c subunits. For example, in cyanobacteriathe c ring can be anywhere between 13 to 15 monomers (Pogoryelov et al., 2007) and they are known to vary in the range of 8 to 17 in diverse organisms (Davis & Kramer, 2019). Therefore, in the F-ATPases, there is a serious symmetry mismatch of 3-stroke rotation in F 1 whereas variable number of monomers in c-ring (wherein both odd and even numbered, without being a multiple of 3). This is also seen in the most primitive life forms of archaea, as shown in Table 1. Such a reality questions the evolutionary relevance of the logic of rotation and counters the basic notion of rotability within the protein complex as a criterion for vital function. It is clearly discernible that the number of monomers within the c-ring decrease as the habitat temperature goes up because of issues associated with spontaneous aggregation. In physiology, the purported stator is not placed in a true firmament (Item 1, SI) and therefore, the fundamental comparison of Complex V with a motor or generator is inapplicable. The analysis of the articulation of the purported stator protein-a with the rotor c-ring by Rubinstein's group (Mazhab-Jafari & Rubinstein, 2016; Schep et al., 2016) is re-interpreted and attached as Figure 2. We find that the structural features (number of helices, relative orientations, etc.) and overall dimensions of the protein-a varies significantly across species. Further, although there are a few acidic (glutamate/aspartate) and basic (arginine) residues near the peripheries to both the phases' (say, matrix and inter-membrane space in mitochondria) and within the interior of protein-a, the depths/separation/relative alignments of these residues vary significantly between the various ATPases ( Figure  2). Rather than enabling a rotary activity, this reality appears more congenial for their role as stabilizers for binding of protein-a with the c-ring, as shown in the bottom panel of Figure 2. A functional stator should be anchored in a stable firmament, and in the fluidic membrane, this is only possible with a confinement in four (separated by 90 degrees) or more protein-a units. Minimally, three a-subunits separated by 120 degrees would be required to accommodate the spatial strains introduced by torsion/rotation of the gamma and c-ring combine. (Since the various flexible and elastic components of both membrane and soluble phases' modules is a reality, this outcome cannot be avoided.) The evaluation of V/A-ATPases clearly shows that the peripheral stalks are not tethered to the "firmament", as needed and solicited for rotation (compare Figure 5 of Stewart et al., 2013 and the expected mooring solicited in our Figure 2).
v. An inspection of the gamma stalk shows it to be a rather frail structure, and it is also known to have significant structural flexibility ( (Abrahams et al., 1994;Stock et al., 1999;Giraud et al., 2012). This is not expected if the cring has to articulate with the gamma and both rotate in two distinct phases. Several elastic (non-rigid) connectives are found in components that link the rotor and stator functions of F 1 and F o modules (Uchihashi et al., 2011). Such a system would be destabilized if torsional forces were generated in the system. None of these real structural aspects were put to test in the Noji type of experiment. This experiment was not explorative, but was powered by the established mandate of demonstrating rotary movement. When a dog laps up water, we tend to imagine that the upper surface of tongue the scoops up water towards the buccal roof. Actually, it is the lower surface of the tongue that is used as a garden spade to spool water into the lower mandibular cavity. Analogously, in this context, the enzymatic mechanism of Complex V can be understood if we steer clear of earlier primed and pre-charted ideas, and approach with a clean slate. vi. There exists significant structural variability in the structure of the F-type ATPases, abundantly found in mitochondria and chloroplasts of eukaryotes, and also in the periplasmic membrane of eubacteria (von Ballmoos et al., 2008). A comparative view of the assembled and dismantled components of the F-V-A ATPases in Figure 5 of the excellent review by Stewart et al. (2013) clearly shows the discerning thinker that such structures cannot afford any sustainable rotary motion. vii. Till date, to our knowledge, naturally assembled sustainable rotary modules are extremely rare (although some Figure 2. The top row images of the a-proteins from the various classes of ATPases are from Mazhab-Jafari and Rubinstein (2016). The images have been rendered against a black background to enable relative size delineation. In the images, the spheres are negatively charged residues present in the lumenal, membrane-core and cytoplasmic sides of the a-protein, as demarcated by three respective ellipses (from left to right, respectively) within each image. The bottom row presents an interaction scheme based on structural evidence suggesting that the c-ring and a-protein could be held together at various loci (at any surface of contact) via hydrophobic, H-bond and other canonical interactions. Given the fluidic nature of the lipid membrane, for efficient role as a stator, protein-a must be multimeric and linked together, and the whole structure should also enclose/encircle the c-ring.
helical or gyrating movements are seen) in nature. All "willfully man-made rotary machines" (whether a rotary combustion engine, electrical motor or turbine power generator) need precision engineering, deterministic assembly with synchronized articulation of components at multiple points, in a phased out manner. Also, the principle of electromagnetic induction operates in the motors, wherein the rotor gets to rotate without contact, due to changing direction of the electromagnetic fieldand this could give reversibility in rotation. No such thing exists in the purely mechanical system advocated by Boyer. In the mechanical rotation systems such as generators/turbines/windmills, reversibility in direction cannot be achieved with the same arrangement of modular components and a mere change in the direction of a 'force' (here, protons analogous to wind in a gas turbine). The c-ring cannot rotate in the lipid environment and be attached to both gamma stalk and protein-a also. We come to the inevitable and evident deduction that structurally, Complex V is incapable of sustained rotation. The structures available only show that the various components are relatively immobilized with respect to each other. Also, a thoughtful comparison of Complex V activity to all the examples of the three manmade rotary machines discussed in this writeup would show that none of the three workable machines have a complete set of equivalents with Complex V functioning. Therefore, the rotary functionality of Complex V is just wishful thinking and only the wildest of possible imagination (at least in the ways projected/conceived till date) can it be seen as a wholesome rotary nano-machine.

