Cellular Respiration

https://www.youtube.com/watch?v=AvJgkNZapsw

1. Here are the arguments why the current/prevailing textbook concepts (for which Peter Mitchell and Paul Boyer received Nobels) are misplaced.

Schematic of the textbook ideas

A. Quite simply, the finger-countable protons in the mitochondria cannot serve the tens of thousands of protein complexes to generate a proton-gradient across the inner mitochondrial membrane.

B. The concept of Chemiosmosis is thermodynamically non-viable because if the protons move across the inner membrane as an exothermic/exergonic process, the inward movement across the same membrane would be an endothermic/endergonic process, which cannot power the endothermic/endergonic ATP-synthesis by Complex V.

C. Consequentially, the purported electron transport chain, therefore, serves little purpose! (Why should it generate water at the expense of redox equivalents?) Also, there is little probability that several decades of electron transfer steps can be "deterministically" coordinated in the time-frame of the physiological reaction, with the available structure and distribution of mitochondrial Complexes.

D. Rotary ATP synthesis by Complex V is a thermodynamically and kinetically non-viable option under the physiological conditions. This is because Complex V has more than ten million folds affinity for ATP compared to ADP.

E. In toto, none of the four components of the prevailing explanatory paradigm (rotary ATP synthesis, chemiosmosis principle, proton pumps, and electron transport chain) are viable independently or comprehensively cohesive/compatible with each other.

2. What is the alternate hypothesis proposed (for which evidence and arguments are available)?

Schematics for the murburn proposal

A. Mitochondrial membrane protein Complexes generate Diffusible Reactive Oxygen Species (DROS), in the presence of redox equivalents and oxygen.

B. DROS serve to attack ADP/Pi and generate ATP. In case that the DROS react among themselves, they lead to water and heat formation.

C. Therefore, DROS is an obligatory requirement in the murburn explanation, not an undesired/toxic waste product as deemed in the earlier perspective.

3. A comparison of xenobiotic metabolism (in which murburn concept has been experimentally and theoretically established) with cellular respiration.

Theoretical aspects

Experimental observations

4. A comparative analysis or the erstwhile versus new perceptions on mitochondrial oxidative phosphorylation (mOxPhos).

Working model for the Mitchell/Boyer ideas

Working model for the murburn perspective

In toto

Supplementary Information: Discussion for the non-experts

(Addendum to Manoj, Biochemistry Insights 2018)

#A: In a hydroelectric power plant, the generator’s turbines would only rotate if a large volume of water rushed through at high speeds (by opening the gates at the catchment on top). Further, the water that flows across the turbines should also have a free exit at the bottom. If the water trickled through a crack on the wall to stagnate at the bottom, the turbine would rather get rusted, and not rotate! Somehow, the analogy of mitochondrial proton pump approaches that of the latter scenario. Seeing it differently and comparing with a bacterial flagellum also does not give any suitable model. A bacterial flagellum works with ~10³ protons (per rotation) unidirectionally rushing into a relatively open cytoplasm whereas here, we have ~10¹ protons (per rotation) cycling through the cytoplasm-periplasm (inter-membrane space). This fundamental nature of mitochondria cannot be overlooked to ratify any theories. If one assumes a falsity to be true, then one can generate more false truths based on the false assumption. For example- If 0 = 0.000000001, then, one can use known and accepted mathematical operations to demonstrate that 0 = any random number!

#B: Osmosis is the movement of solvent molecules from the region of lower osmolarity to the region of higher osmolarity, when the two phases are separated by a semi-permeable membrane which does not permit the free movement of solutes across. Osmolarity is a colligative property, which depends on the number (and not weight, charge, shape, etc!) of dissolved species within a solution. On the other hand, diffusion is the equi-distribution of any solute within a solvent, which “appears” to be a movement of solutes from regions of higher concentration to regions of lower concentration. Such an outcome results due to Brownian motion, and it could occur in the presence or absence of membranes, and is dependent on several variables (temperature, viscosity, etc.). Since protons are “solubilized” in water (the solvent and they are also in an intricate equilibrium with them, forming aquated ions and also dissociation paradigms), the theoretical phenomenon (of trans-membrane unidirectional and cyclic proton movement) hypothesized by Mitchell has no bearing with diffusion or osmosis. It is a surreal concept, and not based in any factual phenomena or any known or ascertainable theories of science. Instead of finding a coherent idea to explain observations, Mitchell had resorted to finding observations and methodologies to fit an assumed idea. To make you see the Moebius strip that Mitchell wove, I must present you some simple analogies first. I hope you can see that the Mitchellian chemiosmotic accounting is similar to the following banker & baker story.

Let us imagine a society that works on a barter basis and therefore, there is no cash or credit involved. (This is analogous to saying that the mitochondrial system has little protons to pump out, and therefore, there is no question of cashing in on the protons’ re-entry!) In such a justifiably accountable world, because one doesn’t owe the banker any money, one need not pay him in cash and he is not entitled to debit a sum from one’ non-existent account too!

