Synthesis of theories on cellular powering, coherence, homeostasis and electro-mechanics: Murburn concept & evolutionary perspectives

Abstract

If evolution were to be a fact, a simplified and unifying approach to explain cellular physiology is warranted. Such a perspective should agree with the thermodynamic, kinetic, structural and the operational-probabilistic considerations; without invoking overt intelligence or determinism, and must enable a synthesis from chaos. In this regard, we first list salient theories in cellular physiology for: (i) powering (generation of chemical/heat energy), (ii) coherence (interconnectivity and workability as a unit), (iii) homeostasis (metabolizing and expelling of unfamiliar/unwanted materials, maintaining concentration/volume), and (iv) cellular electrical-mechanical activities. While doing so, we discuss the scopes and limitations of: (a) the classical active-site affinity and recognition-based modality of lock-key and induced-fit enzyme-catalytic mechanisms established by Fisher/Koshland, (b) membrane-pump hypothesis acclaimed by biologists-physicians and historically championed by the British Nobel-laureates like Hodgkin-Huxley-Katz-Mitchell, and (c) association-induction hypothesis advocated by notable physicists-physiologists from various parts of the world, e.g. Gilbert Ling (China-USA), Gerald Pollack (USA), Ludwig Edelmann (Germany), Vladimir Matveev (Russia), Hirohisa Tamagawa (Japan), etc.]. We apply murburn concept (the thesis that diffusible reactive species play vital roles in maintaining life-order) to amalgamate several core cellular functions and further discuss the prospects for establishing the continuum of the principles of physics in biology.

Full text

Synthesis of theories on cellular powering, coherence, homeostasis and electro-mechanics:
Murburn concept & evolutionary perspectives
Kelath Murali Manoj*1& Laurent Jaeken*2
(*corresponding author)1Satyamjayatu: The Science & Ethics Foundation,
Kulappully, Shoranur-2 (PO), Palakkad District, Kerala State, India-679122.
Email: murman@satyamjayatu.com (ORCID: 0000-0003-4515-994X)
(*corresponding author)2Karel de Grote‐Hogeschool, Antwerp University Association,
Department of Industrial Sciences and Technology, Salesianenlaan 30, 2660, Belgium.
Email: laurent.jaeken@hotmail.com
Abstract: If evolution were to be a fact, a simplified and unifying approach to explain cellular
physiology is warranted. Such a perspective should agree with the thermodynamic, kinetic,
structural and the operational-probabilistic considerations; without invoking overt intelligence or
determinism, and must enable a synthesis from chaos. In this regard, we first list salient theories
in cellular physiology for: (i) powering (generation of chemical/heat energy), (ii) coherence
(interconnectivity and workability as a unit), (iii) homeostasis (metabolizing and expelling of
unfamiliar/unwanted materials, maintaining concentration/volume), and (iv) cellular electrical-
mechanical activities. While doing so, we discuss the scopes and limitations of: (a) the classical
active-site affinity and recognition-based modality of lock-key and induced-fit enzyme-catalytic
mechanisms established by Fisher/Koshland, (b) membrane-pump hypothesis acclaimed by
biologists-physicians and historically championed by the British Nobel-laureates like Hodgkin-
Huxley-Katz-Mitchell, and (c) association-induction hypothesis advocated by notable physicists-
physiologists from various parts of the world, e.g. Gilbert Ling (China-USA), Gerald Pollack
(USA), Ludwig Edelmann (Germany), Vladimir Matveev (Russia), Hirohisa Tamagawa (Japan),
etc.]. We apply murburn concept (the thesis that diffusible reactive species play vital roles in
maintaining life-order) to amalgamate several core cellular functions and further discuss the
prospects for establishing the continuum of the principles of physics in biology.
Keywords: electrophysiology; bioenergetics; evolution; mobile/fixed-water; murburn concept
Layout
1. A socio-philosophical introduction
2. Survey of some salient views on ‘cellular functioning’ floated till date
3. Cellular powering leading to coherence, homeostasis and electro-mechanical activity
4. Origin and unifying principle of the murburn mechanism
5. Summary and future prospects

1. A socio-philosophical introduction
With respect to our mandate, it is opportune to float salient quotes of some eminent persona:
In science it often happens that scientists say, 'You know that's a really good argument; my
position is mistaken’, and then they would actually change their minds and you never hear that
old view from them again… It doesn't happen as often as it should, because scientists are human
and change is sometimes painful. But it happens every day….
Carl Sagan (1987)
A discovery must be, by definition, at variance with existing knowledge... In science, the
Apollonian tends to develop established lines to perfection, while the Dionysian rather relies on
intuition and is more likely to open new, unexpected alleys for research…. The future of mankind
depends on the progress of science, and the progress of science depends on the support it can
find. Support mostly takes the form of grants, and the present methods of distributing grants
unduly favor the Apollonian.
Albert Szent-Gyorgyi (1972)
A new scientific truth does not triumph by convincing its opponents and making them see the
light, but rather because its opponents eventually die, and a new generation grows up that is
familiar with it… Science advances one funeral at a time.
Max Planck (1948)
Optimism is the faith that leads to achievement. Nothing can be done without hope or
confidence.
Helen Keller (1903)
Albert Szent-Gyorgyi and Max Planck, original and acclaimed scientists from biological and
physical sciences (respectively) portray the realistic stature of scientific advancements in society.
Few would question the genuinely communicative spirit of Carl Sagan, who inspired young
minds into scientific thought and exploration of cosmos with an open mind. He too addresses the

subjective issues in the tussles between inertial and novel thought strings; and projects optimism.
Our guiding beacon is the passion embodied in the disadvantaged (blind and deaf) Helen Keller.
With a trumping determination to express and be heard, and with the support of Anne Sullivan
and a few that cared in the society, Helen had successfully championed her cause. Inspired, we
believe that the deontological purpose of advancing truth/reason serves humanity, albeit it might
be disadvantaged and unwelcome at its birthing. For, the leadership of scientific bodies might
find it more convenient to maintain a status-quo. If the leadership entertains a continued
adherence to demonstrably untrue and unreasonable ideas, it fails the spirit of science which
requires paying tribute to the authority of logic. Mutely serving testimony to the logic of
authority that disciplines the bulk into consensus forming exercises caters to the vested interests
and the ‘projected stardom’ of only a select few. It is imperative that probing and dissemination
of the fundamental rationale for life-sustenance supersedes other myopic agenda. Therefore, at
one hand, our endeavour herein should be seen as an attempt to discredit some acclaimed ideas
that must now be considered redundant, for advancing education/research. At the other hand, we
propose a simple set of ideas that could form the pivot for renewed explorative fervour towards
explaining cellular and biological phenomena. With this profession of faith and opening up our
own writing to further critical dissections, we also add two important disclaimers:
(i) A crucial aspect of pursuits in biology is defining the usage/context of terms like dogma,
theory, hypothesis, model, etc.; and differentiating them from concepts and facts. The ‘central
dogma’ is a highly evidenced “rule” of sorts, which essentially prescribes that: (a) cells have a
deterministic governance of cellular functioning and perpetuation, and (b) this occurs via the
maintenance and decoding of genetic material, their decoded protein products (via replication-
transcription-translation and catalysis) and via the interactions such key protagonists with other
cellular components. The ‘cell theory’ posits that cells are the structural and functional unit of
living systems; and that all cells arise from pre-existing cells. Therefore, a cell is bounded by a
membrane and encloses the organelles (if any) distributed within the cytoplasm. This ‘theory’ is
quite unlike the mathematical theorems that are unquestionable derivations from axioms. Any
hypothesis, such as ‘chemiosmosis’, is merely an explicatory paradigm based on some
assumptions/ and postulates, and in no way has it any authoritative stamp of accuracy. When
exceptions (theoretical or experimental) are found against any hypothesis, its value is lost or

diminished, and must be revised in favour of a more comprehensive explanation. A concept (like
‘murburn’) is a naming or reasoning-trail directly deduced from a quantum of elementary
facts/observations, and is usually ratified. A model (can be based on a concept or hypothesis) is a
scheme of mechanistic processes or equations, which may be accurate or inaccurate, and should
be testable and/or disprovable. Therefore, discrediting a hypothesis or model is a natural course
in the progression of science and such inclinations and pursuits should not be considered as anti-
social or disruptive.
(ii) With utmost humility, we quote Robert Frost: “We dance around in a ring and suppose,
but the secret sits in the middle and knows.” Werner Heisenberg had thus cautioned us: “We
have to remember that what we observe is not nature in itself, but nature exposed to our method
of questioning.”, lest we end up like the six blind men from the Indian Parable (slightly
rephrasing the much celebrated John Godfrey Saxe’s beautiful verse):
So oft in scientific wars,
The disputants, we ween,
Rail on in utter ignorance
Of what each other mean,
And prate about an Elephant
Not one of us has seen!
It is important to register that proposing a mathematical model and making statistical analyses
with data to infer that a phenomenon fits the equation’s curve is merely the mid-point in the
pursuit of accuracy/precision for arriving a probable explanation, and not the culmination of the
journey. There could be pitfalls in multiple planes: (a) the initial assumptions used for
theorization may be misplaced (as they may not have any realistic correlation with the actual
system), (b) diverse mathematical forms/equations can be transformed and fitted into a given
experimental data with good linear or non-linear regression fit, (c) a given mathematical form
can be achieved via quite two or more different theorizations/variables, (d) data acquisition or
processing may be skewed and not covering the entire sample space adequately, and (e) even if
all the above boxes were to be ticked, the inferences drawn may still end up being short on
accuracy, owing to the way how probability works in the real world. Therefore, it pays to be
continuously critical, creative, constructive and cosmic (open-minded to dissenting voices and
data out there!) while forming and advocating any scientific theory. As holistic tracing and

accounting of each and all cellular activities is impossible to achieve simultaneously, cellular
functions are studied by scientists in bits and parts; and a holistic picture is integrated thereafter.
In this endeavor, it is opportune to restate that the data available till date and interpretations
thereof are subject to: (i) experimental constraints posed by the minute dimensions of cellular
systems which distort physiology when investigated with probes given by even the most recent
technologies, and (ii) the subjective disposition of the researcher and receptive outlook of the
community at large. The physical and chemical sciences are usually more exact and
discussing/advocating a new observation or explanation does not lead to the
“ostracization/shunning” seen in biological sciences, when it comes to “anti-establishment”
ideas. It is in this context that the universal theoretical foundations in physics take precedence
over questionable biological data or misinterpretations thereof. Thermodynamics, the classical
branch of physics, uses derivable theorizations, macroscopic measurements and adheres to the
“top-down” logic. Detailed observations/explanations at the molecular level (“bottom-up”
rationale) in biochemistry have their own value as long as the conclusions drawn are able to fit as
integral parts within the top-down logic. The molecular data/interpretations thereof may never be
in contradiction with the thermodynamic approach, otherwise it follows straightforwardly that
they are wrong (Einstein, 1950; Kaufmann, 1989a-b; Drukarch et al., 2021). Another aspect of
understanding life processes is that although very complex at the core and in the overview,
explanation of natural phenomena is aided by sticking to Ockham’s razor. That is- the principles
of evolution dictate that a simpler mechanism is more likely to be true than a complex one,
because it is easier for life to evolve with fewer requisites. Besides these two aspects, kinetics
and structure-function-distribution correlations are yet other key considerations for explaining
cellular processes.
Herein, with an evolutionary perspective, we address the topic of how a simple cauldron of
biomolecules could achieve an apparently concerted outcome. That is: how does a mixture of
deterministic and stochastic processes (occurring at distinct loci and via diverse mechanisms
within a cell) converge to a concerted result in a reproducible and constructive manner, aiding
the sustenance of life? In short, we address the question: How do diverse proteins embedded on
the biological membranes and the biomolecules and ions present around come together to enable
chemical powering and heat generation, disposing of unfamiliar molecules and toxic wastes,

continuously carrying out homeostasis and working as a well-knit and immediately responsive
electro-mechanical unit? When an individual like Wim Hof demonstrates extraordinary ability to
regulate body functions in extreme cold for prolonged time frames, it is evident that genetic and
neuronal intelligent controls can deterministically influence cellular physiology. We do not
negate such effects and do not intend to focus upon the complex aspects of how cellular
responses are contingent upon the slower genetic/hormonal/neuronal and other such internal or
external “intelligent” cues. Herein, we would only deal with the immediate molecular and
physical ‘auto-coordinations’ that ensue with the onset of a trigger (from within or outside), and
which proceed without overt deterministic governance dictates (whether internally from the
nucleus or externally from the brain). Towards understanding and elaborating such cellular
processes, we first undertake a survey of the multiple viewpoints, parsing them; briefly
identifying their strengths and weaknesses. Thereafter, we attempt to synthesize a unifying
perspective, with an ab initio approach. The strategy would be to begin from molecular-
metabolic events, leading to nano-micro regime phase changes, which in turn integrate into
macroscopic cellular physiological outcomes. We advocate that besides the acclaimed and
deterministic central dogma, murburn concept is yet another founding principle of life (as shown
in Figure 1), which explains the origin and evolution of all life forms from a chaotic concoction.
Also, we seek that the coacervate nature of the bulk-phase in cellular physiology be recognized,
along with the pursuits of researchers who campaigned for this aspect all along. With this, we
hope to bring diverse feuding factions in cellular physiology to a platform of agreement and
provide a constructive way for futuristic academic and research enterprises.