Energetics
In this essay, we shall not delve into all of the thermodynamic misadventures of ETC-CRAS proposal, but focus only on the stark inadequacies of Boyer's ideas. In retrospect, Boyer's revolutionary claims (of Complex V using energy from oxidation of respiratory substrates to push out the tightly bound ATP via a rotary movement) can be equated to the falsity of Mitchell's idea of membrane proteins pumping out protons and harnessing their energy upon return across the same membrane. Both had little foundations in reality and no real purpose (in the context of the problem at hand), and originated due to a poorly prioritized categorization of agenda. In Mitchell's case, the mirage was woven on the premise that ample protons existed in matrix and that they could be pumped out and the PoPs (pumped out protons) could somehow conserve the energy. (All three proposals are false.) In Boyer's theorization, ATP formation was a de-facto unaddressed agenda, as he assumed that on the enzyme surface, the energy of transition state complexes of "E-ADP þ Pi" or "E-ATP þ water" were more or less similar, and energy was required primarily to push out tightly bound ATP. This does not address the fundamental problem (the predicament of reversing a reaction of $35 kJ/mol was feasible?) at all, and just attributes some miraculous ability to the enzyme.
Suggesting that a high affinity of Complex V for ATP could somehow overcome the energy barriers of reaction in the reverse direction is unheard in enzymology and untenable in a thermodynamic purview, under physiological conditions. Moreover, the way Boyer connected the rotary activity to movement of protons was also energetically and mechanistically unviable, as there is no support for the same. There was no real accounting for the source of energy. That is-in the abridged Mitchell model's equation, the major conserved energy was of electrical nature. Then, how could Complex V (which lacked any electrically active components) serve as a transducer of the electrical potential? How could a steady-state macroscopic non-variant electrical potential be tapped specifically at the active site? How are the rotary movements connected to phosphohydride bond formation? It is bewildering to consider how a proton of mere 1 Da present in one phase can move a massive and non-rigid protein assembly of c-ring and ce combine (>107 KDa) through three phases (the 'rotor' is wedged in membrane þ protein-a, exposed to aqueous ambiance, and wedged in the alpha-beta bulb). Another big error was/is considering that cellular reactions mediated by enzymes are always reversible. In the energetics perspective, the CRAS model does not bear any merit whatsoever. For the ATP synthase activity, the rotor movement requires significant energy input, which must be much higher than 35 kJ/mol (which is the energy required to give the synthesis of an ATP molecule). As per the rotary mechanism, a single proton must be able to move the c-ring, so as to enable the attachment of the next proton at the junction of membrane-c-ring-protein-a, to enable the movement of the sigma rotor and move the "pre-synthesized ATP" out. In physiology, there is no pH gradient and the water movement against the turbine analogy for moving the c-ring is inapplicable. The point is to see that despite all these stunts, Boyer's rotary mechanism still does not explain the phosphohydride bond formation and the respiratory substrate oxidation is still not connected to ATP-synthesis. Item 3, SI provides the mechanistic-energetic non-viability of Boyer's proposal, with a simple comparative perspective.

Kinetics & probability of reactions:
The premise that pure Complex V can only hydrolyze ATP and the fact that Complex V exhibits much higher affinity for ATP than ADP (Lehninger et al., 2004) condemn Complex V to serve as an ATPase, i.e. the kinetics of the enzyme-mediated equilibrium is heavily tilted towards hydrolysis (which is also in accordance with the thermodynamic disposition). It is quite commonplace that reactions that are not thermodynamically favorable are at times kinetically viable (particularly so in the living cells). However, there is no rationale to confirm that physiological ATP synthesis is mediated by Complex V. If the apologist's argument is that the membrane-embedded F o subunit is also needed for ATP-synthase activity, the critical retort is that demonstration of rotary ATP-hydrolysis activity by a truncated and modified version of Complex V (Noji experiment) cannot be used to support physiological rotary ATP-synthesis. Using a single molecule experiment's demonstrative outcomes for drawing similar conclusions in physiology is akin to making trained lions jump through flaming loops in a circus ring and saying that wild lions do the same to sustain themselves in the jungle. The most important insight of kinetics unleashed in this field was by Mildred Cohn in 1950s (e.g. Cohn & Drysdale, 1955and citations therein). This was at a time when it was known that ATP synthesis was accompanied with oxidation of respiratory substrates but the exact nature of chemistry was not figured out due to the restrictive perception that enzymes always work solely via the Michaelis-Menten paradigm. Cohn revealed that when provided with inorganic phosphate (Pi) with heavy-labeled 18 O atoms, multiple 18 Olabels were rapidly incorporated into ATP in mitochondrial preps. The label was also rapidly lost from Pi, and water was also found to have high amounts of 18 O. The extent of incorporation and fast kinetics of the process led Cohn to infer that the synthesis reaction must also involve water. Boyer knew that these findings were important and worked extensively with such isotopic labeled experimental approach. He explained Cohn's key observation as one resulting from the reversibility of the ATPase-ATPsynthase reaction, via covalent catalysis mediated at the beta monomers. We believe that this is no explanation at all, as all this shows is that the enzyme works via futile turnovers.