A man goes to a bakery and takes a cake and asks its price. The baker says- 10 bucks. Upon this, the man puts the cake back on the counter and takes a couple of croissants with two hands and asks for their price. The baker says again- 10 bucks. Now, the man drops the croissants to cup in palms-full of biscuits and asks for the price. The baker says yet again- 10 bucks. Now, the man eats the biscuits. The baker asks for the price of the biscuits. The man says that instead of the biscuits, he had put down the equivalent worth in croissants. To this, the baker asks for the equivalent money’s worth of croissants then. To this query, the man says that he had put down the equivalent worth in the cake. Frustrated, the baker asks for the 10 bucks for the cake. The man says- “I never took the cake at all, and therefore, I see no reason why I should owe anything to you! Now, you need to pay me for the croissants.”

Further, let us consider that a balloon holds some gas and it is allowed to escape through its mouth (which is analogous to pumping of protons by ETC by the inner mitochondrial membrane). In this case, the gas does not spontaneously re-enter the balloon if we provide a pinhole elsewhere (analogous to Complex V) on the balloon. (Similarly, even if the few finger-countable protons were pumped out, why should they spontaneously come back into the matrix when the outer mitochondrial membrane equilibrates with the cytoplasm?) Now, if I cupped my hand over the mouth of the balloon along with an adjacent pinhole (analogous to the crowding effect that Mitchell projected, due to the existence of the outer mitochondrial membrane), there is a remote chance that a few gas molecules might clutter and re-enter the balloon. However, this cannot be a forceful re-entry either, because the balloon is already pumping out gas at a positive pressure. This relatively forceless re-entry of a few molecules (brought about by the cupping of the mouth/pinhole) cannot be attributed another force term that can support the gas for re-entering the balloon to do some usable work. (If the external membrane was impermeable to protons, we cannot have the reaction proceed to water formation because NADH gives two electrons but is proton-limited. Please study my comprehensive analysis of the overall process, particularly in Table 3 of my J Biomol Str Dyn paper.)

Quite simply, the mechanistic/energetic scenario that Mitchell proposed violates the fundamental premises of thermodynamics, as I have amply demonstrated it mathematically in the third point of my Biomed Rev paper (2017). If protons are pumped out, there is no way that they retain or acquire a “high energy” status through the “mirage” of crowding in the inter-membrane space. This, in banking ledger terms, would be analogous to entering a sum of money withdrawn from an account into the account’s credit side, and not debit side!

When Mitchell realized that the recycling protons’ energy terms (22 kJ/mol) by “energy conservation” (a faulty supposition!) was inadequate for explaining the energetics of ATP synthesis (which is another magical and inexplicable step), he added the chemical potential, which is just another term that results owing to the purported proton imbalance. One can only avail such a potential to do some useful work in a connected system, not perform another operation by moving the very protons across the same membrane! That is the double encashing of a non-debt (that never existed in the first place!) and eating the cake and seeking to get paid for it too, per the banker-baker stories earlier!

#C: An analogy would serve to explain things here. If a rope is pulled by two equivalent but opposing parties, the rope does not get displaced and there is no net useful work done. This is a analogous to the “chemiosmotic steady-state functioning” that does not afford useful work. If/when the rope gets displaced because one force is stronger, then useful work gets done. But if the “tug of war” is conducted in a closed room, even if a party exerts stronger force (to displace the rope), steady state functioning cannot be achieved because the room’s dimensions restrict continuity of operations in time. Here, the “rope-pull” operation is analogous to proton-movement in and out of the inner membrane. Else, to explain the scenario, a mitochondrion could also be seen as a dough-nut type building with two chambers. The outer dough-ring would be the inter-membrane space and the inner circular space would be the matrix. Regardless of the number of pumps and generators that connect these two regions (employing the same “workable” force-agent), steady state would need a “workable gradient” to be sponsored by an external source. The closed system perspective for mitochondria (with respect to protons as the force-enacting agent) does not give “workable machine” or logic for power generation.

A relevant analogy would point out the derailed pursuits in contemporary bioenergetics- when a petrol engine runs, it produces smoke and noise. It is the burning of fuel that generates the force to run the vehicle. It is the burning of the fuel that produces smoke and it is the working of the engine that produces the noise. It is not the smoke or noise that drives the wheels. Similarly, it is the oxidation of NADH and the transient generation of DROS in the matrix that generates a trans-membrane potential. The electrostatics of finger-countable protons are in comparison to the electrical/ionic balance of the other ions. It is evident that subscribing to chemiosmosis concept, led to an improper correlation of the cause and consequence correlation in mOxPhos.