Figure 1: The core aspects of cellular functioning (the six vertices) are founded stochastically in murburn
concept at short time-scales and deterministically governed by the central dogma over the long time-scales. Cells
are physically coacervates and bear catalytic abilities chemically.
2. A snapshot of some salient ideas and schools of thought in core cellular physiology
All chemical properties and interactions/reactions thereof are ultimately determined by the
governing principles/laws of quantum physics. Even at more superficial levels (e.g. the treatment
of voltage fluctuations in the HHK or Hodgkin-Huxley-Katz model of action potential),
significant mathematical treatments of processes are required to understand the intricate
functioning of the various models. Herein, to keep matters appealing to most readers, we shall
not delve into the sophisticated equations and treatments thereof, but only highlight the various
theorization principles involved. The attributions to classical and acclaimed schools of thoughts
are taken from mainstream biochemistry and cell biology textbooks (Alberts et al., 2008; Berg et
al., 2002; Nelson & Cox, 2004; Pelczar et al., 2001; Voet & Voet, 2011); and common
awareness in this regard may not be explicitly cited within the manuscript text.
If we accept that cell(s) is(are) the fundamental structural and functional unit(s) of life, then it
implies naturally that: (i) the cell must power itself with chemical energy linked to heat output,
(ii) different parts of the cell need to be seamlessly networked and the cell as a whole must
maintain its integrity (with respect to composition of its components), and (iii) the cell would be
capable of demonstrating other features of life like responding to stimuli with electrical and
mechanical activity. Let’s take a brief survey of the salient theories out there, with respect to
these attributes of cells, as shown in Figure 1. Herein, the classical approach would be deemed as
one in which solely deterministic and topology-affinity based recognition/adsorption/catalysis
logic governs cellular functions (and undermines the effects of stochastic interactions/collisions).
[Please refer Table 2 of Manoj et al. (2022c) for an item-wise distinction and discussion of the
classical schools (MPH and AIH) vis a vis murburn concept, particularly in the context of
electrophysiology.]
I. Bioenergetics (large-scale synthesis of ATP and generation of heat) & redox homeostasis:
Chloroplasts and mitochondria are cellular organelles that produce the energy currency of ATP

and generate heat. Since the mid-part of 20th century, three major schools were witnessed in the
field.
A. The classical enzyme-centric phosphorylating intermediate (or chemical coupling) was
proposed and advocated by Edward Slater (1953) and it postulated the following modality (quite
similar to substrate level phosphorylations) for ATP synthesis:
DH2 + A + E → D~E/D + AH2/AH2~E
D~E/AH2~E + Pi → D/AH2 + E~P
E~P + ADP → ATP + E
-----------------------------------------------------
DH2 + A + ADP + Pi → D + AH2 + ATP
In the above, DH2 is the electron-rich reduced substrate, E is a respiratory enzyme, A is the
electron acceptor and E~P is the enzyme-bound phosphorylating intermediate. When this
hypothesis was more popular, Paul Boyer proposed phosphohistidine as the E~P intermediate
(Suelter et al., 1961; Boyer, 1963; and 10 more publications from 1961 to 1964 in leading
biochemistry journals). Although exhaustive search was conducted for several decades, the
search for such an E~P species did not materialize into fruition, and therefore the classical
enzyme-theory based approach was sidelined by the community. Gilbert Ling, the advocate of
association-induction hypothesis (AIH), had proposed an idea quite similar to the classical view,
but also involving ion-adsorption mechanisms (Ling, 1981). In these mechanisms, the affinity
based recognition of substrates at the active site was an essential mechanistic requirement, and
the mechanism was still 2-electronic.
B. The currently in-vogue Chemiosmotic rotary ATP synthesis or CRAS (chemiosmotic and
conformation-change coupling) is a highly modified and fused version of two quite distinct
mechanisms. Peter Mitchell (1961) proposed the chemiosmotic coupling wherein redox reactions
supposedly resulted in pumping out of protons, causing a proton-gradient and electrical potential
that enabled ATP-synthesis when protons returned inward. This was a scion of the membrane
pump hypothesis (MPH, which espoused that membrane proteins possessed the abilities to
specifically bind and pump ions), advocated by other contemporary British scientists like Alan
Hodgkin, Andrew Huxley, and Bernard Katz. After this, Paul Boyer (1964, and several
publications thereafter until the next ten years) made a volte-face and floated the “conformation-

change coupling” model in which reduction of proteins (particularly, involving the thiol groups)
by electron-transfer processes changed their conformations, which in turn enabled ATP
synthesis. While several of Mitchell’s ideas like proton motive force/chemiosmosis and Q-cycle
have been debunked (Manoj, 2017,2018a; Manoj et al., 2020b; Manoj et al., 2022d), the
vectorial nature of cell/organelle membrane remains valid even today. In the mid-1970s, after
Howard Berg’s purported discovery of “rotary function” in bacterial flagellar basal module and
after Mitchell’s ideas gathered traction with the community, Boyer found it opportune to make
several major shifts, yet again. With the initial reports on structural information on Complex V
coming in, Boyer (1975) added proton-centric rotability and periodic rotational conformation
change of subunits of Complex V. (Now, in Boyer’s model, Complex V did not have to be
reduced and even the proton did not have to get to the active site where the reaction was
supposed to occur!) It is noteworthy that Mitchell did not agree with Boyer’s proposal regarding
the fate of the inward-moving protons (Prebble, 2013). However, although Boyer’s ideas have
been dissected and rendered redundant now (Manoj et al., 2022b), he could convince those that
mattered at the helms of the scientific community then about the merit of the merger of his ideas
with that of Mitchell’s and their compatibility with the newly emerging structural information.
These facts are presented to the readers to show that the much acclaimed ideas that purportedly
claim to explain cellular bioenergetics were enforced via a top-down consensus, which proved
counter-productive. [Herein, not only did such ideas fail to convince the original promulgators, it
could not bring an agreement even among the enforcers; please refer (Manoj, 2020b). Therefore,
rather than pushing agendas via clandestine authoritative means, it is better to debate it out in the
open, as was common during the ‘OxPhos wars’ of the 1960s!] Under the CRAS purview, heat
production is when the protons re-enter into the matrix or cell without going through Complex V,
but when they come in via the uncoupling protein. Not only does the popular mechanism entail
active site recognition of ADP and Pi, the mechanism also solicited that proteins could
specifically pump protons out. Also, since the ‘chemiosmotic-coupling’ has only protons cycling
within the mitochondria, there is no concept of redox homeostasis in this proposal, and it is
impossible to explain the formation of water from NADH, without breaking stoichiometry and
thermodynamic criteria.
C. The murburn model (Manoj, 2017-till date to Manoj et al., 2018-till date) demonstrated that
oxygen-centric diffusible reactive species (DRS) were the actual agents that catalyzed ATP-

synthesis in mitochondria via simple chemical reactions, thereby explaining the failure of
scientific community in the quest/search for a high energy phosphorylating enzymic
intermediate. The murburn reactions mediated by DRS (and the reactions among themselves) are
exothermic, and this is the reason for thermogenesis. The details of this model shall be discussed
in another section of this manuscript and compared with the more popular Keilin-Mitchell-Boyer
model. The murburn model does not necessitate an efficient binding of the substrate at a unique
active site, but it advocates that low affinity binding at various regions of the proteins in
proximity to the DRS generating centers enhanced catalytic outcomes. Further, since the
mechanism is a simple exergonic chemical reaction and it entails the usage of extraneous
protons, it justifies the mandates of architecture of mitochondria and structure/distribution of
components, redox homeostasis, stoichiometry and thermodynamic premises (Manoj & Bazhin,
2021).
II. Coherence (how a cell is networked into a whole unit): An event transpiring at one end of
the cell cannot be immediately experienced or assessed by another disconnected part. Yet, the
unitary functioning of a cell requires that the components of cells must have some modality to
enable spontaneous chemico-physical connectivity.
A. The classical view of deterministic outcomes within cells rests controls at the genetic (in the
nucleotide sequence) and protein (in the topography of the active/allosteric sites) levels. These
perspectives vest the control in the individual protagonists (or in organelles like nuclei or organs
like brain) and there is little logic available for rapid connective linking or holistic networking
among the components. Although the concept of ‘molecular messengers’ is present in the
classical purview, it is still based in specific binding-based recognitions and subject to
deterministic controls via the central dogma. Advocates of AIH perceive that proteins’ binding to
the cardinal ligand of ATP enable a systemic ordering of ions around their vicinities, which get
perturbed due to diverse events (Ling, 2001). This adsorption-based model is a binary response
system, that can enable a distinction based on presence and absence of ATP alone and cannot
afford a spectrum of outcomes.
B. The murburn purview of cellular functioning calls upon the obligatory involvement of DRS
that serve in moving electrons and moieties; thereby forming immediate connectivity and

networking across cellular nano/micro-domains (Manoj et al., 2021c; Manoj & Gideon, 2022;
Manoj et al., 2022c,h). Such DRS-mediated processes can lead to significant changes at
molecular/ionic levels at one hand, and phase-alterations/mobilizations at the other.
III. Xenobiotic metabolism, waste disposal and continuous cellular homeostasis: It is
inevitable that xenobiotics may enter the cell and the cell itself may produce significant amounts
of wastes by routine metabolism. A living and functional cell must continuously deal with such
intakes and agendas to expel the unwanted/wasteful molecular species, and maintain its integrity
(both overall architecture and real-time composition, with respect to discrete amounts of
components distributed in a given volume).
A. Within the classical enzyme active site-centric purview and MPH, the intake and efflux of
materials must be primarily seen as regulated by recognition via membrane/embedded proteins
(Manoj & Tamagawa, 2021). For xenobiotic metabolism, tetra-molecular complexes (between
the heme cytochrome P450 + oxygen + xenobiotic with the flavin reductase bound/reduced by
NADPH) are thought to be deterministically formed (Ortiz de Montellano, 2015). This fails to
explain the physiological abundance of oxygen-centric DRS in cells and biomolecular structures
that are evidently hardwired for DRS production. Since the classical scheme necessitates long-
term binding of substrate at the active site, it is difficult to justify how this can be achieved as
cellular systems do not have evolutionary memory or means of acquiring a topological
complementation of diverse xenobiotics that have not entered the system earlier. (This is quite
unlike how immune system develops antibodies when antigenic epitopes are presented!) Further,
it is difficult to see how a serial scheme of events would be kinetically viable for such
diverse/generic molecules. The advocates of AIH have typically not addressed this aspect of
cellular physiology.
B. As per the Murburn model (Manoj et al., 2016a; Parashar & Manoj, 2022), the cell functions
as a simple chemical engine (SCE) and the redox proteins serving as generators, modulators,
sustainers and utilizers of DRS enable it to work in this capacity (Manoj et al al., 2022h). Since
the generic format of the interaction of DRS with the substrate is core of xenobiotic metabolism,
it obviates the need for protein-inducibility and topology complementation between enzyme

active site and xenobiotic. Therefore, the realities are better explained with murburn concept
(Manoj et al., 2016a).
IV. Electrical activity, as seen in ‘action potential’: In the later evolved life-forms like
vertebrates, functional connectivity of various parts of an organism is made with the nervous
system. First, we deal with a listing of the explanations available for the directional signal relay
along the neuronal axon, which is an example of cellular electrical activity. Although the
physical form of the action potential trace was first mathematically modeled by Hodgkin &
Huxley (HH), quite different and more recent theoretical models appear mathematically more
compatible with HH’s experimental observations, and also physically fit better. For example: the
mathematical form of Goldman-Hodgkin-Katz equation was derived from a totally different set
of variables, starting from Ling’s adsorption theorization (based in his association-induction
hypothesis or AIH) (Tamagawa & Ikeda, 2018). Very sensible ideas floated by quite inquisitive
scientists continued to enrich the field.
A. The HHK model (Hodgkin and Huxley, 1952; Hodgkin et al., 1952) is advocated in the
textbooks, and falls within the context of membrane-pump hypothesis (MPH). It is an
exclusively ionic model of a propagating voltage-pulse driven by currents. These currents result
from bidirectional ion-fluxes mediated via ion-channels and ion-pumps located in the axon
membrane. The sole actors taken into account are voltage-driven protein ion-channels for Na+
(depolarising) and K+ (repolarising); and NKA (or Na,K-ATPase or sodium-potassium pump) for
restoring the resting state (during the refractive period). In short: the voltage across the
membrane is set up by the protein channel-regulated fluxes of ions and the ion-channels are in
turn controlled by voltage changes that the ion-fluxes create. This theorization requires that
proteins must have high fidelity to bind and differentiate efficiently ions such as Na and K.
B. The semi-conduction model (Cope, 1968, 1977a,b) postulated and could quantitatively
explain the data of the HHK experiments better on behalf of semi-conductive Na+ and K+
currents and holes flowing through the membrane and at the membrane surface(s), than by
assumed free ion currents.
C. The surface or phase-boundary potential model (Edelmann, 1973; Ling, 1982, 2001) is
conceptualized within the larger framework of the association-induction hypothesis (AIH). It

enlists the participating cations’ adsorptive properties on the surfaces (membrane/proteins) and
the ability of such events to induce conformational changes in proteins and thereafter influence
the potentials measured.
D. The exclusion zone model (Pollack, 2009,2013) unravels that water is organized in different
modalities within cells, at membrane surfaces and other interfaces, and is directly measurable at
the outer surface of the cell membrane at the EZ/bulk phase boundary, which is shown to exhibit
charge separation.
E. The murburn model (Manoj & Tamagawa, 2021; Manoj et al., 2021; Manoj et al., 2022a,b)
posits that the temporal voltage variation profile noted along the axon is a complex phenomenon
resulting from several contributing factors, including DRS formation and its dynamic
equilibriums with water/ions thereof. Rather than focusing on the variation of electric potentials,
this model stresses on the electron relay along the length of the axon. The murburn model is not
incompatible with any of the other ideas in this field (i.e. it does not negate ion-differentiations,
semi-conductive effects, ion-adsorptions, water/lipid properties that contribute, etc.) but is
encompassing of them. The ion differentiation at the interface is based on thermodynamic profile
and not necessarily owing to differentiation based in binding affinity alone.
Besides the models above that consider the electrical facets of the system, there also exist several
other explanations such as that for the soliton properties of the action potential (Heimburg and
Jackson, 2005, Drukarch et al., 2021, Heimburg, 2022), which consider the top-down large scale
thermodynamic properties of action potentials. They showed that membranes prepared from only
lipids can produce action potentials from which the characteristics are almost indistinguishable
from those of cell membranes, indicating that the lipid components in real membranes are under-
emphasized. Models based on other mathematical foundations such as: relaxation-oscillator
(FitzHugh, 1961), pressure-wave (Rvachev, 2010), electromechanical wave (El Hady &
Machta, 2015), unifying model (Engelbrecht et al., 2020), etc. continue to populate the literature
in the field. This clearly shows that although the HHK model’s platforms were laid in 1950s
(when the molecular structures were not explored well), there are several lacunae in the early
theorization which were identified by later scientists (Faraci, 2013). It is a real pity that such
reflections are not seen in textbooks and the inadequate HHK model is prescribed as if it were
the sole manifestation of truth/reason!