Where goes the directionality of rotation, in this context? When it is necessary to explain the isotope effects, the substrates can do whatever they want at the active site and the rest of the protein's activities are discarded conveniently. A similar analogy existed in Cytochrome P450 (CYP) research wherein to explain kinetic isotope effects, researchers reasoned that the substrate was free to rotate in the active site; but to explain substrate-binding based conformation and redox property changes, they invoked a strong binding-based mechanism (Manoj et al., 2016). Quite clearly, Boyer's explanations do not account for the insightful original findings of Cohn. She had also pointed out the incompatibilities of Walker's deduction with her elaborate findings in subsequent works (with labeled phosphate in normal water and normal phosphate in labeled water). However, the research community overlooked them as Boyer seemed to have more sway over the community. When the issue of protons are concerned, the Boyer explanation falls under the broad purview/format of chemiosmosis, and therefore, falls flat for the sheer fact that the closed-looping of protons and the thermodynamics advocated by Mitchell are fallacies-there are practically less than 10 protons in a mitochondrion. (Smaller bacterial systems don't have free protons.) How can such a mechanism serve the thousands of Complex V? Another important aspect is the kinetics of protons' trans-membrane movement, whose pseudo-first order rate is roughly about 10 3 /second. Although they can move in quite faster through pores/channels, it must be remembered that protons have to push their ways through the space between c-ring and a protein stationed in the membrane. We don't think that such a premise can account for the rates of 7000 rotations per second. Assuming a c-ring of 10 subunits, it would mean that 70,000 protons have to move across the inner mitochondrial membrane per second at a single Complex V. We consider such proposals highly far-fetched and incompatible with the rest of CRAS postulates.
Regardless, let us try to understand the dynamics of one reaction cycle at a Complex V molecule. While spinning at $1000 rotations per second, the three sites should bind two molecules of substrates (ADP at 10 À4 M and Pi 10 À2 M) deterministically at 1/3 cycle frequencies. This must happen when ATP present at 10 À3 M could bind to the very same sites with higher binding affinity. Regardless, let's compute the probabilities assuming equal chances for the exclusive events. So, for a full rotation of the c-ring (assuming it to comprise of 9 subunits, so as to remove the mismatch in symmetry), at each proton binding step, the three outcomes are binding of a proton at the matrix side, binding at the IMS (inter-membrane space) side, and no binding (wherein only binding at the IMS is fruitful). For the ADP/ATP and Pi sites, we have two ligands in one site and only one in the other, and there could be bound and unbound states at each site. Therefore, the conservative probability of a successful Boyer rotation with the given set of events is: (1/3) 9 x (1/3) 3 x (1/2) 3 ¼ < 10 À7 . Even taking the 'unbound state' out of the assessment, and overlooking affinity and availability issues, the favorable Boyer event would be ( 1 = 2 ) 9 x ( 1 = 2 ) 3 ¼ < 1/4000. We find neither rhyme nor reason for entertaining such improbable ideas, particularly given that mitochondria are practically aprotic (making it kinetically unviable) and the thermodynamic cost for water-dissociation/proton-pumping is unaccounted in the CRAS proposal. Other associated issues with the kinetics of Complex V have already been discussed earlier in our works (Manoj, 2018b(Manoj, , 2018cManoj et al., 2019aManoj et al., , 2019b.

Evolutionary considerations:
If Complex V were to be a rotary enzyme, it would mean that 8 $ 15 different proteins were needed to make a simple and fundamentally obligatory reaction possible. Further, we find that the components needed to spontaneously assemble a functional rotary system such as Complex V would be an impossible target to achieve. Also, the diversity of these many components does not augur well for a feature of conserved evolutionary focus. The rotary explanation for Complex V is a classical example of Michael Behe's anti-evolution argument of "irreducible complexity". We have recently compiled multitudes of evolution-based rationale for why F-A-V ATPases cannot be the original ATP-synthase for the LUCA or the earlier ancestors of life (Manoj, 2021). In fact, there are several examples living even now which do not have full or parts of Complex V. In this context, the comparison of Complex V to a rotation of 'bacterial flagellar motor' (BFM) is akin to comparing the potentials of ant and elephant, just because both end in "ant". Here are the reasons why: (1) There are structural components that indicate the scope for a rotary function in the BFM assembly and the components that are supposed to rotate are not located in the membrane. (2) Bacterial body proper has been observed to rotate around the flagellar axis by a tethering of the flagellum. Although this is a non-physiological demonstration, there is some wholesomeness to this observation. (3) Thousands of protons are supposed to be involved in a single rotation of the flagellum. (Also, the bacterium is observed to move by gyrations around its longer axis. So, the ability of the BFM to rotate and change directions is yet an unresolved and uncertain aspect.) Currently, it suffices to say that workability considerations of BFM and (CRAS mediated-) Complex V are dissimilar, and comparisons can be charted only for demonstrating disparities.