#D: In terms of logic, chemiosmosis is analogous to Maurits Cornelius Escher’s much famous work, “The Waterfall” (and the outcome is akin to the maestro’s work- “Ants on a Moebius Loop”). Escher clearly portrays in “The Waterfall” how certain perceptions can be deceptive. By making a few impossible connections and drawing with a few inappropriate angles, the water always seems to flow downhill at any particular locus in the image. This scenario is akin to what the chemiosmosis hypothesis affords us. By assuming unreal postulates as founding assumptions, and thereafter, employing flawed protocols to reaffirm the faulty assumptions, the fabric of logic and reality were broken and resealed to give a Moebius loop. The following discussion would demonstrate why.

Let us hypothesize that the semipermeable (which should theoretically mean “impermeable to protons”) inner membrane of mitochondria serves as a molecular machine that does the useful work of synthesizing energy-rich ATP molecules, using the free energy available from the oxidation of reducing equivalents from NADH/succinate. I think that anyone would buy into this hypothesis/analogy, given our current status of awareness.

Now, I would invite you to make an analogy between the module of mitochondrial membrane (that serves as the proton transfer conduit) and a watermill (that serves as water conduit). This is a simple exercise because the watermill is also a simple machine that uses the free energy (the potential energy of water) to do the useful work of driving a pump or serving as electricity generator. Most would think that this analogy is appropriate. But soon enough, we shall note that it is not that simple! (We shall find that there are quite a few logical catch 22s and zugzwangs in this assumption!)

The three fundamental and necessary attributes of the watermill (working machine) are-

(i) free energy should be expended spontaneously (item 1),

(ii) item 1 enables the mill to perform useful work (item 2) and

(iii) item 2 justifies “man’s benefit-seeking perspective” of getting some useful work done, albeit at an operating cost (item 3).

The three attributes are interconnected and each step must be relatively accountable and must also abide by the fundamental laws of physics. We can see that a single unidirectional flow of water through the mill achieves all the three attributes of the machine.

Now, let us assume the watermill analogy scenario when protons are taken from the matrix to be pumped outward to the inter-membrane space by the respiratory complexes. Starting at time t₁, in the after-bay located at the bottom (in the matrix), energy is expended to pump the water (protons) into the fore-bay positioned upwards (inter-membrane space), through a locus in space; and let us grant it that by the end of this process, the time has become t₂. Let’s see if our machine satisfies the three requirements of the analogy. The membrane spontaneously expended free energy available from oxidation of NADH/succinate in the matrix (item 1), to pump protons out into the inter-membrane phase (item 2). Is this the useful work that we required (item 3)? One can’t really say, because the protons are not useful just because they are pumped out, and therefore, the analogy does not hold yet!

Let’s take the scenario a bit further. In a second step (starting at say, time t₂), the water (protons) from the fore-bay (inter-membrane space) comes back into the after-bay (matrix), through yet another locus in space, where we made it go through an electricity generator (analogous to ATP synthase). Let’s say that the time after this exercise is t₃ and let’s accept it that the “machine” has gone through one full cycle (and that the overall process is repeatable).

Please see that it is only because the second step was continued after the first step, we have retained the terminologies of fore-bay and after-bay (and this is just a semantic connectivity). Here is the important catch- Since the membrane is impermeable to protons, there are no equilibrium seeking forces that the membrane could tap. Therefore, the second step could not be spontaneous and would have to consume energy! This statement would be true no matter how efficiently one arranges the vectorial transport in the membrane. Say- the membrane pores/pumps are funnel shaped, with a narrow entry at the matrix side and wide exit at the inter-membrane space. However, if the return of protons is going to be energetically viable, then the membrane cannot be called semipermeable. If it was permeable, no potential would ever develop in the first place!

Once again, let’s see if our membrane machine satisfies the terms of analogy. In the current scenario, the membrane expended energy (this is not derived by NADH oxidation in the matrix, but avails another independent source) by moving protons from inter-membrane space into the matrix (item 1). Is this what we required of the membrane machine (item 3)? We cannot really say until we find what work was done by the water (protons) at the generator or pump (item 2). If the generator did not yield any electricity, then items 2 or 3 are not justified and we do not have a machine. On the other hand, if the generator/pump yielded more electricity/work than permitted by the laws of physics, then we have been sold an impossible machine. If very little work was done, the machine is poor in efficiency. Regardless of the outcome, the second step had no functional obligation/connection with the first step. Therefore, the first step was a mere waste and as a consequence, even without knowing the outcome of item 2, we can safely conclude that inner mitochondrial membrane could not serve as an effective machine, with the RCPE hypothesis. This is because the same membrane serves as the conduit and the same force is operable. Now (notwithstanding that we do not have any information whether the inward proton movements did any useful work), a logical exercise is to ask if you would use such a watermill scenario where you have to pump water up and then pump water down and make that water do work when it comes down. Clearly, this is not a way of getting work done spontaneously in real life! Why? Because we are doing relatively more work to get a lesser work done; or we have little tools at our disposal and therefore, we are destined to employ a very wasteful machine. Surely, living beings could employ better logic!