V. Mechanical displacements in contraction/transport/motility: A macroscopic signature of life
is the ability of a cell or organism to move its parts. In vertebrates, muscles attached to bones
enable the movements of hands and legs, serving as crucial elements in survival. Within cells,
kinesin and dynein proteins move along microtubular molecular tracks, some even delivering
payloads. Unicellular bacteria, cyanobacteria and algae are known to make chemotaxis and
phototaxis respectively, and even the cytoplasm of plants shows swirling movements. All these
examples from various levels of life and living beings involve a mechanical displacement of
components as a key aspect of cellular physiology.
A. The sliding-filament model of muscle contraction was proposed via two seminal papers by
Huxley & Niedergerke (1954) and Huxley & Hanson (1954), based on microscopy techniques.
The current view proposes that upon availing a suitable neuronal stimulus, thick myosin
filaments and thin actin(+tropomyosin) filaments slide against each other to contract a muscle
cell. The sliding is supposedly achieved by the binding of calcium ions to troponin, after which
myosin heads use ATP-hydrolysis as a cue to bind, release and rebind and thus move ahead on
the thin filament. This view places significance only on protein conformation-change based
movements resulting from ATP hydrolysis. The advocates of classical perspective, Volkmann
and Hanein (2000) expressed this view in the thermodynamic terms of: “muscle contraction is a
disorder- to-order transition” without considering the importance of water. The AIH school
(Ling, 2001; Edelman, 2005) considers that “muscle contraction is an order-to-disorder phase
transition” with ATP adsorption to install the resting state as the energizing mechanism (not
ATP hydrolysis!), and with water phase transformation responsible for both contraction and
thickening (Ling, 1984). When assessing thermodynamics, all involved plyers must be taken into
account, and particularly in cells the entropic factor may not be neglected (Schrödinger, 1944).
In an entirely independent way Oplatka (1994, 1997) also arrived at the conclusion of water
movements as a rationale for contraction. Another inability of the classical explanation is the Z-
disc movement for multiple adjacent sarcomerers (Mungal et al., 2015). Further, since it was
difficult to explain eccentric contractions and force enhancement during stretch (Montesano et
al., 2020), winding filament theory was proposed (Nishikawa et al., 2012) and the big titin
protein has also been mooted to be a participant in the contraction (Herzog et al., 2015). Two

models of ATP production in muscles exist following exertion (Lai et al., 2008). One is where
more usage of ATP generates more ADP and Pi in situ ; another model is where supply and
demand chains of ATP production and utilization are simultaneously activated. The former is
supported by the murburn purview while the latter requires elaborate sensors and networks for
operation.
B. The hand over hand model for the movement of transport movement of kinesin (Vale et al.,
1996) towards the plus end of microtubules (made of tubulin), although supported by evidence,
appears too deterministic and does not account for entropy or water-based effects. A diffusion
biased movement aided by the soft neck linker was recently proposed (Xu et al., 2021).
C. The rotary model for the functioning of bacterial flagella-aided movement (Berg & Anderson,
1973; Berg, 2003) mooted that the bacterial flagellum was a motor like machine that used the
flagellum as a propeller to move with a cork-screw type movement through water, in a regime of
low Reynolds number. The evolution of a rotary setup appears highly improbable in bacteria and
the structures merely have a radial symmetry only for ensuring a snug-fit of the flagellar filament
that pierces the membrane.
D. The murburn model of motility in bacterial system proposes that flagella merely serve as
stabilizers in fluid dynamics and it is a jet propulsion type water-mobilization at the flagellar
basal module (which is a Type 3 injection system) that actually explains bacterial motility
(Manoj et al., 2022i). Ling (1984) and Oplatka (1994, 1997, 1998a,b) had reasoned earlier that
water displacement must be a key to mechanical movements within cells. Although their ideas
did not acknowledge the role of the core 1e murburn respiratory processes involved (and the
latter erroneously invoked rotary motions in bacterial flagellar system), it is noteworthy that
three independent approaches lead to the conclusion that mobilization of water forms the crux of
mechanical movements in cells.
VI. Overall bioenergetic/coherent/homeostatic functioning of erythrocytes: Red blood
corpuscles are quite akin to other cells in their ability to maintain cellular components (e.g. Na/K
ion concentrations). However, they lack a nucleus to intelligently coordinate and govern various
activities for protracted time frames and they lack mitochondria that could power their activities.
The glycolytic enzymes cannot produce enough ATP to sustain them and ensure the Na/K
distribution (Parashar et al., 2021), if classical perspectives alone were to be operational.

A. The classical purview (in both MPH and AIH schools) does not afford any tangible
explanation for the survival and functioning of erythrocytes.
B. The murburn models for ATP synthesis by hemoglobin (Parashar et al., 2021) provides a
viable explanation for powering and for ion-differentiation by Na,K-ATPase and by cytosolic
Na+ excluding and K+ accumulating proteins like actin (Manoj et al., 2022h). It is a tangible
means to explain RBC’s ability to enable bulk ATP-synthesis and maintain its cellular
electrophysiological features.
In summary, although the Fisher-Koshland models of enzyme affinity-based activity is highly
evidenced and explains several metabolic outcomes, the murburn model of DRS-based murzyme
interactions complements and supplements this with a mechanism of primary significance. While
the fundamental theorization of MPH and some premises of AIH are entirely built on the
deterministic principles (topology-affinity, stoichiometry, etc.), the murburn models bring in
stochastic elements that could also support life. Also, AIH presents important/interesting facets
worth exploring further. Therefore, it is opportune to consider how a synthetic approach can be
adopted in such a scenario.
The fundamental fact of a cell is that it is made of diverse molecules and ions, and this must have
been the case during the inception stage of cellular origins too. It is natural to accept the
postulate that the molecule-ion interactions and equilibriums that were operative then must be
operative now also. In this regard, murburn concept, a simple interactive network of 1e and 2e
equilibriums, must have been in prominence/operation even then. Therefore, murburn concept
would be a fundamental contributor to cellular coherence. It can be seen that chemically tangible
and thermodynamically viable murburn models are proposed for bioenergetic, thermogenic,
xenobiotic-clearance, homeostatic, electrophysiological, and mechanical/motility mechanisms.
Therefore, murburn concept is a general principle of cellular functioning (although, it is quite
probable that the murburn models may need improvements!). In this article, we further stress on
the importance of murburn concept based on the fact that till date, this is the only perspective
that gives a tenable cellular powering (ATP-synthesis and heat generation) explanation. Since all
other physiologies would be directly and/or indirectly dependent on powering and coherence, we

perceive that the mandate to found and ground life in murburn concept can be visualized via a
detailed exposition of its relevance in bioenergetic and evolutionary perspectives.
3. Cellular powering leading to coherence, homeostasis and electro-mechanical activity
ATP’s function as universal energy currency in all known life forms strongly suggests that the
mechanisms and bio-energetic principles for the re-phosphorylation of ADP, once established
(say in an entity like LUCA, or the Last Universally Common Ancestor), were perhaps
perpetuated/conserved as such. They are those of substrate phosphorylation in glycolysis,
oxidative phosphorylation in all its forms with O2; N-compounds, S-compounds, CO2 etc. as
weak oxidizers in methanogenic bacteria, and those of anaerobic and aerobic photo-
phosphorylation. Apart from substrate phosphorylation, the other more complicated mechanisms
depend on the structure and working of so-called ‘energy (energized) membranes: bacterial cell
membranes, thylakoid membranes, and mitochondrial inner membranes. If they were to work
according to pump/pmf-based mechanism, it would necessitate that not only all the diverse
components should be present a priori, the detail of such protein complexes should be elaborated
into perfection before “the whole complicated network” can start to work. This necessarily
translates to creationist ideas and we opine that there must be a much simpler evolutionary
feasible mechanism. An overview of the classical principles, which met with huge controversies
from more than half a century ago up to now, and its comparison with the new murburn model is
necessary from an evolutionary perspective.
A sophisticated and essentially proton-centric proposal stitched up over time from distinct ideas
mooted by ‘David Keilin (1930s) – Peter Mitchell (1960s) – Paul Boyer (1980s)’ and came to be
the widely accepted explanation for bioenergetics (Braun, 2020; Nolfi-Donegan et al., 2020).
With reference to mitochondrial oxidative phosphorylation, this proposal vouches that some of
the ‘electron transport chain’ (ETC) proteins (Complexes I, III & IV) serve as proton pumps,
leading to the establishment of ‘proton motive force’ (pmf) consisting of a chemical pH gradient
and an electrical trans-membrane potential (TMP), which in turn, enables ‘chemiosmotic rotary
ATP synthesis’ (CRAS) by Complex V. We had provided elaborate critique of the pmf-based
explanation for bioenergetics (Manoj, 2017; Manoj, 2018a) and proposed an idea in lieu called

murburn concept. The insights derived from murburn concept were applied to explain the
observations in various core physiologies involving oxygen (Manoj et al., 2022a CBI; Manoj &
Gideon, 2022), such as: xenobiotic metabolism and anomalous dose responses (Manoj et al.,
2016a; Parashar et al., 2018; Parashar & Manoj, 2021), aerobic respiration, thermogenesis and
homeostasis (Manoj et al., 2018; Manoj et al., 2019a,b; Manoj et al., 2020a; Manoj & Bazhin,
2021, Manoj et al., 2022b), photo-transduction in vision (Manoj & Jacob, 2020, Manoj et al.,
2022c), oxygenic photosynthesis (Gideon et al., 2020; Manoj et al., 2020b; Manoj &
Manekkathodi, 2021; Manoj et al., 2021a,b; Manoj et al., 2022d-f), ionic differentials and
electrophysiology (Manoj & Tamagawa, 2021; Manoj et al., 2021c; Manoj et al., 2022c,h), etc.
In the context of aerobic respiration or oxygenic photosynthesis, the murburn model advocates
that DRS produced during oxidation of NAD(P)H activates ADP/Pi producing ADP* and/or Pi*
radicals, which spontaneously react for the direct formation of phospho-anhydride/phospho-ester
(ATP), as it is the case we demonstrated in vitro (Manoj et al., 2019a, 2020a). This simple
exergonic mechanism will be called ‘murburn phosphorylation’ in order to discern it from the
impossible traditional mechanism of oxidative phosphorylation. Thus, murburn model provides
the first tangible chemical rationale for ‘directly coupled’ ATP synthesis in mitochondria and
chloroplasts, which is executed by the redox complexes themselves and is aided by the newly
discovered murburn activity of complex V (Manoj et al., 2022b).
The ETC and chemiosmosis/pmf proposals were mooted and rooted at times when there was
inadequate information on mitochondrial architecture or membrane-embedded protein structures.
Our critical publications in recent times and the citations therein (Manoj, 2018a; Manoj et al.,
2019a-b; Manoj, 2020a-b; Manoj et al., 2020a-b; Manoj & Bazhin, 2021; Manoj et al., 2022e)
have brought to the fore that: (i) As pointed out in the authentic biography of Peter Mitchell and
elsewhere (Prebble, 2000; Prebble & Weber, 2003; Roskoski 2004), the proposal of
chemiosmosis and pmf was floated on pure imagination and little evidence. (ii) In spite of strong
opposition from leading scientists of yesteryears (‘oxphos wars’ fame), the chemiosmosis idea
came into vogue only because the implicit unrealistic theoretical assumptions were not
identified. (iii) Several disconnected observations were unfortunately misinterpreted as supports
for chemiosmosis or pmf. (iv) Whenever theoreticians or experimentalists discovered flaws, such
aspects of chemiosmosis proposals were conveniently morphed over time. (v) There exists no

consensus amongst its advocates nor is there any substantial evidence to support it even today.
(vi) The structure-function correlations of the pertinent proteins and even the fundamental
composition of reaction milieu do not agree with the ETC-pmf-CRAS proposal. (vii) Since
researchers of the past got tired of searching for enzyme-linked “high energy phosphorylating
species” like that of the erroneous proposal of phosphohistidine (Boyer, 1963), ‘chemiosmosis’
soaked into the parched community. and (viii) The DRS-based mechanistic eluded the scientific
quests because of aesthetic stigma/disposition that DRS would be too chaotic and non-selective.
In spite of the critical developments over the last five years, it is a pity that data in
biochemistry/medicine and philosophical quests in biology are ultimately brought under the
purview of pmf/chemiosmosis. Therefore, it is imperative to reorient perceptions and seek better
explanations for life’s powering principles. To begin with, examples of some fellow living
beings are cited, to weigh in on this debate and cast their veto votes against the ETC-pmf-CRAS
bioenergetic proposal.
A. With ETC concept, cyanobacteria cannot respire and photosynthesize within the same cell.
Prokaryotic organisms such as cyanobacteria do not have the luxury of organellar demarcations
and yet, they carry out oxygenic photosynthesis and aerobic respiration, and both processes are
highly interconnected (Lea-Smith et al., 2016) and generate ATP (Figure 2). The cyanobacterial
system cannot be explained within the classical perspective of bioenergetics with ideas such as:
exciton relays/transfers, electron transport chains (ETC, like Z-scheme), chemiosmotic rotary
ATP synthesis (CRAS), proton motive force (pmf), cycling proton-pumps, etc. Here is why: The
purported membrane-bound ETC complexes for respiration (Complex I, Cyt. b6f, and Complex
IV) and photosynthesis (Photosystem II, Cyt. b6f, and Photosystem I) have the common
component of Cyt. b6f. In the respiratory ETC, PQ (plastoquinone) receives electrons from
Complex I and transports it to Cyt. b6f whereas in the photosynthetic ETC, PQ receives electrons
from PS II and takes it to Cyt. b6f. With the ETC concept, we have a sort of ‘metabolic
zugzwang’ here: electrons from the respiratory substrate of NAD(P)H can go into making the
photosynthetic product of NAD(P)H. This consideration undermines the deterministic circuitry
based outcomes afforded by the classical paradigm. The quinone-recycling protein Cyt. b6f
purportedly gives electron to Cyt. c6, which could then pass it on to either Complex IV or
Photosystem I. Now, Photosystem I’s electron deficient state is a highly transient one and it is

unlikely that the few molecules of Cyt. c6 present could ‘voluntarily decide’ to not pass on the
electrons to Complex IV. Since Cyt. c6 molecules cannot go up and down the two ETCs at the
same time, NAD(P)H synthesis would become an unlikely event in daylight. Again, this is
because electrons coming in from the photolysis of water from PS II would go into the
downstream water-formation at Complex IV, rather than the upstream formation of NAD(P)H
via PS I. Clearly, such an ETC-based metabolic scheme, which necessitates several dozens of
serial electron transport steps (among ~20 odd redox-active participants) for the formation of a
simple molecule like NAD(P)H and cannot prevent the futile drainage of redox power in the
respiratory-photosynthetic cycles, cannot evolve spontaneously! Recent publication from our
group have shown new mechanistic routes for electron transfers in such systems which
necessitate us to outgrow the concepts such as ETC (Gideon et al., 2020, Gideon et al., 2021).
Very importantly, the ETCs and proton pumps working logic is compared in the right panel of
Figure 2. As seen, the Mitchellian proposals violate fundamental concepts of electrochemistry
and feasibility for getting a working/workable chemical system. There is no evidence to say that
the ETCs of the two anti-parallel metabolic schemes are arranged distinctly or separately in the
cyanobacterial thylakoid membranes, in the necessary sequence and orientations. There is no
evidence whatsoever that recycling proton pumps could work in such a system, as the structure
of cyanobacteria like Prochlorococcus merely shows the respiratory/photosynthetic pigments
strewn around on the thylakoid membranes (Partensky et al., 1999).
Figure 2: Left panel- A simple schematic of the anti-parallelism of respiration and photosynthesis in the cell of a
cyanobacterium like Prochlorococcus. While both processes produce ATP and generate heat (not shown),
respiration consumes oxygen whereas photosynthesis liberates oxygen. Right panel- The electrochemistry of
cyanobacterial thylakoid membranes (in yellow) with the ETC of photosynthesis and respiration is shown in the
left and compared to a simple electrochemical setup on the right. The direction of proton pumps is shown in white
arrows and the direction of electron transport across various phases is shown in line arrows. The ETC and proton
pump loops seek the impossible premise of steady state movement of electrons in haphazard manner with respect to
the potential at one hand, and movement of protons too in bidirectional ways across the same membrane! Further,