3.1.5. Failure to meet the fundamental 'control logic' for rotation creates multiple proposals: Even overlooking the shortage of protons and assuming its abundance and accepting a feasibility of rotary function, we do not get any directive logic. That is: one proton binding on the IMS side would rotate the purported c-ring-gamma rotor counter-clockwise. What prevents a proton from binding on the matrix side of the c-ring and rotating the c-ring clockwise? The fundamental premise that Mitchell-Boyer seek is that proton is copious and omnipresent. This leads to a logical stalemate (or disadvantageous zugzwang, in chess parlance). Thus, even overlooking the structural, energetic, kinetic, probabilistic and evolutionary limitations, Complex V can function along a given direction in a rotary mode only with overt intelligent governance, which is unforeseeable in the context of mitochondria/chloroplasts. Such ideas are not needed if considering the simple inescapable FACT that there are too few protons to give PoPs and there is little pH gradient in physiological working ambiance of mitochondria/ chloroplasts. But sticking on to the CRAS premises for explaining the purported ATPsynthase function of Complex V (via the reaction ADP þ Pi ¼ ATP þ Water) has led to so many mechanistic proposals (differing by subtle to high levels of disparities) that one gets a picture of the proverbial Indian blind men clasping at different parts of an elephant. To cite a few examples: Chemiosmotic-rolling well and turnstile (Mitchell, 1985), Conformation changing machine (Boyer, 1997), Electroconformational coupling (Tsong & Astumian, 1988), Plastic network model (Pu & Karplus, 2008), Rocking ratchet (Bartussek et al., 1994;Hayashi et al., 2009) Theoretical and/or experimental evidence suggests that protons exist as hydronium/Zundel/Eigen (H 3 O þ /H 5 O 2 þ / H 9 O 4 þ ) ions in water and their 'desolvated migration' most probably occur via Grotthuss or semiconductor type mechanism, which could be favored by localized/macroscopic electrical effects. These realities are not considered in the Boyer proposal, wherein protons are somehow deemed like hard spherical balls that roll down a gravitational plane (or at best, seen as charged spheres spontaneously passed on across phases aided via some amino acids that work as panes of a turbine) (Item 1, SI). The inspiration for such a thought was perhaps the operating principles of mechanical mills/turbines, whose directionality could be tailored by sophisticated ratcheting mechanisms (as some later proposals advocate). However, the assembly of such components requires high precision and these kinds of machines are seldom formed spontaneously. Therefore, in all possible angles of analyses, Boyer's linking of the purported cooperative 3-site function of F 1 with the supposed rotary movement at F o by the mirage of protons sponsored by Mitchell's pmf must be dismissed as a forgettable chapter in the story of scientific progression.

The murburn model for Complex V
To enable an easier comprehension of the murburn model of the complex group transfer enzyme of Complex V, we present the explanation for the much simpler redox enzyme lactate dehydrogenase (LDH), which was also supposed to work in bidirectional way (earlier, as per classical perceptions). Thereafter, we present the murburn model for Complex V and evidence/arguments to validate the proposal.

LDH is a murzyme; and is not a fully/freely reversible enzyme in physiology
In the classical textbook explanations, LDH was supposed to serve as a reversible redox enzyme, via the equation: CH 3 (CO)CO 2 -(Pyruvate) 1 NADH 1 H 1 $ CH 3 (HCOH)CO 2 -(Lactate) 1 NAD 1 . Such a perception did not provide any rationale as to why lactate gets transported to liver (Cori cycle), when muscle had much higher concentration of the same isozyme of LDH. We showed that the backward reaction (pyruvate reduction, as written in the equation above) and the forward reaction (lactate oxidation) that occurs in liver are quite distinct (Manoj et al., 2021d). Although mediated by the same enzyme, they use different equations and mechanisms. This is necessitated by the premise that while the classical pyruvate reduction is energetically favorable (about -450 kJ/mol), the reverse reaction is not. Liver cells have a high density of DR(O)S in the cellular ambiance, by virtue of cytochrome P450 and its reductase (CYP-CPR) activities. The DRS is effectively stabilized by LDH and subsequently this aids the oxidation of lactate, which gets presented at an auxiliary binding site on the enzyme surface. Thus, LDH is a redox enzyme that has the ability to convert both its Michaelis-Menten substrate (pyruvate) and product (lactate), but the product is reacted upon via an alternative mechanism, wherein LDH serves as a murzyme. This explanation also gives a tangible rationale as to why the enzyme is a monomer when embedded in the membrane and a tetramer in soluble state. Since the reaction is essentially propagated via a radical mechanism, having a cluster of reaction sites helps better harvesting of the reaction potential of DRS. Thus, LDH can both make and break lactate, but it is not a truly reversible enzyme (Figure 3 and its legend). For details in mechanism and analogy (and misperceptions in enzyme catalysis/thermodynamics), please peruse the details in our publication (Manoj et al., 2021d). In short, we propose that Complex V is akin to LDH in the way that both these proteins work in the thermodynamically unfavorable direction by using different sites and different mechanisms. This is quite distinct from the classical view, which considers both directions to be freely reversible, using the same equation.

The murzyme model for Complex V, in analogy with LDH
Quite similar to LDH, Complex V acts as a Michaelis-Menten enzyme in the ATPase direction and murzyme in the ATPsynthase direction, using two different reaction routes. The murburn proposal is given in Figure 4, and it can be noted that the new proposal does not invoke any rotary function at all. The F o subunit of Complex V can act as a proton pore, activated either by ATP binding at the e or ab subunits, letting in nH þ via the pore of c-ring. The utilization of ATP would give a mechanical change in the epsilon monomer, or this could also occur owing to a twanging type of motion of the gamma protein, both of which could open the pore. This proton-inletting activity would support the murburn function of Complexes I-IV in steady-state, where bound ADP is activated by DROS, leading to proton deficiencies in the matrix, as NADH is a 2e but single H þ donor. (This premise would lead to the formation of negatively charged diffusible intermediates, which results in an overall negative charge, relative to outside.) The murburning in matrix can also lead to scenarios wherein the phosphorylation substrates or intermediates can also be transiently bound at Complex V. Since the matrix is practically aprotic, proton-driven equilibrium assisted ADP-Pi bond formation is only possible if there is a proton-excess near the active site (which is not appreciated by the Boyer model) or if there is an electrical charge disparity (which is possible and explained physiologically in the murburn model, and TMP (trans-membrane potential) has duly been noted in the active phase of oxidative phosphorylation). Complex V may aid secondary ATP synthesis by enabling a packaging type of function of binding ADP and/or its activated intermediates generated in situ. This could also enable an attack of bound ADP by activated phosphate/ DROS.