Regardless of the discussion in the earlier two paragraphs, let’s do a stock-taking of the process till now. The membrane machine requires at least two distinct steps involving two different “pores on the membrane”. (The first one was supposed to use a bigger original source of free energy and the second one was touted to be a transiently generated quantum that resulted because of the first step. We have now debunked this falsity.) Further, the two events must transpire through three non-exchangeable and non-overlapping points in time for the potential to build and dissipate. Only then, the “retuning pore” can do some useful work and the inner membrane could qualify for a machine! These summations shall enable us to assess the energy used at the pumping pore and work done at returning pore.

Regardless of the spatial arrangements into respirasomes or any other type of distribution or connection on the inner membrane, the overall system can be seen to comprise of two fundamental modules: proton pumps and ATP synthases (return channels). They must work through at least three distinct time points (t0, t1 & t2; t3 can be considered equivalent to t0), as shown in Figure D.

The simple rendition (with the assumption that the outer mitochondrial membrane is impermeable to protons and protons are not consumed or lost in the overall process) could help the reader see what the hypothesis achieved in real space/time. (In this image/at this stage, we shall not consider the energy requirements at all.) Please note how the same locus was used both as permeable and non-permeable point (in time) for the temporal build-up of potential. The setup cannot work in steady state because there is no mechanism to synchronize the outward pumps and stagger their operation with respect to the inward channels. Also, there is no way the setup can “do useful work” in a “proton-pump + proton-inlet” mode using the same membrane, with the same driving force. This setup is not like a hybrid car that uses an engine to burn hydrocarbons (a source of power) and battery (yet another source of power) to reduce hydrocarbon consumption. From the energetics perspective, the hybrid analogy would seek that mitochondrial Complex V uses protons from two sources- from the matrix and from the cytoplasm. Then, what would keep the machine going (if we allow Mitchell’s postulate to hold good), are the protons that come from cytoplasm! In this case, we are still left with the catch 22/zugzwang that the outer mitochondrial membrane would need to temporally secure the permeable-impermeable feature, energy equation would be unaccounted and also, the ETC-proton pumping would be rendered a redundant exercise anyway! By now, it is also evident that the inner mitochondrial membrane cannot be called semipermeable with respect to protons.

Figure D: Schematic diagram chronicling the events at a 2-dimensional chemiosmotic mitochondrial membrane

The best way to catch a fallacy is to assume that it is true and go on verifying it left, right and centre. Surely, it is bound to blurt out its own falsities. We have seen that affording the best case scenarios for ETC-chemiosmosis-rotary synthesis did not achieve the desired quantitative or qualitative outcomes that these explanations were supposed to give. Therefore, it is now conclusively shown that the ETC + proton pump is disconnected from the useful work step (making the up and down transport of protons a totally futile exercise!), and there is no way that the Steps 1 & 2 OR Steps 2 & 3 would have same signs for the energy terms!²⁴ If it had the same signs, then we would have a super-efficient machine that would violate the laws of physics. (Herein, an analogy from the banking sector would hold good- An entry in a bank ledger that had to be made in the debit side was inserted into the credit side; and that too, of a totally disconnected account. This is no wealth generation but just a case of poor accounting!)

Coming out of the loop of the discussion above: While comparing the chemiosmosis logic with a recycling hydroelectric power-plant (Figure 3 of main manuscript), one pivotal aspect stands out. The most important aspect is that in the Mitchellian system, the forces that work at the inner mitochondrial membrane are the same, electro-chemical force! Further, if the water was just pumped to another reservoir at the same level as that of the after-bay, the gravity-based gradient cannot be formed and the pumping out process cannot be harnessed. This scenario is more analogous to the mitochondrial spaces (matrix and inter-membrane region). In both the automobile engines and hydroelectric power-plants, the existence of an “externally open sink” is a must (the fume exhaust or after-bay outflow, respectively) for ensuring the spontaneity and thermodynamic feasibility for the system to do work. The constrained inter-membrane space that Mitchellian model proposes cannot be deemed as an open outlet (for the expulsion of protons by Complexes I, III & IV), and this would increase the cost of pumping protons out with increasing potential. Else, if the outer-membrane was freely permeable, then why should the protons go back into the matrix? Even otherwise, protons have been pumped out of one macroscopic phase (matrix) to another (inter-membrane space) and they are disconnected by the semipermeable membrane. Why should the protons feel any pressure to go back in now? From a functional perspective, the chemiosmosis supposition keeps asking for too many mutually contradictory requisites. We want a low proton density at the matrix side to serve the proton gradient and we want a high proton concentration at the same locus to serve the proton pump. Very interestingly, while the trans-membrane potential is what seems to be sponsoring the work, the actual commissioning agents (the physical connective) are the protons that go through Complex V. Since Mitchellian concepts seek a closed perspective of the mitochondria, the sheer lack of proton availability within mitochondria completely implodes the proposal! Also, even if we concede that steady state does not lead to equilibration of protons within matrix and inter-membrane space (and accept that a huge proton differential exists between the two phases), the workable proton flux would be constant (assuming the constancy of all other factors). Then, the fluxing miniscule amount of protons (that keep going through Complex V) cannot be scaled up with excess NADH or oxygen input. Such a setup could function continuously to generate only very miniscule amounts of work (in the best possible scenario) and that too in a modularized and synchronously phased setup, which should be very "intelligently" coordinated. Such setups and governance are lacking in the mitochondria. Therefore, chemiosmotic machine cannot explain physiology from a multitude of perspectives.