while electrons rise in the cytoplasm and sink therein itself within the respiratory ETC, the photo-electrons are
sourced from lumen and sunk in cytoplasm. Quite simply, these loops and routes violate all known concepts of
electrochemistry and working logic of chemical systems.
B. Proton pumping/fluxes are challenged by organism/organelle dimensions
Prochlorococcus is a coccoid (sphero-cylinder) photosynthetic organism, of approximately 0.6 x
0.4 x 0.4 micron dimensions. The amount of free (dissociated) protons in any such a cell would
be = (volume of cell) x (concentration of protons) x (Avogadro’s number) = (0.085 x 10-15) x
(10-8) x (6.023 x 1023) = ~0.5! Within a cyanobacterium, the number of photo-respiratory
proteins would be ~105 and the cycling proton pump concept would require at least two orders
excess of this amount for efficient chemiosmotic cycling. Similar calculations for normal sized
mitochondria show the intra-organellar availability of only 5-6 protons. If someone argues that
Le-Chatelier’s principle or buffering would supplant the necessary protons, it would imply the
denial of the fact that heterolysis of water requires >79 kJ/mol! Therefore, the concept of cycling
proton-pumps and proton motive force (pmf) fails, negating the ETC-pmf-CRAS proposal, and
wishing for such systems to violate laws of thermodynamics (Bal et al., 2012) does not support
scientific fervor. In stark contrast, murburn bioenergetic model recruits free electrons and
diffusible reactive species (DRS), which can explain the cyanobacterial system well.
C. Some aphids can respire and photosynthesize without complex ETC.
Alain Robichon’s group reported the light-induced electron transfer and ATP synthesis in a
carotene-synthesizing insect, Acyrthosiphon pisum (Valmalette et al., 2012). These animals
perhaps acquired the gene for carotene synthesis from plants via horizontal/lateral gene transfer.
However, they do not have photosystems and chlorophyll binding proteins or light harvesting
complexes arranged to function in elaborate ETC, such as the Z-scheme. In the insect,
carotenoids are found either distributed freely in hemolymph (attached to hydrophobic patches of
proteins) or bound to hydrophobic membranes of cells. In the classical view, carotenoids have
only photon-harvesting or exciton transfer roles in photosynthesis. In the murburn model of
photo-transduction, carotenoids can give out electrons and replenish them from various sources
in the photosynthetic cycle (Manoj & Jacob, 2020; Manoj & Manekkathodi, 2021). The
delocalized nature of NADH and ATP synthesis advocated by murburn model can explain the
outcomes in the aphid system whereas the classical model falls flat. The outcome observed in

aphids is akin to the observations of enhanced ATP-synthesis with low-level laser therapy
(LLLT) in humans. LLLT is associated with increased diffusible reactive oxygen species (DRS)
observed in these systems (Freitas & Hamblin, 2016) and it was also shown that the
photomodulation’s primary target is the mitochondrial water pool (Sommer, 2019). As per the
murburn model, upon photo-excitations, suitable chromophores (like hemes, flavins, melatonins,
etc. in mitochondria) of human cells can give out electrons (which gives rise to DRS). These
DRS, in turn, can directly aid ATP synthesis and the electron deficiency can be replenished from
other sources thereafter. This murburn perspective explains the overall observations.
D. The chemiosmosis perspective suggests distinct bioenergetic origins for archaea.
Along with the cell/protoplasm theory (cell or protoplasm is the basic unit composition of life –
we don’t consider viruses and prions as living beings!) and central dogma (genes code for
proteins and these two govern key perpetuating attributes of a living species), the bioenergetic
principle (chemiosmosis or murburn concept?) would form the three pillars of life on earth. In
this regard, it is interesting to note that archaea have very distinct bioenergetic membrane-
machinery. While bacteria/eukarya have fatty acyl chains esterified to glycerol-3-phosphate,
archaea have isoprenoid chains etherified to glycerol-1-phosphate. While bacteria/eukarya have
ubiquinone/plastoquinone with unsaturated isoprenyl chains, archaea have methazophenazines or
menaquinone or thiophene bearing quinones with saturated alkyl chains. The enzymatic
machinery for making these components is distinct and not connected/conserved (Sojo et al.,
2014). Archaea like Ferroplasma have a bolamphiphilic monolayer (Macalady et al., 2004) and
only a single bi-layer is present in bacteria (wherein the periplasm is in equilibrium with the
environment). These facts are incompatible with the ETC-CRAS requirement of two bilayered
membranes. Although Mitchellian postulates seek that both mitochondrial membranes present in
eukarya must be impermissible to protons, the reality does not meet the expectations. Further, it
is also known that the periplasm of prokaryotes equilibriates with the external environment and
thus, cannot support chemiosmosis (Manoj et al., 2019a). While it is believed that
bacteria/eukarya possess a conserved FoF1ATP(synth)ase, archaea lack the genes completely and
have a significantly different V-ATP(synth)ase. The structures of these two protein complexes
are significantly different and it is evident that they cannot work in an identical manner (under
the classical purview). Researchers have tried to reason out such grass-root level dichotomy with

LUCA, within the purview of chemiosmosis (Sojo et al., 2014). This is an impossible errand, as
the proposal of chemiosmosis is a thermodynamically unviable concept with no parallels in the
physical world outside the bioenergetic community dealing with mitochondria/chloroplasts.
Archaea are known to inhabit a wide range of habitats: some thermophilic species of
Methanopyrus can reproduce at 122 °C, halophilic species of Halobacterium can grow at NaCl
concentrations of 25%, alkaliphilic species of Natronomonas can thrive at pH values of 11 and
acidiphilic species belonging to Picrophilus can live at pH 0. Under the chemiosmosis purview,
the key players are “smart membranes/proteins” and they must function in cooperative unison
across these diverse habitats to maintain pumps and gradients of H+ (and/or Na+, as this ion also
seems to have come under the purview of chemiosmosis now). Any skeptic would question this
premise because protons are known to have critical equilibriums with various components in
milieu and proteins can easily change their attributes in such variable ambiances. The
mitochondrial system is finicky, which changes its metabolic profiles with a few folds variations
of ionic strength, pH and temperature. Also, it is just a perception that biological membranes
(like that of mitochondria/chloroplast) are impermeable to protons. If it were, the Jagendorf
experiment (demonstration of ATP synthesis with an external pH gradient: erroneously taken as
support for Mitchell’s hypothesis!) would not have given any results! We know that protons
cross phospholipid membranes in millisecond time-frames (Biegel & Gould, 1981). Most
importantly- chemiosmosis requires the maintenance of events in three disconnected phases for it
to be operative; and it is not perceivable with any stretch of imagination how the archaean
habitats and infrastructure would permit this seemingly impossible mandate. It is easy to see with
the murburn model that organisms can recruit ‘various molecules – unbound ions – reactive
species’ interactive equilibriums for harnessing energy, across the diverse habitats.
E. Some proteobacteria show violation or irrelevance of ETC-pmf-CRAS mechanism.
The genes of ATP(synth)ase are considered obligatory and conserved in bacteria/eukarya.
However, the genes of this protein complex are located clustered at two different/distant loci in
Rickettsiae prowazekii, which signifies its low evolutionary significance for the organism
(Andersson, 1998).

In aphids, the proteobacterial strains of Buchnera aphidicola reside as endo-symbionts in
specialized gut cells called bacteriocytes. A Buchnera strain isolated from the aphid Cinara cedri
showed the absence of all the eight genes of FoF1ATP(synth)ase (Charles et al., 2011). It is
difficult to think that the aphids-bacteria symbiotic association dating back more than a hundred
million years could be viable for the two species if one of them was an energy parasite over the
other. Also, it is difficult to see how these bacteria could take in ATP from its host when they
have three membranes separating them, have little ATP-dependent transporters and their cells are
present in several layers (Perez-Brocal et al., 2006). These findings suggest that the bacterium
must have some efficient ways to synthesize ATP, that is independent of FoF1ATP(synth)ase.
Proteobacteria comprise of a wide variety of organisms with an equally wide metabolic
repertoire, and they are also found as extremophiles, with respect to temperature, pH, salinity and
nutrient requirements (quite like the archaeans). They could be chemolithotrophs, phototrophs,
heterotrophs, etc. or aerobic and anaerobic or free-living and parasitic/pathogenic, etc. While we
can consider the archaeans’ unique structural components to aid their adaptability to extreme
environments, the proteobacteria have only the usual biochemical options available to help them
in the extreme environments.
The metabolic diversity of one campylobacterium species is presented here, with respect to the
nature of electron acceptors usable in the respiratory reaction. Sulfosprillum multivorans (van der
Stel & Wosten, 2019) can use a wide array of species like: neutral diatomic gas like oxygen;
organic anion like fumarate; halocarbons like perchloroethene and trichloroethene; neutral or
charged organics like dimethyl sulfoxide and trimethylamine oxide; inorganic anions like
perchlorate, nitrate, nitrite, tetrathionate, arsenate, selenate, thiosulfate, etc. These species have a
wide range of redox potentials, have various molecular dimensions/geometries, topologies, bond-
rotation abilities and electrostatics/H-bonding, partitioning effects, etc. How could such diverse
species be bound at the active site of the protein to relay multiple electrons to it? (The mandate
of passing on four electrons to molecular oxygen to make two molecules of water is referred
here! Can one imagine the same fate for all these molecules, with the same protein or with new
protein for each case?) Also, it is impossible to imagine that such an organism can accept
electrons from a wide range of anionic or neutral organics (like formate, pyruvate, lactate,

ethanol, glycerol, etc) and inorganics (gas like H2S or ion like sulfide, etc.) through some
unthinkable specific affinity-based mechanisms and pass it on to the diverse final electron-
acceptors through the affinity-based mechanism yet again! All this, only to deterministically
relay the electrons through an elaborate scheme of ETC to generate a non-harvestable and non-
existent pH gradient! While the ETC-CRAS proposal cannot explain the diversity of metabolic
repertoire, the chemical reactivity based rationale of murburn concept (which does not
necessitate a binding-based recognition of a protein’s active site) accommodates the versatility.
F. Some eukaryotic protists lack parts/whole of FoF1ATPase/mitochondria altogether.
Several ciliated pathogens belonging to the Apicomplexa and one strain of free living oxymonad
Tetrahymena thermophila were found to lack key components of the Fo module of the Complex
V (Balabhaskaran Nina et al., 2010). Plasmodium berghei is a malarial parasite in rodents which
does not possess Complex V in the blood-stage of its life cycle (Sturm et al., 2015). Although
several eukaryotes were known with mitochondria related organelles (MROs), the first true
microbe lacking any mitochondria or MROs was first reported in Monocercomonoides (a tetra-
flagellated protist found in respiratory tract of insects and vertebrates) few years ago
(Karnkowska et al., 2016). Therefore, there are several examples of single-celled eukaryotic
systems that show ATP synthesis without the essential components of ETC-CRAS mechanism.
In the murburn model (Manoj et al., 2019a-b), Complex V is not the ATP-synthase and
therefore, these reports are accommodated.
G. The multicellular cnidarian Henneguya lacks the ‘ETC/proton-pumps’ of chemiosmosis.
Recently, it was reported that the multicellular cnidarian parasite of salmon (living in low-
oxygen conditions), Henneguya salminicola, does not possess mitochondrial Complexes I, III &
IV (Yahalomi et al., 2020). These are the purported proton pumps of the ETC-CRAS proposal.
Since murburn concept can explain ATP synthesis even with Complex II (which is not believed
to enable trans-membrane proton pumping) and generate DRS with other heme/flavin proteins of
the milieu, the ATP synthesis in this multi-cellular organism can be explained. Since Complex II
is not considered to be a proton pump, the physiological functioning of Complex V is rendered
redundant in this organism, thereby annulling the ETC-CRAS proposal.

H. Alkaliphilic bacteria speak against proton gradient as bioenergetic rationale!
Alkaliphilic bacteria (which also move around aided by flagella) live in environments of pH 11-
12 (Krulwich, 1986; Aono et al., 1992; Olsson et al., 2003; Padan et al., 2005; Preiss et al.,
2015). Since it is known that the periplasm equilibrates with the external environment (Wilks &
Slonczewski, 2007) and since these bacteria have an internal pH of 7-8, the physiological
premises in such organisms counter the proton-gradient centric rationale for bioenergetic
phosphorylation and flagella-aided motility (Manoj et al., 2022). The pmf-primed and ion-flux
fixated community interpret such organisms as “Na-gradient” utilizing systems (which would
imply that the bacterial flagellar module uses proton-centric functionalism in acidic pH and
sodium-centric functionalism in alkaline pH!) or some have even introduced yet other terms into
the flawed Mitchellian equation (Saeed & Lee, 2018) to offset such issues.
I. Explanation for electro-mechanical activities
The ETC-CRAS mechanism connects electrical activity with a cyclic logic and does not include
1e-active species as contributors to TMP. Also, the mechanical activities are not direct connected
to the redox processes within the cell. In contrast, the murburn concept perspective affords a
seamless integration and interconnectivity of powering redox processes with coherence,
theremogenesis, homeostasis, electrical and mechanical activities (Manoj- group’s works cited in
this paper).
J. Comparison & verifiability of the CRAS versus murburn bioenergetic models
Besides the above quoted naturally occurring life forms, a plethora of mutation and knock-out
studies carried out in various prokaryotic and eukaryotic experimental models show results that
cannot be accommodated within the ETC-pmf-CRAS proposal, which definitely appears to be
‘irreducibly complex’ (an anti-evolutionary and pro-intelligent design coinage by Michael Behe,
1996). Quite simply, the Keilin-Mitchell-Boyer idea requires each and all of the components of
the mitochondrial protein complexes to be present for oxidative phosphorylation and there is
little way that such a system could evolve. However, reality is quite otherwise, as shown in the
multitudes of examples earlier. Even otherwise, there are other examples: E.coli with mutated
and dysfunctional Complex V survived (Klionsky et al., 1984). Complex I deficient mutants
survive in Arabidopsis thaliana (Fromm et al., 2016) and the European mistletoe (Viscum album)

lacks Complex I altogether (Senkler et al., 2018). Mutants lacking plastocyanin and cytochromes
c553/f can live almost like the wildtype plants/cyanobacteria (Zhang et al., 1994; Fernandez-
Velasco et al., 2001; Pesaresi et al., 2009). Therefore, the ETC-pmf-CRAS proposal fails the
definition and mandate for a valid (non-redundant and non-superfluous) and universal/ubiquitous
bioenergetic routine logic. Proton-gradients that could give any meaningful rightward tilt to the
equilibrium (ADP + Pi + H+ → ATP + H2O) don’t exist in physiology and therefore,
chemiosmosis is a mirage. The classical active site binding mechanism fails to explain the small
lethal dosage or acute toxicity of cyanide (Manoj & Soman, 2020). The chemiosmosis model
does not provide any viable exhaust logic or explanations for thermogenesis/homeostasis
whereas the murburn model is comprehensive in its package (Manoj/Manoj et al., 2018-2021). It
does not make sense evolutionarily that substrate phosphorylation seems to have higher
efficiency than the classical model for oxidative- or photo- phosphorylations; and yet, nature
chose the latter over former. The evolutionary significance of DRS in plant physiology is
underlined by the fact that leaves showing high rates of photosynthesis shed/die quicker than the
rest (Matsuki & Koike, 2006). Further, molecules like NADH and ATP would be needed quickly
and at high demand in cells, which the chloroplasts are mandated to furnish. It does not make
evolutionary purpose that a simple hydride transfer to NADP+ or phosphoryl transfer to ADP
would require several dozens of electron transfer steps, as mandated by the ETC concept. One
would expect evolution to work in the reverse way: to simplify the bioenergetic chemical
reactions, and not to increase the steps and components! The reaction logic of murburn model of
bioenergetics requires only: (a) a molecule/ion (like NADH or sulfide) with low redox potential,
serving as a source of electrons + (b) a d-orbital/π-electronic system to serve as catalyst (like
heme or flavin) + (c) a molecule/ion (like O2 or Fe3+) that can accept electrons. The ETC-pmf-
CRAS proposal requires not only the above items (a) through (c), but also: (i) several
sequentially acting electron donor/acceptor proteins/molecules embedded in a closed
phospholipid membrane, which is in turn enclosed in yet another membrane + (ii) electron
transfer/transport catalysts working in intricately woven deterministic pathways across three
different phases, recognizing each other with topological affinity + (iii) proteins that can couple
electron transfer to trans-membrane proton pumping and TMP generation + (iv) the mutually
exclusive options of ‘high concentration of protons in a given phase’ to serve proton pumps and
‘low protons in the same phase’ for generating TMP + (v) sophisticated membrane-embedded