One example of this would be the "ADP þ Pi Ã þproton" reaction, giving ATP. The proton could aid the release of a formed ATP and or a DRS, by virtue of an O-H bond formation. This 'stochastic' function would be enabled by ANBS on both alpha and beta monomers, as proximity would be the only criteria. Greater the number of sites, greater is the efficiency. Therefore, the multimeric nature of of the alpha-beta bulb and its simple connectivity with the c-ring pore can be explained. The protons coming in could be relayed to the alpha-beta bulb, wherein the DELSEED loop could recruit protons. There is no proton gradient in physiology and the electrical disparity in physiology is generated via murburn. Also, ATP hydrolysis by Complex V is associated with both acidification and proton exchange with the connected phase; and thermodynamics and in vitro studies point out that ATP synthesis is facilitated by proton consumption. Therefore, the murburn proposal shown in Figure 4 is justified. The events in murburn model occur in parallel and are not serially ordered. Therefore, the mechanism fares high on probability scales. The cooperativity interpretation can result due to the favorable effects of DRS released in the reaction occurring at a nearby site. (On the other hand, Boyer's proposal that such sites could all go about changing their conformations suiting to ADP and ATP in one direction and the other is highly fastidious.) Since ions form a part of the reaction cycle in the murburn ionradical equilibrium, and they also come in through the pore, the effects of various ions like K/Na/Ca/Mg etc. can further be explored under this purview, particularly in the light of our recent works published (Manoj & Tamagawa, 2022;Manoj et al., 2021e). The current explanations satisfactorily account for the apparently motor-like 'complicated appearance' of Complex V. In the murburn model, Complex V may work with a low efficiency as a physiological ATPsynthase via the reaction ADP þ Pi þ protons ! ATP þ Water. This is not the outcome of a Mitchell or Boyer model either, but one murburn consideration that helps explain the in vitro observations (in the Jagendorf/Racker experiments) misinterpreted to support Mitchell's pmf proposal. The protons/cations that enter the matrix do not come in merely like the opening of Figure 3. The Janus reaction mechanism of LDH. In the backward reaction (left), pyruvate and NADH bind at the deep-rooted active site with the nucleotide moiety adjacent to the reactive carbon of pyruvate. In the murzyme forward oxidation, the nucleotide binds differently (wherein the postitively charged nicotinamide moiety juts out of the classical active site) and the superoxide stabilized at the arginine residues on the enzyme enables an attack on the lactate presented on the murburn site on the enzyme surface. These outcomes also include re-arrangement of water clusters around the enzyme. Also, while the stoichiometry is standardized in the backward Michaelis-Menten mechanism, the murburn forward oxidation could give diverse products of varying stoichiometry (owing to delocalized mechanism and multiple competing reactions). a flood-gate. Rather, this results due to a complex interactive equilibrium between molecules, ions and radicals (the core idea espoused in murburn concept), both in and out of the matrix. (This aspect is a focus of discussion in a future article.) Thus, although group transfers are involved in Complex V, the overall scheme is quite like that of LDH (both systems involve intricate molecule-ion-DRS equilibriums, giving solvent rearrangements), as shown in Figure 5.
If one were to make an analogy, Complex V could be analogous to a turbocharger in a vehicle that enables better efficiency in energy cycling. Its function is relevant only if there is a fundamental engine already present. The primary driver is the battery of Complexes I -IV that synthesize ATP (and generate TMP), which opens the pore through which protons usher in (aided by the TMP). This in turn, sponsors O-H bond formations and release of ATP at Complex V. While the ATPase reaction is stoichiometric, the murzyme reaction is not (owing to a delocalized stochastic scheme). This is the reason researchers in the field could never reach a consensus on various ratios like P/O or H þ/ ATP for oxidative phosphorylation (Hinkle, 2005). If it were as ordered and deterministic as the 2e group transfer advocated in Boyer's model, such a situation would not have arisen. The overall interactive scheme of NADH utilizing Complex I and ATP utilizing Complex V is shown in Figure 5. Upon presenting NADH and oxygen, a small amount of ECS occurs (leading to a small TMP) which is exaggerated by the presence of substrates (ADP þ Pi), as the electrons find a 'sink' as phosphorylation leads to O-H bonds (water and peroxide) formation. The murburn sequence leads to a high accumulation of negative DROS, which gives higher TMP and they can also react among themselves to give heat. To overcome the limitation posed by membrane barriers, Complex V serves as a proton inlet.

Support for the murburn interactive mechanism
We have demonstrated that the available information on mitochondria adequately supports the murburn model of oxidative-/photo-phosphorylation in bioenergetic organelles/ membranes (Manoj et al., 2021c). Herein, we shall focus only on the matters relating to the structure-function aspects of Complex V, as seen from several perspectives.