A short story to point out the lesson learnt- A guy named Mr. Hypothesis Cook wanted to light up a house that he had never seen. He hatched up a plan that he would pump water up to the roof, and then let the water flow down using gravity, and harvest the energy using a turbine generator and use that to light up the house. He put his plans up to the authorities and some guys bought into it and supported his plans. This inspired Mr. Cook, who bought some crayons and made altered plans to improve the appeal. And lo and behold! When the house was visited, it was lit up! All, including Mr. Cook continued to believe that the house was lit up because of his impeccable scheme! (He thought the authorities had put his plan into action and the authorities trusted the zeal of Mr. Cook, and assumed that he had executed it himself.) But one Mr. Explorer Find eventually turned up to report that the so called house was already lit up because of street lights; and that the house was actually a picket fenced property and had no roof, there was no water-body in the vicinity, and there was neither a pump nor any turbine generator anywhere around! Mr. Find was mocked by all first, but they too found the truth later in Mr. Find.

Therefore, it is better to stick to time-tested methodologies of science, and not to foray into Mitchellian model of problem-solving wherein ideas are made first and facts matter later.

#E: This is analogous to the following scenario- two marbles roll down the hill, one gets stuck in a valley whereas the other rides all the way back up the hill to the starting point on the hill top! Quite simply, this cannot happen in reality.

#F: Further, several researchers perceive the redox centers’ connectivity within the proteins as a “wire” (as exemplified by the linear connectivity drawn within matrix-ward projections of Complexes I & II). This might be a misplaced perception because a wire “conducts electrons” when/where there is a potential difference. If there is no applied field, no electrons would flow through a metal wire. Potentials are created or exist, when electrons or negative/positive charges accumulate at a given point. Clearly, the wire-analogy is inapplicable here. Seen in another way, in biological systems, there cannot be “freely-transferable high or low energy electrons or protons”. An electron in a particular orbital of an atom/molecule may have higher/equal energy term when compared to/with another electron in another orbital within the same/similar atom/molecule. But, if the electrons are mobile through the system, they should be seen as “similar”. In other words, when an electron (or proton) changes its “address” within a delocalized system, it retains little information regarding its previous “residence”. The physiological conditions, there is no external source to enhance the electrons’ thermal energy or electric field input to accelerate electrons. This argument is very important because in several cases, an electron at certain junctions of the “wired ETC” can move further only by going against the “resting potential” and they seem to “somehow upgrade their stature” at their new locus. Therefore, the plots which show that ATP-synthesis occurs at “energy differentials” (when electrons are transferred through two definite carriers within such a “roller-coaster” of an ETC) are theoretically incorrect. The above statements need to be rearticulated again. How could an electron move from a lower to an upper rung of potential via a junction, and suddenly gain energy at the higher energy locus? If it is freely mobile, energy profile must be similar. If energy profile is important, the electron cannot move freely against gradient in a connected system. Comparing electron flow with water flow is erroneous in one context because water can log at a point (because of a dip in the contour) and the water column at a given locus may also gain height owing to capillarity. In the proteins, there is no way for several electrons to reside at a junction (similar to water-clogging) and thereby raise the electron’s potential at that locus to a higher status. The junctions in context are one-electron donors/receivers and the next stop in the relay is usually too far for an electron to “tunnel” through against the gradient. Also, it is a very constrained aesthetic and deterministic notion that electrons would be transferred in pairs or only across narrow potential differences, as the prevailing ETC concept seeks. Therefore, it is incorrect to say that “mobile protons or electrons retain or lose energy/potential” just because they move from one macroscopic system to another.