molecular motors triggered by protons/TMP + (vi) intelligent membranes that regulates the
in/out flux of several components including protons + (vii) molecules behaving in ways alien to
their inherent nature (oxygen must bind only at Complex IV and remain bound until it receives
four protons and four electrons, Complex V must work as an ATPsynthase against the mandate
of equilibrium and its own nature-destined disposition as an ATPase, etc.), (viii) the overall
system must violate the laws of thermodynamics and generate energy from endergonic
processes, and so on and so forth. Therefore, the murburn model makes the Okham’s razor cut.
While Michael Behe called it correctly that the ETC-pmf-CRAS mechanism is irreducibly
complex and cannot evolve spontaneously, the realities do not mandate that there is any
‘deterministic creative hands working behind the scenes’ either. To our knowledge, the known
structural aspects on mitochondria and its components fit well within the statistical reaction
mechanism offered by the stochastic murburn paradigm, which gels well with the consideration
that life evolved from chaos. While murburn concept’s principles are ubiquitous and well-
grounded, the ideas of chemiosmosis, ETC, pmf, rotary ATP synthesis, etc. violate fundamental
notions of chemico-physics and are unheard of in any realms other than bioenergetics. A
comparison of the two models is made in Figure 3. The ETC-pmf-CRAS model cannot justify
the wayward locations of several redox centers in mitochondrial proteins (or photoactive centers
in chloroplast complexes) of inappropriate redox potentials. Also, the large bulky protrusions
with/without redox centers and multiple ADP binding sites are inexplicable in its purview. The
ETC-pmf-CRAS mechanism is analogous to a man-made hydroelectric power generation system
or automobile whereas the murburn model functions similar to a spontaneously formed hearth or
nuclear reactor (Manoj, 2018a-b). DRS mediated formation of peroxide in the murburn scheme
allows the synthesis of ATP at mM levels (Manoj et al., 2019b), which, in turn, enables ATP-
hydrolysis mediated endergonic physiologies. In this context, it is imperative to register that a
steady trans-membrane potential (TMP) is not the directive/causative principle of oxidative-
/photo- phosphorylation, it is the consequence of the same. In the era before the popularization of
ETC-pmf-CRAS mechanism, researchers had explored other modalities of phosphorylations in
prebiotic systems (Schwartz & Ponnamperuma, 1968). It is natural to imagine that murburn
mechanisms would have been prevalent in the ancient times too, as it is even today in biospheres
(for example: the degradation of lignocellulosics and recycling of halogenated organics) (Manoj,
2018b). Also, the formation of peroxide is not a mere wasteful/toxic reaction (as deemed by the

classical perspective), as it can further be recycled for peroxidative phosphorylations. Contrary to
popular perceptions, recent research has unraveled that peroxide formation is facile from water
(Lee et al., 2019) and it can spontaneously exist in equilibrium with superoxide and hydroxyl
radicals in water (Stoin et al., 2013). Further, not just the highly recognized NO, peroxide is also
nowadays seen as a key molecular messenger (Di Marzo et al., 2018; Rashdan & Pattilo, 2020;
Tena, 2020). Therefore, murburn model offers a tangible and comprehensive chemico-physical
explanation for the bioenergetics of life. This is when pmf has no basis in physiology, as cells
and organelles function with little proton gradient and some even work against the required
proton gradient. Many such cells do not even have protons, so as to build a pmf!
Figure 3: A comparison of the proton/TMP-centric ETC-CRAS and the oxygen/DRS-centric murburn model for
bioenergetic phosphorylations. On the left is shown the ETC-CRAS elements, which includes a closed cycling of
protons to build TMP and harness the same via the synthesis of ATP at Complex V. There is no direct chemical
connectivity between the electron transfers or TMP formation or ATP-synthesis. On the right is the essential
features of murburn model, which involves the activation of oxygen via redox center on the proteins (denoted by
star, on Complexes I through IV), which enable effective charge separation (ECS) and substrate presentation (ADP
bound on the protein). The DRS produced in oxidation of redox equivalents directly activates ADP/Pi, and thus
leads to the formation of peroxide in milieu, along with ATP and water. In this model, Complex V serves as pH/ion-
chemostat. Formation of radical species (hydrogen atom, superoxide, hydroxyl, etc.) would be facile and they could
directly mediate the coupling of the oxidative process with the phosphorylation group transfer activation chemistry.
(For convenience, although ATP formation is shown as a condensation of two radicals, the actual reaction may
proceed with the formation of a phosphate radical, which could attack ADP, to for ATP and liberating a DRS.)
While advocating the new theory, we have faced majorly two dismissive comments over the last
four years. (1) “ROS is chaotic and will mess up life in an uncontrollable way. So, it is unlikely
that such an uncontrolled reaction can give anything meaningful.” (2) “ROS is only in oxic
systems. Since anoxic systems evolved before oxygen concentration became higher in earth’s
atmosphere, the murburn model is irrelevant.” At the outset, it must be said that such comments
stem purely from aesthetic disposition or inadequate awareness on the new theory. Most

importantly, the first objection statement is invalid because the existence of ROS is a reality of
cellular functioning and its presence cannot be wished away. If ROS were only toxic wastes,
evolution need not have resorted to one-electron active agents that ensure ROS production. Safer
two-electron alternative modalities like substrate-level phosphorylations alone could have been
used. But the observed realities and theoretical premises mandate the utilization of DRS in life
processes. The murburn model is a simple chemical theory well-founded in thermodynamics,
kinetics, probability, mechanistic, and evolutionary considerations. An example of selectivity of
radical mediated oxidative reactions is when a cloth dipped in water+oil or a paper dipped in
water+alcohol gets burnt. The fabric/paper gets charred only when the oil/alcohol supply runs
out. Similarly, the collateral damage occurs over long periods in cellular systems that employ the
murburn principles. Also, the fact that there are several DRS utilizing enzymes in the cells
speaks of their utility, not of their danger (Jacob & Manoj, 2019)! A suitable analogy is- if we
find gloves, cutting boards and knife-stands and knives in the kitchen, it does not mean that
knives are weapons of combat that are unused in kitchen. Also, quite contrary to perceptions,
redox enzymes are not easily denatured by DRS and could be stable to them (Manoj & Hager,
2001). DRS mediate chaotic reactions only when present in large amounts at a given locus (at
any instant), whereas they are very selective/specific in discretized generation and sustained
release scenarios (Manoj, 2006; Manoj et al., 2020a). If a highly unstable DRS like nitric oxide
can evolve to carry out the agenda of sensitive molecular messenger, why cannot other such
species serve positive roles in routine physiological scenarios? The perception that ‘DRS are
dangerous!’ is a preconceived disposition, and lately this perception is changing, with more
constructive functions emerging for the same (Mittler, 2017). Although, there is some collateral
damage, the pros of the spontaneous and stochastic murburn scheme far outweigh the cons.
Further, shift from anoxic to hypoxic or oxic environments (or vice versa) would be facile with
the murburn bioenergetic principles (Manoj & Bazhin, 2021), as the functionalism is dependent
primarily on stochastic interactions/reactivity and is not contingent upon deterministic outcomes
based on intermolecular recognition/affinity. While the formation of oxygen-centered radicals
would be facile in oxic systems, there could be H/N/S centered reactive species in the anoxic
systems. This gels well with the presence of quinones in oxic systems in contrast to molecules
like methanophenazines in anoxic methanogenic archaea (Schoepp-Cothenet et al., 2013). While

the ETC-CRAS is a deterministic and essentially multi-molecular proposal, murburn model is
based on stochastic/statistical principles and is bimolecular at the core. Thus, it can better explain
the spontaneous origin from chaos, as the analogy it offers is akin to a hearth or spontaneous
nuclear reactor (Manoj, 2018b). DRS mediated reactions can afford temporal
selectivity/specificity and thus, sustenance amidst chaos (Manoj, 2006; Manoj et al., 2020a).
Continuous evolution and trait differentiation through generations is aided by virtue of mutations
and epigenesis induced by DRS. Eventually, termination of life occurs due to the accumulation
of unfavorable collateral/side reactions introduced by DRS, and thereby life ceases and
disintegrates, returning to the larger domain of chaos. It is quite rational to consider that chaos
can support a limited reign of order within, as seen manifested in fractal structures of several
biological systems. If life can originate from/within chaos, it is logical to consider that chaos
would have favorable elements for life-sustenance too! That is: several spontaneous reactions or
stochastic processes that occur in a cell may be favorable to life, and murburn concept can be
deemed as a compendium of the hitherto lesser considered (but favorable!) aspects of DRS
mediated outcomes. It is evident (and also partly accepted now) that DRS also enabled trans-
phase or organelle-organelle or cell-cell communications, thereby leading to systemic coherence
or multi-cellular assemblies and control systems.
Murburn model is the only standing mechanistic proposal that can explain the dosage, kinetics,
thermodynamics effects of low levels of cyanide, which generates an acutely debilitating impact
in the porphyrin/flavin mediated physiologies within membrane systems, including aerobic
respiration and oxygenic photosynthesis (Parashar et al., 2014, Manoj et al., 2016b-c; Manoj &
Soman, 2020; Manoj et al., 2020a; Manoj & Bazhin, 2021). It is now imperative that life
scientists address the quantitative arguments that support the murburn model, as it mandates an
overall one-electron chemistry (for effective charge separation), which justifies the presence of
several one-electron redox active agents on membrane-embedded proteins. The murburn model
successfully explains why Complexes I-III have long matrix-ward pendulous arms with redox-
active centers, whereas the ETC-CRAS model cannot justify the evolutionary mandate for large
apoprotein bulbs with multiple redox centers outside the trans-membrane region (Manoj et al.,
2019a-b). Also, super/mega-complex or aggregate formation of redox proteins on endoplasmic
reticulum or mitochondrial or chloroplast membranes and multimeric nature of the soluble

hemoglobin are not explained within the classical perspective. Murburn concept explains these
structural evolutions as stochastically selected events that enabled the proteins to better peruse
the DRS generated in each other’s vicinity (Manoj et al., 2019a, Parashar & Manoj, 2021,
Parashar et al., 2021; Manoj et al., 2021b; Manoj & Gideon, 2022). Another success of murburn
concept is explaining the ‘bioenergetics-coherence-homeostasis’ paradigm for red blood
corpuscles or erythrocytes, which form a major portion of human cellular counts. Lacking
mitochondria and nucleus, they function for ~four months and sustain mM levels of ATP.
Calculations show that glycolysis cannot explain such high concentrations of ATP and there is
structural evidence to project that the outcome could owing be to hemoglobin serving as an ATP-
synthesizing murzyme (Parashar et al., 2021). Yet another success story of murburn concept is
the explanation of lactate dehydrogenase function, as to why blood transports a major portion of
lactate to liver for recycling (Manoj et al., 2022g). We present some simple and direct agendas
that delineate and ratify the murburn model for bioenergetic phosphorylations (particularly,
within the lesser complicated and experimentally facile aerobic system):
1. Provision of NADH/oxygen would lead to an increase of DRS in the system. This
observation would be an anti-thesis to the evolutionary relevance of ETC-CRAS model.
2. Increase of DRS within the system would correlate to increase in TMP/ATPsynthesis.
Such an observation would correlate TMP build-up as an outcome of ECS and DRS production
and ATP synthesis within mitochondria; and not the other way around!
3. In situ/in vitro, provision of DRS could lead to the formation of ATP (from ADP/Pi).
This observation would directly implicate DRS in ATP synthesis, while it would remain
inexplicable how TMP can be tapped to give rise to an O-P bond in ATP.
4. As the model involves DRS, it is destined to be stochastic, non-integral and variable
reaction outcomes are expected. This justifies the murburn equation of oxidative
phosphorylation: NAD(P)H + H+ + O2 + nADP + nPi → NAD(P)+ + H2O2 + nATP + nH2O. 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.
5. Each one of the Complexes I-IV would have DRS-production abilities. Such an
observation would be incompatible and inexplicable with ETC-CRAS model.