(i) Structural aspects: The overall architecture of Complex V (Item 1, SI and Figure 1), albeit apparently complex, is really simple. It is essentially a membraneembedded proton pore (c-ring) that is stabilized with respect to multiple ANBS (on alpha-beta bulb) adjacent to the membrane. This consideration is fully justified by the structural assembly and the diversity of the Complex observed across diverse species. The frail and non-rigid nature of the central and peripheral stalks, the simple articulation of the single protein-a block with the c-ring and the large central pore of the c-ring support the murburn proposal. Conservation of DELSEED loop adjacent to the pore opening locus appears to be a deliberate ploy for proton recruitment. Our proposals are strongly backed by the findings of Alavian et al. (2014) who reported that the c-ring serves as a permeability transition pore. (ii) Docking and binding studies: Multiple ANBS were known to be present on alpha and beta monomers since long (Khananshvili & Gromet-Elhanan, 1984;Berden, 2003). Even Boyer knew that consensus data had indicated that the ATPases showed at least six nucleotide binding sites of varying affinities. He had admitted in his Nobel lecture that ever since the awareness of the trimeric nature of ATPase bulb, he had focused on a tri-site cooperative model. Also, ATP binding sites on epsilon (Krah & Takada, 2016) were reported in bacteria. We explored these putative binding sites using in silico docking methods ) and a salient sample of the detailed results given in Table 2 is depicted in Figure 6. We ratified that non-specific ANBS are present on both alpha and beta monomers. The Walker-motif bearing sites were not necessarily the ones giving higher affinity ANBS. There was no consensus in the alpha-beta monomers as to which site was conserved to be high affinity ATP-site or ADP-site. There exists no conserved consensus either on the ATP/ADP binding preference for the various sites across the diverse species either. Therefore, the cooperative conformation change mechanism can be discarded. (There can always be the induced fit argument, but that would be a nonevidenced proposal. If that were to be true, one would need to imagine that these multitudes of sites undergo periodic changes in affinities or else, they exist only to mess with their own fundamental purpose. It is quite possible that some of the surface residues would be high energy and they could change conformations in physiology. If so, one would not expect them to deterministically affect the selectivity strongly.) The pore valve (epsilon-delta protein) is clearly an ATP binding locus, as the ATP binding is higher than ADP in all four of the F-ATPases explored. The conserved Walker motifs ensure a high phosphate concentration at the alpha-beta bulb and the generic ANBS (adjacent to the DELSEED loop and Walker motifs) make facile the reactions shown in Figure 6. (iii) Energetic/kinetic/probability considerations Since the mitochondrion is practically aprotic, this reaction does not occur naturally. As per the murburn model, protons are ushered in by Complex V, using ATP binding at epsilon or alpha-beta bulb. This extraneous proton's inward movement is facile because the desolvation energy goes up with the increase in the hydration shell of proton, at least within the series of hydronium, Zundel and Eigen ions (in the respective order of -8.6, -23.2, -34.5 kJ/mol . The origin of this energy lies in the increase in entropy due to the de-structuring of the water-lattice outside. That is: the de-structuring of lesser hydrated protons at the interfaces occurs via a facile relay in conjunction with the highly hydrated structures in bulk. This could explain the in vitro synthesis of ATP aided by pH gradients. However, it must be remembered that this energy is not sufficient for ATP synthesis in physiology, as in the non-acidic ambiance, resetting water hydrolysis equilibrium in the bulk would also require energy, at >79 kJ/mol (for heterolytic proton formation from neutral water molecule). The point to note is that in the physiological process, NADH and FADH 2 oxidation provides ample energy for this outcome (-586 kJ/mol for NADH and -112 kJ/ mol for FADH 2 ; in which the latter reaction does not need solvent-lysis), which is used for bond formation, heat generation and also availed through solvent re-arrangements (via the continuum that Complex V's pore opening offers). The details of such solvent rearrangement and its interaction with the ion-solvation/exchange are subjects of discussion elsewhere (in a forthcoming communication). If we take the input of -586 kJ/mol (oxidation of NADH to peroxide; , getting 4 ATP molecules' synthesis (needing 4 x 35 ¼ 140 kJ/mol) would be very facile. This fundamental thermodynamic accountability overcomes the 'consensus' imposed by Walker's group which poses the restriction of 2.5 ATP/NADH (based on poor accounting with protons and cring stoichiometry) (Watt et al., 2010). Murburn model supports the researchers who have asserted that even 3.7 ATP were made in mitochondria per NADH (Hinkle, 2005). So, can Complex V also make ATP via murzyme process in physiology? We believe it could be possible, given the thermodynamic scope afforded by murburn model shown above. (But the thermodynamically unaccountable CRAS cannot explain it, as protons are not involved in the active site of alpha-beta monomers.) When protons move in, species like superoxide would tend to drift towards Complex V's regions near the DELSEED loop, and ADP and Pi are presented at the murburn site and Walker motif, quite nearby. Therefore, Complex V serves the role of chemostat cum turbocharger. This consideration also explains the independent distribution of Complex V (sometimes at the end of cristae), not clumped with the other respiratory complexes.
Because of the aprotic nature of the matrix, there cannot be proton-pumping in the matrix and therefore, there is no bulk phase delocalised Dm H . When an efflux is observed in non-physiological setups, the observation must be due to proton dissociation from fixed charges on mitochondrial macromolecules (i.e. proton exchange occurs with other cations). It must be born in mind that this efflux would negate "the proton-crowding within IMS" postulate of Mitchell. During respiration, negative potential results due to ECS (formation of DROS) and this allows an influx of protons from the outside. Salt-linkage formation or cation-anion solubilization or dissociation among matrix and inner-membrane associated macromolecules is the only available explanation for the tight coupling between ion fluxes, oxido-reductive state of the redox complexes, and swelling-contraction behavior of the mitochondrial matrix. This is a more complex phenomenon, including 'dissociative swelling/associative shrinkage', osmotic, colloid and colligative properties. There are numerous indications that membranes are on both sides lined by structured water (Pollack, 2013, Jaeken, 2021: specifically for the mitochondrial matrix side: L opez-Beltr an et al., 1996). . The murburn proposal for Complex V: DRS can be a radical or an ion derivative or even an atom such as O 2 Ãor Mg þ /Pi Ã or H, respectively. DRS could also be a high localized ambiance of protons, enabling equilibrium driven ATP synthesis. Although we believe that the epsilon-based pore-opening is more relevant, a potential pore-opening via the modulation of ATP-binding to alpha-beta bulb is included as a possibility to account for reported movements of gammastalk post ATP-binding. Even in the large pores of aquaporins membrane-associated water, though being highly permeable for water itself, is selective for protons (Benga et al., 1986, Kuchel, 2006, Pollack, 2013. The cause for this effect may lie in 'polarization of water', as revealed by detailed structural studies of aquaporins showing that the two halves of the pore contain oppositely polarized water (Stroud et al., 2003, Fu & Lu, 2007. Thus, the murburn model allows for explanations of other accompanying phenomena like thermogenesis (Manoj et al., 2018), TMP generation and proton/ionic/solvent homeostasis Manoj et al., 2021aManoj et al., , 2021bManoj et al., , 2021cManoj et al., , 2021d. Kinetic and probabilistic considerations favor the murburn model as binding of ATP or ADP/Pi on each site is not crucial to the next step. Only if the ambiance has ATP, the pore opens. When the pore opens, sites with adjacent phosphate and ADP could find a higher ambiance of protons, which could enable ATP synthesis. Else, DRS activity in milieu could generate species like Pi Ã or H or Mg þ , which could also mediate murburn phosphorylations. All these processes would be highly facile and probable around the vicinity of the alpha-beta bulb, owing to the high density of ANBS. (Contrast this with the Boyer model wherein binding of a proton must move the c-ring, which should be able to relay a torque to the alpha-beta bulb, which in turn, must have ADP and Pi already bound … . And this must go on deterministically while the rotary movement goes on.) Most importantly, the earlier perceived cooperativity in outcomes results due to the involvement of proton-assisted one-electron equilibriums, as we have highlighted in several multimeric redox proteins exemplified in cytochrome P450 system (Manoj et al., 2016;, hemoglobin , respirasomes (Manoj et al., 2019a), photosynthetic complexes (Manoj et al., 2021a(Manoj et al., , 2021b, LDH (Manoj et al., 2021d), etc. It is now facile to imagine that this complex protein could have evolved at later times, for increasing the efficiency of oxidative phosphorylation.