#G: The concepts in statements above can be verified with a simple thought-experiment again. Assume a resting state (t0) where the matrix has 10 protons and the inter-membrane space has 10,010 protons, so that the necessary proton gradient is served. (Let us just be gracious here.) The enzyme activity now kicks in and in a second, 10⁴ protons move into the matrix through the ATP(synth)ase. This would be a simple staggered arrangement and it would help us understand the scenario better. Then, the enzymatic processing (esterification/hydrolysis) at the first second, t1, would be as shown in Table G. With the current model, the rotary synthase can only work futile cycles, releasing or binding pre-formed substrates or products. If we must imagine this system to recycle; either through the ETC-proton pumps (t2) or through F_o portion of ATPase itself (t2’), the futility only intensifies. Therefore, ATPase’s “rotary synthase” role must be discarded as an unreal proposition. There is no evidence or logic to the supposition that the direction of rotation (if it rotates, that is!) dramatically alters the affinities of the enzyme. A realistic scenario in which a rotary ATPase would catalytically “work” is given in the alternate option (after the backslash). Please note that once again, in t2’, it did not matter which direction the protons flowed through F_o portion. This is because ATP hydrolysis is the horse that could potentially drive the cart of protons in a given direction; not the other way around. Even otherwise, the three binding sites of ATPase cannot be equivalent in strict terms because the α-β dimer of at least one site must interact with b-δ units (on top and the sides) and ε unit at the bottom. Therefore, movement of the shaft is unlikely to give exactly identical effects in all three sites, in both directions. So, imagining Complex V as a perfectly reversible enzyme would be a grave misunderstanding, given its known structural attributes. Even if a large amount of protons came in through the F_o portion (in the synthetic mode), it would, at best, lead to futile revolutions of the ATPase (given the binding limitations imposed by ADP + Pi). The most sensible logic for a rotary catalysis works only in the hydrolysis direction. An ATP molecule needs to bind to ATPase, which introduces a movement in the shaft, inducing a movement of 3-4 protons through the F_o module. (Besides, the aspartate on the F_o part of ATPase looks too trivial a sensor/hook to latch on to a proton, or for serving as a bidirectional pedal. If it were a relay of amino acids involving at least some crucially located histidine residues, perhaps things could have been better, though not still cutting the acceptable mark, because we know this to be a low proton realm!)

Table G: Understanding Complex V functionality in the best case scenario: The rotating stalk primarily serves to liberate ATP or ADP+Pi. At t0, there are equal numbers of molecules of ATP & ADP in milieu. Immediately after t0, disregarding binding affinities and only going by probabilities (for the “ligand-on” process), a molecule each of ATP and ADP are bound to Complex V and the third site is vacant. Ten protons are used for one rotation of Complex V and the rotor spins a thousand cycles per second, with three distinct rounds of binding/liberations per rotation of the γ stalk. (* ± connotes rotation, clockwise or anticlockwise directions)

#H: I compared the Complex V setup to a medieval hand-driven (purely mechanical) flatstone grain-grinder. Whole grains introduced from the top into a small hole (analogous to protons’ inward movement) leads to the grains entering a gap between two flat stones. The grains are ground between the two stones to give powdered flour (analogous to ATP formation) by mechanical movement of the stone at the top (enabled by the operator’s application of a torque). The operational principle herein is that the weight of the top stone works (gravity, friction, torque, etc. all combined!) on the grains held against the stationary bottom. In comparison here, it is difficult to envisage that the mechano-chemical changes brought about by the inward trickling of a few protons (I could not see this process as a chemical reaction or electrical field or mechanical push!) would afford enough torsion to move the stalk (γ) through the αβ bulb. Even if the huge excess of pumps met with success in their vitally deterministic mission (that is- let us suppose fallaciously that they found and pumped every single one of the protons out and created very high potential across the small mitochondrial membrane owing to a disparity in these small amounts of protons), I felt that it cannot perform a major work across the same membrane because the amount of charge would be too little. But was it the charge or the numbers+mass or all of it combined that was doing the trick? How does the contraption work? Let’s now imagine that we have a very easily squishy fruit and when the fruit is placed at the intake portal of a juicer, we get pulp and juice at the bottom outlet. By changing the direction of the blades of the juicer and/or by inverting the setup, one cannot get the squishy fruit (at the new bottom) by adding the pulp and juice (at the new top)! Otherwise, keeping the juicer in the same configuration and applying suction at the top, does not yet give a fruit. No amount of complexity incorporation can give bi-directionality with the same/similar “instrumentation” modality/approach. The fruit can definitely be synthesized, but via a different route altogether. This premise exists in reality because there is a practical limit to reversibility, induced by free energy expense and increase in entropy associated with a given scenario. In the current case: Boyer’s proposal cannot overcome the imbroglio that in physiological conditions, (i) ATP hydrolysis in mitochondria is associated with a significantly negative free energy term, (ii) Complex V has high affinity for ATP (in comparison to ADP) & (iii) ATP is present at much higher concentrations than ADP. The overall Complex V reaction involves at least three distinct phases (matrix, membrane and IMS) and needs to recruit several molecules/ions, etc. While a simple redox reaction is easily reversible (for example: electron transfers mediated by an enzyme like reductase that could equilibrate electrons to and fro between NADPH and ferredoxin, depending on the environmental dynamics), a complex mechanistic scheme like rotary ATP-synthesis/hydrolysis cannot be deemed freely reversible under most realistic scenarios.

#I: This scenario is untrue, quite like the case that the following mathematical assumptions cannot lead to the final deductions, as shown-

If A = B, C = D; B = C + 1, Then, A ≈ D!

If A, B, C & D >> 1, then perhaps the assumption could be practically OK. But yet, such assumptions and deductions are best avoided in scientific practice.