6. Each one of the Complexes I-IV would have ADP-binding sites. This is a necessary
mandate of murburn model, whereas it is a superfluous aspect in ETC-CRAS.
7. Provision of ADP+Pi would increase NADH/O2 consumption and DRS/ATP production.
In the ETC-CRAS mechanism, since there is no direct coupling or thermodynamic pull concept,
presence of ADP+Pi does not affect the ETC. Therefore, this is a simple way to delineate the two
proposals.
8. ATP synthesis can be achieved or demonstrated in vesicles/mitochondria/bacteria without
pH gradient and even countering the necessary pH gradient. This would demonstrate the
physiological irrelevance of equilibrium-assisted synthesis via the equation: ADP + Pi + H+ →
ATP + H2O.
In our earlier publications (e.g. Manoj et al., 2019b), we have listed 30 comparative agendas
where the murburn model pips the pmf narrative. Regardless, this article lists 8 distinct agendas
that disclaim the pmf-based viewpoint and 8 points of ratifying the murburn model. In other
instances wherein paradigm-shifting explanations have been given (e.g. photosynthesis,
xenobiotic metabolism, vision, electrophysiology, etc.), the pertinent publications make dozens
of comparative points that show the trumping nature of murburn proposals. Quite simply, the
larger picture is that most cellular organelles and systems work under physiological pH, with
very little pH gradients. When a lipid vesicle or organelle occludes a miniscule aqueous volume
within itself, it could have the same pH but with the murburn activity inside, consumption of
extraneous protons might occur (owing to proton deficiency arising due to chemical reactions,
and not due to any gradient resulting out of pumping). Now, this consumption of protons cannot
be conveniently branded as pmf (which is a mere flux of protons owing to a gradient) in yet
another shape-shifting exercise, which would be a travesty of all boundaries!
4. Origin and unifying principle of the murburn mechanism
In the anaerobic environment of the young earth H2, CH4, NH3, HCN, H2O, and H2S were
perhaps the main molecular species to start the organic pot leading to the living world, with
phosphate getting involved only at later times. In the intermediate metabolism of current-day
cells carboxyacids, aminoacids, hydroxyacids and ketoacids take a central position. They all

contain oxygen atoms. In pre-biotic times the O-atoms could be sourced from water, as it is still
today in all photosynthetic systems known. Oxygenic photosynthesis is highly endergonic and
needs light for photo-reduction and photo-phosphorylation. Under the earlier anaerobic
conditions, as atmospheric oxygen was lacking, oxidation of water was needed to synthesize the
simple organic acids mentioned above. A probable candidate is Fe3+, also because it still forms
the active component in a large number of present-day cellular redox systems. Under the early
reducing conditions Fe2+ would have been the stable form. Aided by UV (during the day), it can
easily photo-oxidize to Fe3+, making the conditions during the day more/slightly oxidative and
during the night reducing. Timoshnikov et al. (2015) demonstrated that Fe3+ reacts with water in
presence of light, forming Fe2+ + H+ + HO*. The HO* can serve as one of the products necessary
to introduce oxygen atoms into carbon-containing compounds. Two HO* radicals may also form
H2O2, which can react with the Fe2+/Fe3+ mixture leading to other radicals, which could originate
the first organic compounds and thereafter chemical interactions leading to life. Iron-sulfur
clusters of different constitution now found in ferredoxins might have been the early catalysts.
They are the reduction equivalents with the lowest potential in present-day metabolism, and the
relevant protein structure in anaerobes such as Clostridium pasteurianum is very simple, derived
from a repetitive tetrapeptide (Eck & Dayhoff, 1966). The above reasoning puts oxygen-
containing radicals derived from water at the oldest and the very centre of metabolism, which is
also the essence of murburn concept. Therefore, the murburn bioenergetic principles have
groundings from the earliest prebiotic times, initially as a free process in the sea or some aqueous
pools. Once Fox-like microspheres came to existence, they could act as an interesting micro-
environment, a first start of ‘muring’, which could be further narrowed during later evolution.
During the early period, presence of a membrane is not required. Figure 4 presents a time-line
for the origin of major molecular species, and principles/processes considered ‘pillars of life’.
Thus, the earliest incorporated Fe-containing systems and heme (and also other extended pi-
electron containing organics) could serve as a melting point of 1e murburn reactions. Products
that could serve many purposes are particularly advantageous at different stages of evolutionary
build-up, serving to interconnect processes. For instance, ATP got elaborated to serve as a
universal energy currency, and as a building block (together with other nucleotide-tri-
phosphates) for RNA synthesis, and later indirectly for DNA-synthesis. The ATP (NTP) “energy

currency” would enable cells to achieve some non-spontaneous objectives (and to act with a
“will”). The easy to obtain NTP’s allowed the Central Dogma to come into existence later on.
Figure 4: A simple rendition of the origins of cellular life and its various operating principles
In the current context, it can be understood that murburn concept (in the short time scale and
with stochastic outcomes) and central dogma (in the longer time scale and with deterministic
mandates) interlink the six essential physiological requisites of a cell (the six vertices of the
hexagon shown in Figure 1). Earlier, these aspects were not seen to closely inter-connected and it
was all left to the control of central dogma. Physically, coacervation is a key observable
nature/feature of cell and chemically, catalysis is a cell’s essential attribute. Murburn concept is
an integrated representation of the elements of chaos that supports life (and quite paradoxically,
it is also the reason that all living beings must die), which enabled the origin, evolution and
sustenance of life. Central dogma comprises of the deterministic and directive principles (DNA
encodes the information for RNA, which in turn codes for proteins) that ensure affinity or
topography-aided catalysis. It was not the most important criteria during pre-biotic times but it is
DETERMINISTIC ORDER
Aerobic Cell
O2
Anaerobic Cell CELL THEORY
DNA CENTRAL DOGMA
RNA, Membrane lipids
TIME
Polypeptides / Oligonucleotides
Amino acids / Bases / Sugars CATALYSIS COACERVATION
MURBURN Random chemistry
Small molecules/ions: CH4 CO2 CO C1 H2/H H2O NH3 H2S Pi Fe2+/Fe3+ …
CHAOS BUILDING BLOCKS OPERATING PRINCIPLES

the most salient and governing feature of life today. (A demarcating boundary is not conceivable
during prebiotic times but now this is a definition of life.)
The cell theory, as generally taken, implies a higher focus on the membrane and does not pay
tribute to the coacervate nature of the cell and biologists erroneously perceived cytoplasm as a
simple dilute aqueous solution. To correct this perspective, coacervation must be recognized as a
fundamental prebiotic event that was necessary for the realization of life (Figure 5). Quite
simply, if cytoplasm behaved as a simple solution, we would not have had life because some
necessary aspects of murburn concept like ECS and partitioning effects would not have
materialized. Therefore, the cell theory must get a fourth attribute that cells are membrane-
bounded coacervates capable of catalysis (Figure 5). This measure pays tribute to the
marginalized scientists who were on the right tracks! To explain the mechanical displacements, it
is important to accept that water does not merely serve as the cellular solvent, but also as a
physical actor organized in discrete phases in cells in a manner that permits both ordering and
mobility. With respect to the roles and properties of water, Denys Wheatley’s (2003) diffusion-
perfusion-exclusion model is a significant way of seeing the dynamics in cells.
Figure 5: The difference in the classical and coacervate nature/perception of cells is shown. In the classical view,
water behaves as the same everywhere within the cell. The coacervate view recognizes multiple phases and ordered
arrangements of water in the cytoplasm, in and around organelles and proteins. Some water molecules may be
highly mobile whereas others may be relatively bonded to form ordered structures with other molecules and ions.
Most biologists believe that life originated from precursor stages that could form by random
chemistry upon which chemical selection could act on behalf of affinity and longevity/stability
of formed products and larger assembles. Some aminoacids, hydroxyacids, ketoacids, carboxylic
acids, nucleotide bases and sugars (like ribose) are relatively easy to obtain in vitro under

prebiotic (anaerobic) conditions, whereas phosphates were present as such. Other kind of sugars,
and particularly phospholipids are more troublesome to obtain. With a limited number of the
easiest to synthesize amino-acids (Ala, Val, Gly, Pro, Asp, Glc) short oligopeptides or proteoids
can be obtained which resemble Fox-microspheres (deDuve, 1991), i.e. extended coacervates.
Such coacervates demonstrate remarkable properties that are akin to cellular features: unitization,
physico-chemical growth and budding, preference for accumulating K+ over Na+, colloidal
osmotic behavior, surface boundary potentials akin to action potentials, etc. It must be seen that
such facets are possible to achieve without ATP, nucleus, organelles, membranes, etc.! In this
regard, it is also interesting to read the ideas on intrinsically disordered proteins and membrane-
less organelles (Jaeken, 2017; Matveev, 2019;2022). Yet with respect to the origins of the
murburn mechanism, one has to look to the period before and ask the question how the
hydroxyacids, aminoacids, ketoacids and carboxylic acids came to existence.
Yet another salient aspects of the biomolecules that constitute life is that the major biopolymers
(whether proteins, carbohydrates, nucleic acids, or lipids) use the ester linkage [-(C=O)-O-C] as
a fundamental building block. In almost all these cases (between any two homo- or hetero-
combinations of amino acids, sugars, etc.), the thermodynamic premises favor hydrolysis over
esterification. That is water formation is favored in water; for the generic case (without
partitioning effects and in copious water):
R-(C=O)-O-Rʹ + H2O → R-(C=O)-OH + RʹOH; ΔG = -ve.
Therefore, the esterification reaction would be favored either with very low water activity nano-
realm and/or via the 1e catalytic route like that afforded by the murburn processes. Later, with
the provision of external oxygen, the reactions leading to water formation are bound to be even
more exergonic, thus forming an excellent powering source. Therefore, we are inclined to infer
that although a physical phase-separation phenomena like coacervation would be an important
step in pre-biotic processes (which could enable some partitioning effects, even without a lipidic
membrane separation), chemical murburn catalytic processes were an earlier necessity, enabling
coacervation and origin of life and its sustenance even today with core roles in routines like
respiration and photosynthesis (Figure 4). We believe that murburn concept also enables the
simplest explanation why the combination of lipid membrane + oxygen became so crucial for
generating the diverse forms of life; along with the closure of sub-micron dimensions that

minimized protons. Our efforts have clearly shown by now that murburn concept serves as the
primordial evolutionary pivot of life (as it explains several redox enzyme functions and
important biological electron transfer processes in crucial life sustaining routines of respiration,
photosynthesis, xenobiotic metabolism, electrophysiology, etc.). Murburn concept forms the
founding pillars for cells functioning as the building block of life and provided the ambiance
wherein central dogma could take shape in proto-cells. We can now understand that oxygen
became the choice of oxidant because of the greater speed in reactions afforded by its
intermediates and the facile modality in which the product of CO2 could be expelled (Figure 6).
Another added aspect is the practically zero-order chemistry of several DRS-mediated reactions,
which ensure steady conversion rate over even several decades of micromolar concentrations.
The murburn model of cell functioning as a simple chemical engine (SCE) by virtue of effective
charge separation (ECS) affords us a simple amalgamation of the basic processes (powering,
homeostasis, electrical activity, coherence, etc.). Here, it is opportune to point out that water in
cells must not be seen as ordinary water but must be seen as a discretized and heterogenous
phase, with rather fixed and mobile formats.
Figure 6: A simplified rendition of the murburn SCE (Simple Chemical Engine) model of cell function, which
depends on Effective Charge Separation (ECS) for cellular powering, thermogenesis, homeostasis, coherence,
electrical activity, mobility, etc. (Please refer Manoj et al., 2022h for more details.) A simple fuel (like methane) can
be burned or murburned in a simple chemical engine or cell (respectively) to produce water and carbon dioxide,
liberating heat. Since the product of carbon dioxide is a gas, it can be easily voided. Since the other product is also
the solvent, this leads to increase in water inside, which must spontaneously go out. In cells, this process leads to
waste disposals and homeostasis. It must be noted that murburn concept does not necessitate oxygen’s involvement;
however, it serves life in a highly efficient way with dynamic roles of oxygen. When oxygen gets reduced in
respiration or other metabolic processes, it transiently generates negatively charged species, which give electrical
activity to the cells, and this process also networks cell and enables its powering (ATP synthesis).
5. Summary and future prospects

A deeply embedded aspect of the classical paradigm of cellular physiology is that membrane
proteins can measure, recognize and control the cellular intake to deterministically set up
solute/ion concentration gradients and electric potentials over membranes. Purportedly, this was
for achieving cellular coherence and homeostasis by concentration and volume control. This is
exemplified by the cases for Na/K-pump, proton-pumping of respiratory and photosynthetic
redox complexes in inner mitochondrial, thylakoid, and bacterial cell membrane producing a
pump-dependent potential gradient for ATP synthesis, ion and volume control. This central idea
has been applied to explain rest/action potentials in excitable cell membranes according to the
Hodgkin-Huxley-Katz (HHK) model, and has influenced thinking about muscle contraction
according to the sliding filament hypothesis, and even about how bacterial flagella enable
bacterial movements. This perception has marginalized other models, and has not addressed key
issues such as that in erythrocytes, where glycolysis cannot suffice to power the Na/K-pump. The
membrane pump hypothesis (or MPH) was originally based on the erstwhile untenable
assumption that the laws of diluted ideal solutions could be used to calculate ion gradients. It
perceived that cellular water behaved exactly like normal water, without phase demarcations or
organizations. This simplistic perception is definitely outdated now because the coacervate
nature of cytoplasm is a non-questionable direct observation. Also, the proposal of deterministic
ion-pumping has been demonstrated to be a thermodynamically untenable proposal (Manoj et al.,
2022h). Therefore, the cell theory (which stressed overt importance on the membrane alone)
must be expanded to accommodate coacervation, so that the internal phases also get their due
importance. Further, it was shown here that murburn concept (Manoj, 2018) is a new
physiological pillar which has full relevance in modern day cells and also played primary roles in
the emergence of cellular life from prebiotic times. This new insight also settles that the
deterministic active site model of enzyme action as a functional mechanism of non-murzyme
proteins has a stochastic and evolutionary elder in the ’murburning’ murzymes.
Holistic biological rationales were provided to show why the classical ETC-CRAS model’s
pmf/TMP-driven phosphorylation mechanism is universally untenable. Also presented are
multitudes of living examples that lack the fundamental requisites sought by the ETC-pmf-CRAS
paradigm. Further, using comparative analyses, the agendas ratifying the murburn proposals have
also been delineated. Our recent works and re-interpretation of a vast amount of data available

from literature disclaim the Keilin-Mitchell-Boyer paradigm of ETC-pmf-CRAS. This
development sets the stage for a serious indulgence of the alternative presented in murburn
bioenergetics model. The murburn theory posits that redox proteins mediate ECS leading to
DRS/ROS, which are the true catalytic agents driving ATP-synthesis at membrane interfaces in
prokaryotes and within organelles (mitochondria/chloroplasts) of eukaryotes. ECS-based
mechanisms afforded by DRS enabled cells to function as SCE and allowed the origin of life,
and they continue to sustain life and serve as the driving forces for the evolution and termination
of life. Murburn activity enabled the channelization of thermodynamically facile one-electron
reactions for building life systems, on which the more selective two-electron and ionic processes
were built. The murburn activities in cell ultimately power the ionic fluxes and not the other way
around! The earlier “cyclic logic” perception was erroneous in that deterministic ionic fluxes
alone contributed to TMP (which drove ATP synthesis!) and that ATP utilization also enabled
ionic fluxes. The murburn purview correctly posits that murburn activity within cells can affect
TMP, but that ATP synthesis occurs independently and in parallel. ATP could be used to affect
ionic concentrations/fluxes thereafter. That is: TMP is a signature of redox activity inside, which
is the cause, with ion differentiation as a result. Since the earlier analyses focus only on ion
fluxes as the bioenergetic causative (and overlook the redox mandate completely!), they wrongly
prioritize the cart as the one driving the horse! Therefore, any work (including the movement) of
a living being, whether ancient or current would rest on the fundamental bioenergetic and
coherence mechanisms inherent within the cell. The generation/utilization of proton/ion
gradients (if any!) in/around the cell should be deemed secondary. That is, the de-facto
necessitation of an ion/proton gradient as the primary bioenergetic causation for ATP-synthesis
or motility is absurd, as such a mechanism would solicit: (i) a complex gambit unthinkable in
evolution, (ii) a miraculous stunt in which TMP can somehow be tapped by a protein to make
phosphoanhydride bonds, and (iii) something that counters the mandates posed by reality, as
systems that don’t have ion fluxes and gradients are also seen to give ATP-synthesis and
motility. It is commonsensical to envisage that ≤micron dimension forms would have been the
first proto-cells (as smaller units are easier to self-assemble, coordinate and homeostasize). No
wonder, micro-dimensioned mitochondria and chloroplasts are considered primitive life forms
that became endosymbionts. In such systems with convoluted/packed membrane assemblies, it
would be easier to avail or to generate DRS in a sustained manner and harness their one-electron

reaction potentials owing to the limited availability of protons, which enhance DRS
lifetimes/utility (Manoj & Gideon, 2022). DRS served as thermodynamic and kinetic agents of
selectivity and specificity before the more deterministic affinity-driven reactions mediated by
later proteins set in. All these premises support the deduction that murburn concept is a
fundamental pillar of life (like cell theory and central dogma), whereas terms/proposals like
ETC, Z-scheme, Kok-Joliot cycle, chemiosmosis, pmf, Q-cycle, rotary ATP-synthesis, etc. have
little physiological relevance and must be jettisoned as elements of a forgettable past. In lieu,
employing murburn concept allows a pan-systemic and holistic scope for comprehending cellular
function, which was lacking till date in the discretized treatments within cellular physiology.
Declarations
Author contributions: KMM identified crucial shortcomings of the classical explanations and
prepped the first draft of the paper. LJ critically discussed various contents of the essay, pointed
out to historical aspects and enhanced the manuscript’s ordering and presentation (particularly in
the text on pre-biotic evolutionary phase).
Dedication: This article is dedicated to all those scientists that disengage from an inertial
mindset, question overtly deterministic ideas and adopt more probabilistic explanations in
biology. In particular, we highlight Gilbert Ling (1919-2019; the advocate of association-
induction hypothesis), who questioned several traditional/acclaimed ideas, reported key
observations and proposed new ways of looking at cellular physiology.
Conflict of interests: There are no conflicts of interests to declare.
Ethics statement: This article preparation did not involve experimentation with any living
systems and the article was prepared along the guidelines provided for authors.
Acknowledgments: The work was powered by Satyamjayatu: The Science & Ethics Foundation.
KMM thanks Vivian David Jacob for proof-reading the article.
REFERENCES
Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2008). Molecular
biology of the cell (5th edn.). Garland Science.