Explaining Inhibitions: Oligomycin is highly hydrophobic and the binding would be at the c-ring of F o , whereas the OSCP is soluble and binds at the distal base of the F 1 . These outcomes are largely unexplained, although it has been postulated that it should be connected with Complex V serving as an ionic pore (Devenish et al., 2000;Giorgio et al., 2019). Oligomycin interrupts the activity of ATP synthesis, as it is known to bind to F o module, and this is subject to OSCP/d at F 1 . This proposal is substantiated with evidence, as ATP hydrolysis is known to be associated with acidification on one hand (generally) and also enables proton movements across phases by Complex V. Other inhibitors like venturicidin, ossamycin, apoptolidin, cytovaricin, etc. all have a cyclic ring structure that could effectively disrupt the pore dynamics. DCCD (dicyclohexylcarbodiimide) is a coupling or dehydrating agent for carboxyl groups, forming dicyclohexyl urea derivatives. It could inhibit Complex V activity by reacting with any of the essential acid groups on the protein's surface (c-ring, DELSEED, etc.). Since bongrekate binds to ADP/ATP translocase, the matrix concentration of ADP goes down and ATP is not available to the cell. This would adversely affect the activity of all Complexes (I through V). A membrane-embedded ionophore like Valinomycin would affect the ion/TMP dynamics, and thus bring about relatively unpredictable effects in mitochondrial physiology. Disubstituted phenolics like dinitrophenol (DNP) are interfacial DROS/proton modulators. They are not proton-shuttlers because it is impossible to conceive DNP to move across the membrane while protons cannot. The effect of the mechanistically important respiratory toxic principle of cyanide is thermodynamically/ kinetically explained by the murburn model . Cyanide catalyzes the futile formation of water in the milieu (using the protons that come in via its pore), without enabling effective DRS-interactions with ADP/Pi. Therefore, all matrix ATP are used up by Complex V (with little formation of ATP by the other Complexes), leading to depletion of cellular energy reserves. The commonality of DRS-based mechanism's operations in respiratory and photosynthetic is conclusively established by the murburn explanation for  cyanide inhibitions, and this crucial outcome is inexplicable in the ETC-CRAS model. Physiological observations: If we start with a clean slate and list out the projections of CRAS and murburn models, we could be in a better position to disconnect the classical model from its inertial advantage. (a) In bacteria, Complex I is actually long-known as a pH stat (Shibata et al., 1992). (b) Ions were found to affect the physiological ATP synthesis and hydrolysis. If the ions moved between the c-ring and a subunit alone, and did not involve in the reaction directly-this is not possible. (Cope & Glynn, 1977;Ling, 1981) (c) Provision of NADH/oxygen leads to increase of DROS in the mitochondrial system (Murphy, 2009). This observation is an anti-thesis to the evolutionary relevance of ETC-CRAS model. (d) Increase of DROS within the system would correlate to increase in TMP/ATPsynthesis in the murburn model. Such an observation would correlate TMP build-up as an outcome of ECS and DROS production and ATP synthesis within mitochondria; and not the other way around. This is in fact the physiological case. (Nicholls, 2004) (e) In situ/in vitro, provision of DROS to mitochondria could lead to the formation of ATP (from ADP/Pi). This observation would directly implicate DROS in ATP synthesis, while it would remain inexplicable how TMP can be tapped to give rise to an O-P bond in ATP. Such observations have also been ratified in physiological premises. (Mailer, 1990;Manoj et al., 2019, 2020) (f) As the murburn model involves DROS, it is destined to be stochastic, non-integral and have variable reaction outcomes similar to those already demonstrated even in the bienzymatic CYP þ CPR system (Manoj et al., 2016). This justifies the murburn equation of oxidative phosphorylation: NAD(P)H þ H þ þ O 2 þ nADP þ nPi ! NAD(P) þ þ H 2 O 2 þ nATP þ nH 2 O. This equation shows that protons serve as a reactant in the oxidative phosphorylation reaction, thereby contributing to the overall energetics, a point completely missing in ETC-CRAS assumptions . Since the ETC-CRAS mechanism mandates deterministic ratios of electrons to protons and to ADP, the two mechanisms can be thus delineated. The diversity in P/O and H þ /ATP ratios across literature (Hinkle, 2005) ratifies the murburn model. (g) As per the murburn model, each one of the Complexes I-IV would have DROS-production abilities. Such an observation would be incompatible and inexplicable with ETC-CRAS model. This ability has been verified experimentally from earlier reports (Bleier & Dr€ ose, 2013;Dr€ ose, 2013;Grivennikova & Vinogradov, 2006;Ksenzenko et al., 1992). (h) Each one of the Complexes I-IV would have ADP-binding sites, and this has been demonstrated experimentally and the putative sites on these proteins have been identified (Manoj et al., 2019a). This is a necessary mandate of murburn model, whereas it is a superfluous aspect in ETC-CRAS. (i) Provision of ADP þ Pi would increase NADH and O 2 consumption, concomitant with DROS and ATP production. In the ETC-CRAS mechanism, Figure 