#J: We have now seen that trying to comprehend oxidative phosphorylation with the RCPE hypothesis does not allow us a cohesive/coherent logic or quantitative account, with respect to mechanistic, kinetic or energetic perspectives. This is besides the fact that there are no available answers as to how the system senses electrostatics or concentration differences and how chemico-mechanical signals are relayed or transduced across the relevant protein networks. (The structures of all five major respiratory Complexes are known now. There is nothing in these that could work towards achieving the sensory/regulatory role.) Since the structures were revealed, consensus seemed to have crystallized more based on extrapolation of structure-function correlation. For example- the F₁ portion has three copies of the α-β protein and the F_o portion has more than 10 subunits of c protein; and the motor rotates in three strokes of 120 degrees. Therefore, the values that seemed to approach the concepts that support these structural details seemed to be favoured by the researchers! Let’s remember that aesthetics has little to do with reality or science. If Nikolaus Copernicus had not challenged the prevailing perceptions that sun circled the earth, if Tycho Brahe hadn’t made those meticulous astronomical observations and if Johannes Keppler had not perused/believed Copernican theory and Brahe’s experimental values, the world (perhaps!) would have been in great darkness now. In the scientific validation process (and ultimately, concrete progression towards understanding a phenomenon), it is more acceptable to disprove a concept and that purpose is the pivotal aim of this manuscript. Science works by negation, a principle which most practitioners of science seem to forget. If someone makes a hypothesis that an object makes a circular motion around a center, all that I need to know is the radius of the circle. If I find one point that does not fall on the circumference that would be dictated by the equation of the circle (traced by, perimeter = 2πr), the hypothesis goes for a toss! In Figure J below, this reality is clearly portrayed. All four images have a circle of the same dimension as “an apparently common” feature. If I knew that the trace of the function (all points!) fell on the pattern as described by the image on the extreme left, then the “circular” hypothesis is beautifully accurate. The image in the middle-left has a small but significant deviation. Though the equation of circle would define the trajectory of the object for more than 90% of the spatial attribute, it would be a crime if someone applied the circle’s equation as the object reached the top. (And who knows how much time is spent on the protuberance at the top, compared to the circular pattern? What if it took a year to traverse the top and just a second at the circular frame? The probability that you will find the function tracing a linear course would be much higher than the circular trajectory!) If you see the image on the middle-right, though more than two-thirds of a circle’s equation can be justified, it would be a disaster to consider the image as one being governed by the equation of a circle. (The object traces several folds more distance than what is permitted by the equation of the circle.) The image on the extreme right is similar to the one in the middle-right in terms of the loci of breaks from the circle’s equation. But though it has some similarities with the third image and looks closer to the circle, in fact, it is technically more complicated and cannot be governed by the equation of a circle at all!

Figure J: The philosophy of science and the practice of verifying hypotheses

This is yet another reason why I campaign to the uninitiated with this part of the write-up. People experienced in the field would continue to trace a circular paradigm and keep their followers flocking together (to show only the areas which apparently are “explainable”), no matter how many discontinuities arise! I hope that the simple arithmetic and forthright and argumentative logic/tone used throughout this article serves the younger researchers’ effort to seek better explanations. Even Einstein had introduced a cosmological constant for a static cosmos model, based on the prevailing information, only to change it later when the expanding cosmos became a more acceptable paradigm. I hope you see that we must let go chemiosmosis as a mistake of the past, just like Einstein jettisoned the cosmological constant when the static cosmos model became redundant. I am confident that if scientists indulge in open-minded discussions, chemiosmosis/RCPE will no longer be taught in schools to our future generations.

#K: Let’s say that a bank has a few transactions annually with a firm called “Protons Inc.” in the order of tens and hundreds of dollars. However, the same bank also holds the accounts of firms called “Potassium & Sons”, “Sodium Brothers Ltd.”, “Chloride & Carbonate Enterprises”, “Calcium Ventures”, etc. that transact billions of dollars annually at the bank. Now, while prepping the annual statement of the bank, the contribution of Protons Inc. can be safely sidelined, as the resulting money would be minuscule compared to the other financial giants.

#L: Let me give a very relevant and simplistic analogy here- Let’s say that four elephants (duly marked with identity tags in Malayalam, the language of Kerala) from Kulappully (my village in the Kerala, the state of elephants) keep going to Lake Manasarovar (a dream landscape in Tibet). A Maruti (a popular brand of sedan/hatchback in India) with Kerala registration plates is also found in Manasarovar, at times. (And, the presence of elephants at Manasarovar coincided with mild showers in Kerala.) Now, just because the car has four seats and both (car and elephants) originally belonged to Kerala, one cannot validate or accept a theory that the elephants drove down to Manasarovar in the Maruti. (And, the elephants’ presence at Manasarovar could not have pushed massive monsoon rain clouds to Kerala, by any “reproducible butterfly effects” either.) Particularly, when we know that the elephants were found in Manasarovar within a couple of hours of their disappearance from Kulappully and the car landed up in Manasarovar only after a couple of days! Even if we don’t have a good measure of the mass and size of the elephants versus the dimensions and horsepower of the car (spatio-temporal considerations and energetics), the time discrepancy and the distance involved (>3000 km) are factors which should tell us that the hypothesis is misplaced. Further, qualitative concerns that the elephants could not have walked or driven there on their own jolly will (and that too, not without a mahout or google-maps to direct them, in that good time!) should set off warning bells. And if we knew a bit more about the terrain, we would also know that the car cannot be driven all the way there either. Therefore, to get to know the truth, one option is to ask the elephants how they went there. (But before indulging in these single elephant cozy chat-up sessions, we must know elephantine communication.) Attributing the onset of seasonal monsoons in Kerala to those “four elephants’ occasional travel to Manasarovar in a Maruti” is a very unintelligible exercise. The best option for solving the issue is to find the “cause-consequence” correlations ourselves. It is unwise to stick to faulty rationales, just because they happen to be around.