Andersson SG. (1998) Bioenergetics of the obligate intracellular parasite Rickettsia prowazekii.
Biochimica et Biophysica Acta. 1365: 105-111.
Aono R, Ogino H & Horikoshi K. (1992). pH-Dependent Flagella Formation by Facultative
Alkaliphilic Bacillus sp. C-125, Bioscience, Biotechnology, and Biochemistry 56: 48-53.
Bal W, Kurowska E, Maret W. (2012). The final frontier of pH and the undiscovered country
beyond. PLoS ONE 7(9): e45832.
Balabaskaran Nina P, Dudkina NV, Kane LA, van Eyk JE, Boekema EJ, Mather MW, Vaidya
AB. (2010). Highly divergent mitochondrial ATP synthase complexes in Tetrahymena
thermophila. PLoS Biology 8: e1000418.
Behe, M.J. (1996). Darwin's Black Box: The Biochemical Challenge to Evolution. Touchstone
book. Free Press. p. 39. ISBN 978-0-684-82754-4.
Berg H C, Anderson R A. (1973). Bacteria swim by rotating their flagellar filaments. Nature
(London) 1973;245:380–382.
Berg, H. C. (2003). The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72, 19–54.
Berg, J. M., Tymoczko, J. L., & Stryer, L. (2002). Biochemistry (5th edn.). WH Freeman.
Biegel CM, Gould JM. (1981). Kinetics of hydrogen ion diffusion across phospholipid vesicle
membranes. Biochemistry 20: 3474-3479.
Boyer PD (1964) New hypotheses about the mechanisms of energy transfer and phosphorylation
in muscle and mitochondria. In: Molecular biology: problems and prospects. Moscow, “Nauka”
(in Russian)
Boyer PD. (1963). Phosphohistidine. Science 141: 1147-1153.
Boyer PD. (1975) A model for conformational coupling of membrane potential and proton
translocation to ATP synthesis and to active transport. FEBS Lett. 58:1–6.
Braun H-P. (2020). The oxidative phosphorylation system of the mitochondria in plants.
Mitochondrion 53: 66-75.
Chance B, Mela L. (1966a). A hydrogen ion concentration gradient in a mitochondrial
membrane. Nature 212(5060):369-372. [DOI: 10.1038/212369a0]
Chance B, Mela L. (1966b) Proton movements in mitochondrial membranes. Nature 1966;
212(5060): 372-376. [DOI:10.1038/212372a0]
Charles H, Balmand S, Lamelas A, Cottret L, Pérez-Brocal V, Burdin B, et al. (2011). A
Genomic reappraisal of symbiotic function in the aphid/Buchnera symbiosis: reduced transporter
sets and variable membrane organisations. PLoS One 6: e29096.
Cope FW (1968) Evidence for semiconduction in Aplysia nerve membrane. Proc. Natl. Acad.
Sci. USA 61: 905-908. Doi : 10.1073/pnas.61.3.905.
Cope FW (1977a) Solid state physical replacement of Hodgkin-Huxley theory. Phase
transformation kinetics of axonal potassium conductance? Physiol. Chem. Phys. 9: 155-160.
PMID: 601107.
Cope FW (1977b) Solid state physical theory of nerve impulse conduction: Elovich kinetics of
axon repolarisation currents. Physiol. Chem. Phys. 9: 383-387. PMID: 613332

De Duve C (1991). Blueprint for a cell: The nature and origin of life. Carolina Biological
Supply.
Di Marzo N, Chisci E, Giovannoni R. (2018). The role of hydrogen peroxide in redox-dependent
signaling: homeostatic and pathological responses in mammalian cells. Cells 7:156.
Drukarch B, Wilhelmus MMM, Shrivastava S (2021) The thermodynamic theory of action
potential propagation: a sound basis for unification of the physics of nerve impulses. Rev.
Neurosci. 33(3): 285-302. Doi: 10.1515/revneuro-2021-0094.
Eck RV, Dayhoff MO (1966) Evolution of the structure of ferredoxin based on living relics of
primitive amino acid sequences. Science: 152: 363-366
Edelmann L (1973) The influence of rubidium and cesium ions on the resting potential of guinea
pig heart muscle cells as predicted by the association-induction hypothesis. Ann. N. Y. Acad.
Sci. 204(1): 534-537. Doi: 10.1111/j.1749-6632.1973.tb30802.
Edelmann L (2005) Doubts about the sodium-potassium pump are not permissible in modern
bioscience. Cell. Molec. Biol. 51: 725-729.
Einstein A (1950) Out of my later years. Philosophical library, p. 54.
El Hady, A., & Machta, B. B. (2015). Mechanical surface waves accompany action potential
propagation. Nature Communications, 6, 6697.
Engelbrecht, J., Tamm, K., & Peets, T. (2020). Modelling of processes in nerve fibres at the
interface of physiology and mathematics. Biomechanics and Modeling in Mechanobiology, 19,
2491–2498.
Faraci, F. (2013). The 60th anniversary of the Hodgkin‐Huxley model. A critical appraisal from
a historical and modeler's viewpoint. Masters research thesis at Leiden Universtiy.
http://www.math.leidenuniv.nl/scripties/MasterFaraci.pdf
Fernández-Velasco JG, Jamshidi A, Gong XS, Zhou J, and Ueng RY. (2001). Photosynthetic
electron transfer through the cytochrome b6f complex can bypass cytochrome f. Journal of
Biological Chemistry 276: 30598-30607.
FitzHugh, R. (1961). Impulses and physiological states in theoretical models of nerve
membrane". Biophysical Journal, 1, 445–466.
Freitas LFd and Hamblin MR. (2016). Proposed Mechanisms of Photobiomodulation or Low-
Level Light Therapy. IEEE Journal of Selected Topics in Quantum Electronics 22: 348-364.
Fromm S, Senkler J, Eubel H, Peterhänsel C, Braun HP. (2016). Life without complex I:
proteome analyses of an Arabidopsis mutant lacking the mitochondrial NADH dehydrogenase
complex. Journal of Experimental Botany 67: 3079-3093.
Gideon DA, Nirusimhan V, and Manoj KM. (2020). Are plastocyanin and ferredoxin specific
electron carriers or generic redox capacitors? Classical and murburn perspectives on two
photosynthetic proteins. Journal of Biomolecular Structure and Dynamics. doi:
10.1080/07391102.2020.1835715
Gideon DA, Nirusimhan V, and Manoj KM. (2021). Mechanism of electron transfers mediated
by cytochromes c and b5 in mitochondria and endoplasmic reticulum: Classical and murburn

perspectives. Journal of Biomolecular Structure and Dynamics. doi:
10.1080/07391102.2021.1925154
Heimburg T (2022) The thermodynamic soliton theory of the nerve impulse and possible
medical implications. Progr. Biophys. Mol. Biol. 173: 24-35. Doi:
10.1016/j.pbiomolbio.2022.05.007.
Herzog W, Powers K, Johnston K and Duvall M (2015) A new paradigm for muscle contraction.
Front. Physiol. 6:174. doi: 10.3389/fphys.2015.00174
Hodgkin AL, Huxley AF (1952) A quantitative description of membrane current and its
application to conduction and excitation in nerve. J. Physiol. London 117: 500-544. Doi:
10.1113/jphysiol.1952.sp004764.
Hodgkin AL, Huxley AF, Katz B (1952) Measurement of current-voltage relations in the
membrane of the giant axon of Loligo. J. Physiol. (London) 116: 424-448.
Doi: 10.1113/jphysiol.1952.sp004716.
Huxley, A. F. & Niedergerke, R. (1954). Structural changes in muscle during contraction:
Interference microscopy of living muscle fibres. Nature 173, 971–973 doi:10.1038/173971a0.
Huxley, H. E. & Hanson, J. (1954) Changes in the cross-striations of muscle during contraction
and stretch and their structural interpretation. Nature 173, 973–976 doi:10.1038/173973a0.
Jacob VD and Manoj KM. (2019). Are adipocytes and ROS villains, or are they protagonists in
the drama of life? The murburn perspective. Adipobiology 10:7-16.
Jaeken L (2017) The neglected functions of intrinsically disordered proteins and the origin of
life. Progr. Biophys. Molec. Biol. 126: 31-46. doi: 10.1016/j.biomolbio.2017.03.002.
Karnkowska A, Vacek V, Zubáčová Z, Treitli SC, Petrželková R, Eme L, Novák L, Žárský V,
Barlow LD, Herman EK, Soukal P, Hroudová M, Doležal P, Stairs CW, Roger AJ, Eliáš M,
Dacks JB, Vlček Č, Hampl V. (2016). A eukaryote without a mitochondrial organelle. Current
Biology 26: 1274-1284.
Kaufmann K (1989a) Action potentials and electrochemical coupling in the macroscopic chiral
phospholipid membrane.1th ed., Caruaru, Brazil. Available at:
https://sites.google.com/site/schneiderslab/research-group/literature.
Kaufmann K (1989b) On the role of the phospholipid membrane in free energy coupling.
Caruaru, Brazil. Available at: https://sites.google.com/site/schneiderslab/research-
group/literature.
Klionsky DJ, Brusilow WSA, Simoni RD. (1984). In vivo evidence for the role of the epsilon
subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli. Journal of
Bacteriology 160: 1055-1060.
Krulwich TA. (1986) Bioenergetics of alkalophilic bacteria. The Journal of Membrane Biology
89, 113-125.
Lai N, Gladden LB, Carlier PG, Cabrera ME. (2008). Models of muscle contraction and
energetics. Drug Discov Today Dis Models. 5(4):273-288. doi: 10.1016/j.ddmod.2009.07.001.

Lea-Smith DJ, Bombelli P, Vasudevan R, Howe CJ. (2016). Photosynthetic, respiratory and
extracellular electron transport pathways in cyanobacteria. Biochimica et Biophys Acta.
1857(3):247-55.
Lee JK, Walker KL, Han HS, Kang J, Prinz FB, Waymouth RM, Nam HG, Zare RN. (2019).
Spontaneous generation of hydrogen peroxide from aqueous microdroplets. Proceedings of the
National Academy of Sciences USA. 116:19294-19298.
Ling GN (1981) Oxidative phosphorylation and mitochondrial physiology: a critical review of
chemiosmotic theory, and reinterpretation by the association-induction hypothesis. Physiol.
Chem. Phys. 13(1): 29-96. PMID: 7022492.
Ling GN (1982) The cellular resting and action potential: interpretation based on the AI
hypothesis. Physiol. Chem. Phys. 14: 47-96. PMID: 6294693.
Ling GN. (1984) In Search of the Physical Basis of Life. Plenum Press, New York.
Ling GN (2001) Life at the Cell and Below-Cell Level. The Hidden History of a Fundamental
Revolution in Biology. Pacific Press, New York.
Macalady JL, Vestling MM, Baumler D, Boekelheide N, Kaspar CW, Banfield JF. (2004).
Tetraether-linked membrane monolayers in Ferroplasma spp: a key to survival in acid.
Extremophiles 8: 411–419.
Manoj KM and Bazhin NM (2021). The murburn precepts for aerobic respiration and
homeostasis. Progress in Biophysics and Molecular Biology 167: 104-120.
Manoj KM and Hager LP. (2001). Utilization of peroxide and its relevance in oxygen insertion
reactions catalyzed by chloroperoxidase. Biochimica et Biophysica Acta (BBA) - Protein
Structure and Molecular Enzymology 1547: 408-417.
Manoj KM and Jacob VD. (2020). The murburn precepts for photoreception. Biomedical
Reviews 31:67-74.
Manoj KM and Manekkathodi A. (2021). Light's interaction with pigments in chloroplasts: The
murburn perspective. Journal of Photochemistry and Photobiology 5: 100015.
Manoj KM and Soman V. (2020). Classical and murburn explanations for acute toxicity of
cyanide in aerobic respiration: A personal perspective. Toxicology 432: 152369.
Manoj KM and Tamagawa H. (2021). Critical analysis of the explanations for cellular
homeostasis and electrophysiology from murburn perspective. Journal of Cellular Physiology
237:421-435.
Manoj KM, Bazhin N, Wu Y, Manekkathodi A. (2022e). Murburn model of photosynthesis:
Effect of additives like chloride and bicarbonate. In Chlorophylls. ISBN 978-1-80355-487-7.
Edited by Ameen S. et al., Intech Open. DOI: 10.5772/intechopen.103132.
Manoj KM, Bazhin NM, and Tamagawa H. (2021c). The murburn precepts for cellular ionic
homeostasis and electrophysiology. Journal of Cellular Physiology 237: 804-814.
Manoj KM, Bazhin NM, et al. (2021a). Structure-function correlations and system dynamics in
oxygenic photosynthesis: Classical and murburn perspectives. Journal of Biomolecular Structure
and Dynamics. doi: 10.1080/07391102.2021.1953606