6. NBS on alpha-beta bulb and epsilon subunit in F-ATPases. A. Nucleotide binding site on epsilon unit. Epsilon (e) motif (magenta) shows interaction with both ATP (red) and ADP (bright yellow), with the former preferred over the latter. gamma-shaft is presented in light pink whereas a and b-bulbs are presented in blue and pale yellow respectively. B. ADP/ATP binding sites on yeast F-ATPase (PDB ID: 2HLD) is presented. aand b-monomers are shown in sky blue and pale yellow, respectively. Walker motifs on aand b-monomers are represented as blue ribbon and green, respectively and are enclosed in a rectangle. Both ADP and ATP binds to Walker motif present in the b-monomer. Also, two murburn sites (selected based on highest binding energies among the murburn sites) are shown in ovals. For clarity, ADP is coloured in bright yellow and ATP in red. Same colour codes are maintained for other subunits as well. Four monomers (two of a and two of b) are faded to maintain clarity.
since there is no direct coupling or thermodynamic pull concept, presence of ADP þ Pi does not affect the ETC. Therefore, this is a simple way to delineate the two proposals, and this observed premise also supports the murburn model. The same observation has been demonstrated in the CYP þ CPR system also, which confirms the murburn model's relevance in physiology (Manoj, 2018a;. (j) A survey of compositional and structural data of diverse life forms and experimental cells (genetically manipulated) supports murburn model's mechanism where Complex V is accorded a secondary status in ATP synthesis (Manoj, 2021).
There are several examples of non-conserved/ non-linked genes in Complex V and even total absence of this protein seen in some life forms (e.g. blood cell stages of the malarial parasite, Plasmodium), validating the murburn model. Further projections for ratification/delineation of the murburn model: Since the basic models of CRAS and murburn are clearly charted out, we propose the following experiments/considerations to delineate and to ratify the physiologically operational mechanism: (a) Complex V constituted in vesicles could make ATP with xanthine oxidase function. Even a stabilized stock of KO 2 would be expected to give similar effects. This outcome would be affected by interfacial ROS scavengers/modulators and SOD activity. (b) Provision of ATP to the reconstituted Complex V system would give ADP and Pi whereas the provision of ADP and Pi to the system would only give a miniscule amount of ATP. Provision of equimolar amounts or physiological amounts of the various components would not give any increase in the amount of ATP. (c) ATP synthesis can be achieved in vesicles/mitochondria without pH gradient. This would demonstrate the physiological irrelevance of equilibrium-assisted synthesis via the equation: ADP þ Pi þ H þ ! ATP þ H 2 O. (d) Inhibition of reaction in some of the setups above (or the ones observed in physiology, listed in the earlier section) by catalytic amounts of cyanide can be noted. Since cyanide is a stoichiometric inhibitor in the classical scheme, this demonstration would ratify the murburn model. In the classical purview, if we add ADP þ Pi to cyanide-blocked mitochondrial Complex IV, we should still see some ATP synthesis in the mitochondrial system because Complexes I & III can still pump protons. To the one conversant in the physiological experimental results with mitochondria, it would be evident that many of these experiments have already been conducted and the observations/premises have already provided support for the murburn model.

Conclusions
In our recent works, we had given murburn-based structurefunction correlations for mitochondrial components (Manoj et al., 2021c). The critical dissection presented herein on Complex V's functionality challenges the cooperative conformation-change based rotary ATP-synthase paradigm. The multiple ANBS on each of the alpha-beta monomers denounce the deterministic Boyer scheme and support the stochastic murburn model. The latter proposes that the ATP binding site on the e-subunit may control the proton flux via c-ring, making it a 'regulated port'. Physiologically, Complex V is an acid-based murzyme, quite like the LDH function that we have recently revealed (Manoj et al., 2021d), using both its substrate (ATP) and products (ADP þ Pi), via different mechanisms. The murburn model also satisfactorily explains how Complex V could serve as equilibrium or pH-gradient/ DRS assisted ATP-synthase in vitro. Although Complex V could enable enhanced efficiency of ATP synthesis in oxidative and photophosphorylation via two modalities, we should no longer call Complex V as ATP-synthase, but it could be called a chemostat or ATPase. As the murburn model involves the interactive equilibrium of molecules and ions via one-electron species within the solvent milieu, the thermodynamic outcomes in mitochondria can be interconnected and explained satisfactorily. Whether the murburn perspective succeeds the CRAS model for explaining cellular bioenergetics is for the earnest researchers in the field to decide. However, it is no longer a matter of choice for all concerned that redundant ideas/terms like-ETC, chemiosmosis, proton motive force, and rotary ATP synthesis-are jettisoned.
based cations. HT and LJ contributed crucial arguments, inputs and literature.