Another analogy, this time with numbers, to give you a picture of what happened: 7 guys hailed a cab and the fare ran to 28 bucks. Now, a mathematical genius in one of them convinced the rest that each one of them had to contribute 13 bucks apiece, with the following protocol for division. 28/7 = 13. How? 7 goes once in 8. So, write 1 on top. Then, 7 is subtracted from 8 to get 1, which is written down. Now, the remaining 2 is brought down to the left of 1, to get 21 as the remainder. Next step- 7 goes thrice in 21 and therefore, write 3 down next to the earlier derived 1 and you have the exact number of 13! A cynic among the bunch felt that there was something wrong in the calculation and 13 seemed too big! He got out a piece of paper and said that if this calculation was true, then 13 added up 7 times should give the same number of 28. He wrote the number 13 seven times, one below the other. Now, the addition was carried out in the following manner: 7+7+7+7+7+7+7 = 21; 1+1+1+1+1+1+1 = 7; therefore, 21+7 = 28. Quod Erat Demonstrandum!

#M: The scenario analyzed above could be analogous to the following “one buck” accounting problem (which can be made out in many formats, of which I present two popular variants).

1. Let me ask you a question after a small story- Three friends go to a bar and have a drink each. The bill runs to 30 bucks and each one of them shares 10 bucks and thus, 30 bucks is pooled. The waiter takes it to the manager’s counter, where the manager gives them a 5 buck discount and puts a fiver back on the check platter, to be handed back to the customers. The waiter knew that the three customers had paid up independently and therefore, he knew they would have difficulty sharing the fiver amongst them. So, he took the fiver and placed three single buck coins on the platter instead, to return to the customers. They duly took one buck apiece. Now, each one of them spent 9 rupees and since there is three of them, the total comes to 27. The waiter has taken 2 bucks. Where did 1 buck go, out of the original total of 30? Did you see how a poorly framed (illogical) question can make a mess of simple accounting principles and take one on a wild goose chase? (This is just akin to have us using the spent energy as “stored energy”. Later, we will see that this spent energy is the trans-membrane potential observed, which is supposed to serve as the stored energy for driving ATP synthesis!)

2. The following entries are made in a guy’s expenses’ diary, as given in Box K. Why does the discrepancy arise?

The conceptual flaw in such a “trick question” appears to be the practical scenario in the bioenergetics field. The overall accounting was erroneously tabulated, owing to the “acceptance of chemiosmotic TMP as the driving principle”. This is demonstrated with another story-

A biologist chose to work on correlating the effect of sound stimulus, using frogs as test animals. The following experimental conditions were adopted and results were meticulously collected and tabulated.

Experiment 1: A sample lot of 10 male healthy frogs of similar age, weight and sizes were taken and the experiment was conducted on the same bunch for 5 days.

Experiment 2: With the preliminary results available, an elaborate experiment was planned for corroborating a hypothesis. A sample lot of 10 frogs were taken each day and subjected to the experiment, and the experiment was replicated on two different days, separated by two weeks.

Thus, by removing all subjectivity that could perhaps result by the thoughtless choice of unmatched experimental conditions and variables, with high precision and accuracy, the experimenter deduced that cutting of the third limb resulted in the frogs turning practically deaf!

Science deals with putting the horse before the cart, i.e. defining the causes that lead to a set of consequences! NADH oxidation is the horse, ATP synthesis is the cart. TMP is the clattering or jangling of tins attached to a horse driven cart (which could perhaps make the horse run faster, but it is the horse which runs faster on its own merit!). It is the legs of the horse that enable the horse to run, which in turn, powers the movement of the cart. Similarly, it is the DROS produced by NADH oxidation within the mitochondria that sponsors ATP-synthesis and the whole process gives rise to TMP. There is a connection between the frog’s jumping and the auditory stimulus (as there is between the TMP and NADH oxidation / ATP-synthesis). However, the primary causative is the DROS that is produced within. The negative charges within could facilitate the inward movement of cations, but that is only because of what happens inside. In the RCPE hypothesis, there is no mechanistic logic that CONNECTS the enhancement of NADH & O₂ oxidation by the presentation of ADP & Pi. This is the most important aspect that researchers in the field overlooked.