Manoj KM, Bazhin NM, Tamagawa H., Jaeken L, Parashar A. (2022b). The physiological role
of complex V in ATP synthesis: Murzyme functioning is viable whereas rotary conformation
change model is untenable. Journal of Biomolecular Structure and Dynamics. DOI:
10.1080/07391102.2022.2060307.
Manoj KM, Gade SK, Venkatachalam A, and Gideon DA. (2016b). Electron transfer amongst
flavo- and hemo-proteins: diffusible species effect the relay processes, not protein–protein
binding. RSC Advances 6: 24121-24129.
Manoj KM, Gideon DA, & Jaeken L. (2022a) Why do cells need oxygen? Insights from
mitochondrial composition and function. Cell Biology International 46:344-358.
Manoj KM, Gideon DA, & Jaeken L. (2022d). Interaction of membrane-embedded cytochrome
b-complexes with quinols: Classical Q-cycle and murburn model. Cell Biochemistry & Function
40: 118-126.
Manoj KM, Gideon DA, and Jacob VD. (2018). Murburn scheme for mitochondrial
thermogenesis. Biomedical Reviews 29: 73-82.
Manoj KM, Gideon DA, and Parashar A. (2020b). What is the Role of Lipid Membrane-
embedded Quinones in Mitochondria and Chloroplasts? Chemiosmotic Q-cycle versus Murburn
Reaction Perspective. Cell Biochemistry and Biophysics 79:3-10.
Manoj KM, Gideon DA, Bazhin NM, Tamagawa H, Nirusimhan V, Kavdia M, Jaeken L.
(2022h). Na,K‐ATPase: A murzyme facilitating thermodynamic equilibriums at the
membrane‐interface. Journal of Cellular Physiology. DOI: 10.1002/jcp.30925
Manoj KM, Gideon DG, et al. (2021b). Validating the predictions of murburn model for
oxygenic photosynthesis: Analyses of ligand-binding to protein complexes and cross-system
comparisons. Journal of Biomolecular Structure and Dynamics. doi:
10.1080/07391102.2021.1953607
Manoj KM, Gideon KM. (2022). Structural foundations for explaining the roles of murzymes
embedded in diverse phospholipid membranes. Biochimica et Biophysica Acta - Biomembranes
1864: 183981.
Manoj KM, Manekkathodi A, Bazhin N, Parashar A, Wu Y. (2022f). Comprehensive analyses of
enhancement of oxygenesis in photosynthesis by bicarbonate and effects of diverse additives:
Classical explanation versus murburn model. Accepted in New Advances in Photosynthesis.
ISBN 978-1-83768-427-4. Edited by Kimatu JS. Intech Open.
Manoj KM, Nirusimhan V, Parashar A, Edward J, Gideon DA. (2022g). Murburn precepts for
lactic-acidosis, Cori cycle, and Warburg effect: Interactive dynamics of dehydrogenases, protons,
and oxygen. Journal of Cellular Physiology 237:1902–1922.
Manoj KM, Parashar A, David Jacob V, and Ramasamy S. (2019a). Aerobic respiration: proof of
concept for the oxygen-centric murburn perspective. Journal of Biomolecular Structure and
Dynamics 37: 4542-4556.
Manoj KM, Parashar A, Gade SK, and Venkatachalam A. (2016a). Functioning of Microsomal
Cytochrome P450s: Murburn Concept Explains the Metabolism of Xenobiotics in Hepatocytes.
Frontiers in Pharmacology 7: 161.

Manoj KM, Parashar A, Venkatachalam A, Goyal S, Satyalipsu, Singh PG, Gade SK, Periyasami
K, Jacob RS, Sardar D, Singh S, Kumar R, and Gideon DA. (2016c). Atypical profiles and
modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest
diffusible radicals' obligatory involvement in such redox reactions. Biochimie 125: 91-111.
Manoj KM, Ramasamy S, Parashar A, Gideon DA, Soman V, Jacob VD, and Pakshirajan K.
(2020a). Acute toxicity of cyanide in aerobic respiration: Theoretical and experimental support
for murburn explanation. Biomolecular Concepts 11: 32-56.
Manoj KM, Soman V, David Jacob V, Parashar A, Gideon DA, Kumar M, Manekkathodi A,
Ramasamy S, Pakshirajan K, and Bazhin NM. (2019b). Chemiosmotic and murburn explanations
for aerobic respiration: Predictive capabilities, structure-function correlations and chemico-
physical logic. Archives of Biochemistry and Biophysics 676: 108128.
Manoj KM, Tamagawa H, Bazhin N, Jaeken L, Nirusimhan V, Faraci F, Gideon DA. (2022c).
Murburn model of vision: Precepts and proof of concept. Journal of Cellular Physiology 237:
3338-3355.
Manoj KM. (2006). Chlorinations catalyzed by chloroperoxidase occur via diffusible
intermediate(s) and the reaction components play multiple roles in the overall process.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1764: 1325-1339.
Manoj KM. (2017). Debunking chemiosmosis and proposing murburn concept as the operative
principle for cellular respiration. Biomedical Reviews, 28, 31-48.
Manoj KM. (2018a). Aerobic Respiration: Criticism of the Proton-centric Explanation Involving
Rotary Adenosine Triphosphate Synthesis, Chemiosmosis Principle, Proton Pumps and Electron
Transport Chain. Biochemistry Insights 11: 1178626418818442.
Manoj KM. (2018b). The ubiquitous biochemical logic of murburn concept. Biomedical Reviews
29: 89-97.
Manoj KM. (2020a). Murburn concept: a paradigm shift in cellular metabolism and physiology.
Biomolecular Concepts 11: 7-22.
Manoj KM. (2020b). In defense of the murburn explanation for aerobic respiration. Biomedical
Reviews 31:135-160. (Also refer to the Supplementary Information in the issue.)
Manoj, K. M., Jacob, V. D., Kavdia, M., Tamagawa, H., Jaeken, L., & Soman, V. (2022i).
Questioning physiological rotary functionality in the bacterial flagellar system and proposing a
murburn model for motility. OSF Preprints. https://doi.org/10.31219/osf.io/p9wua
Matsuki S and Koike T. (2006). Comparison of Leaf Life Span, Photosynthesis and Defensive
Traits Across Seven Species of Deciduous Broad-leaf Tree Seedlings. Annals of Botany 97: 813-
817.
Matveev VV (2019) Cell theory, intrinsically disordered proteins, and the physics of the origin of
life. Progr. Biophys. Mol. Biol. 149: 114-130. Doi: 10.1016/j.pbiomolbio.2019.04.001.
Matveev VV (2022) Membraneless physiology of the living cell. The past and the present. 4open
5 (2022) 15. Doi: 10.1051/fopen/2022013.
Mittler R. (2017). ROS are good. Trends in Plant Sciences 22:11-19.

Montesano, P., Palermi, S., Massa, B., & Mazzeo, F. (2020). From “sliding” to “winding”
filaments theory: A narrative review of mechanisms behind skeletal muscle contraction. Journal
of Human Sport and Exercise, 15(3proc), S806-S814.
doi:https://doi.org/10.14198/jhse.2020.15.Proc3.31
Mungal SU, Dube SP, Dhole A, Mane U, Bondade AK. (2015). New hypothesis for mechanism
of sliding filament theory of skeletal muscle contraction. Natl J Physiol Pharm Pharmacol 5:72-
75.
Nelson, D. L., & Cox, M. C. (2004). Lehninger: Principles of biochemistry (4th edn.). W H
Freeman.
Nishikawa, K. C., Monroy, J. A., Uyeno, T. E., Yeo, S. H., Pai, D. K., & Lindstedt, S. L. (2012).
Is titin a "winding filament"? A new twist on muscle contraction. Proceedings of the Royal
Society B: Biological Sciences, 279(1730), 981-990. https://doi.org/10.1098/rspb.2011.1304
Nolfi-Donegan D, Braganza A, Shiva S. (2020). Mitochondrial electron transport chain:
Oxidative phosphorylation, oxidant production, and methods of measurement. Redox Biology,
37:101674.
Olsson, K., Keis, S., Morgan, H. W., Dimroth, P., and Cook, G. M. (2003) Bioenergetic
properties of the thermoalkaliphilic Bacillus sp. strain TA2. A1. Journal of Bacteriology 185,
461-465
Oplatka A. (1994) The role of water in the mechanism of muscular contraction. FEBS Lett.
355(1): 1-3. Doi: 10.1016/0014-5793(94)01158-3.
Oplatka A. (1997) Critical review of the swinging crossbridge theory and the cardinal active role
of water in muscle contraction. Crit. Ev. Biochem. Mol. Biol. 32(4): 307-360. Doi:
10.3109/10409239709082375.
Oplatka A. (1998a) Are rotors at the heart of all biological motors? Biochem Biophys Res
Commun. 246(2):301-306.
Oplatka A. (1998b) Do the bacterial flagellar motor and ATP synthase operate as water turbines?
Biochem Biophys Res Commun. 249(3):573-578.
Ortiz de Montellano P. (2015). Cytochrome P450: Structure, mechanism and biochemistry. 4th
Edition. Springer Cham. https://doi.org/10.1007/978-3-319-12108-6
Padan, E., Bibi, E., Ito, M., and Krulwich, T. A. (2005) Alkaline pH homeostasis in bacteria:
new insights. Biochimica et biophysica acta (BBA)-Biomembranes 1717, 67-88
Parashar A & Manoj, KM. (2021). Murburn precepts for cytochrome P450 mediated
drug/xenobiotic metabolism and homeostasis. Current Drug Metabolism.
doi:10.2174/1389200222666210118102230
Parashar A, Gideon DA, and Manoj KM. (2018). Murburn concept: A molecular explanation for
hormetic and idiosyncratic dose responses. Dose Response 16: 1559325818774421.
Parashar A, Jacob VD, Gideon DA, and Manoj KM. (2021). Hemoglobin catalyzes ATP-
synthesis in human erythrocytes: A murburn model. Journal of Biomolecular Structure and
Dynamics (in Press). doi: 10.1080/07391102.2021.1925592

Parashar A, Venkatachalam A, Gideon DA, and Manoj KM. (2014). Cyanide does more to
inhibit heme enzymes, than merely serving as an active-site ligand. Biochemical and Biophysical
Research Communications 455: 190-193.
Partensky F, Hess WR, and Vaulot D. (1999). Prochlorococcus, a marine photosynthetic
prokaryote of global significance. Microbiology and Molecular Biology Reviews 63: 106-127.
Pelczar MJ, Chan ECS, Krieg NR. (2001). Microbiology (5th edn.). McGraw-Hill.
Pérez-Brocal V, Gil R, Ramos S, Lamelas A, Postigo M, Michelena JM, Silva FJ, Moya A,
Latorre A. (2006). A small microbial genome: the end of a long symbiotic relationship? Science
314:312-313.
Pesaresi P, Scharfenberg M, Weigel M, Granlund I, Schröder WP, Finazzi G, Rappaport F,
Masiero S, Furini A, Jahns P, and Leister D. (2009). Mutants, overexpressors, and interactors of
Arabidopsis plastocyanin isoforms: Revised roles of plastocyanin in photosynthetic electron flow
and thylakoid redox state. Molecular Plant 2: 236-248.
Pollack GH (2001) Cells, Cells and the Engines of Life. A New Unifying Approach to Cell
Function. Ebner and Sons, Seattle.
Pollack GH (2013) The Fourth Phase of Water: Beyond Solid, Liquid and Vapor. Ebner & Sons,
Seattle.
Prebble J, Weber B. Wandering in the Gardens of the Mind: Peter Mitchell and the Making of
Glynn 1st Edition. Oxford University Press, 2003. Cary, NC, 2003, 324 pp., ISBN 0-19-514266-
7. https://iubmb.onlinelibrary.wiley.com/doi/full/10.1002/bmb.2004.494032019998
Prebble JN. (2013). Contrasting Approaches to a Biological Problem: Paul Boyer, Peter Mitchell
and the Mechanism of the ATP Synthase, 1961-1985. Journal of the History of Biology 46(4):
699-737.
Prebble, J. (2000) The lasting value of Mitchell's mechanisms. Nature 404, 330.
https://doi.org/10.1038/35006257
Preiss L, Hicks DB, Suzuki S, Meier T and Krulwich TA (2015) Alkaliphilic bacteria with
impact on industrial applications, concepts of early life forms, and bioenergetics of ATP
synthesis. Frontiers in Bioengetics and Biotechnology 3:75.
Rashdan NA, Pattillo CB. (2020). Hydrogen peroxide in the ER: A tale of triage. Redox Biology
28:101358.
Roskoski R Jr. (2004) Book review (Wandering in the Gardens of the Mind: Peter Mitchell and
the Making of Glynn 1st Edition.). Biochemistry and Molecular Biology Education. 32(1):64-65.
Rvachev, M. M. (2010). On axoplasmic pressure waves and their possible role in nerve impulse
propagation. Biophysical Reviews and Letters., 5, 73–88.
Saeed H, Lee J. (2018). Experimental determination of proton-cation exchange equilibrium
constants at water-membrane interface fundamental to bioenergetics. WATER 9:116–140.
Schoepp-Cothenet B, van Lis R, Atteia A, Baymann F, Capowiez L, Ducluzeau A-L, Duval S,
ten Brink F, Russell MJ, Nitschke W. (2013) On the universal core of bioenergetics.
Biochim.Biophys. Acta Bioenerg. 1827: 79-93.
Schrödinger E. (1944) What is Life? The Physical Aspect of the Living Cell. Cambridge

Schwartz A, Ponnamperuma C. (1968). Phosphorylation of adenosine with linear polyphosphate
salts in aqueous solution. Nature. 218:443.
Senkler J, Rugen N, Eubel H, Hegermann J, Braun HP. (2018). Absence of Complex I implicates
rearrangement of the respiratory chain in european mistletoe. Current Biology 28:1606-1613.e4.
Slater, E. C. (1953) Mechanism of phosphorylation in the respiratory chain, Nature 172: 59.
Sojo V, Pomiankowski A, Lane N. (2014). A bioenergetic basis for membrane divergence in
archaea and bacteria. PLoS Biology 12: e1001926.
Sommer AP. (2019). Mitochondrial cytochrome c oxidase is not the primary acceptor for near
infrared light—it is mitochondrial bound water: the principles of low-level light therapy. Annals
of Translational Medicine 7: S13.
Stoin U, Shames AI, Malka I, Bar I, Sasson Y. (2013) In situ generation of superoxide anion
radical in aqueous medium under ambient conditions. Chemphyschem. 14:4158-4164.
Sturm A, Mollard V, Cozijnsen A, Goodman CD, McFadden GI. (2015) Mitochondrial ATP
synthase is dispensable in blood-stage Plasmodium berghei rodent malaria but essential in the
mosquito phase. Proceedings of the National Academy of Sciences USA 112:10216-10223.
Suelter CH, Deluca M, Peter JB, Boyer PD. (1961) Detection of a possible intermediate in
oxidative phosphorylation. Nature. 192:43-7. doi: 10.1038/192043a0.
Tamagawa, H., Ikeda, K. (2018) Another interpretation of the Goldman–Hodgkin–
Katz equation based on Ling’s adsorption theory. Eur Biophys J 47, 869–
879. https://doi.org/10.1007/s00249-018-1332-0
Tena G. (2020) Sensing peroxide. Nature Plants 6:178.
Timoshnikov VA, Kobzeva TV, Polyakov NE, Kontoghiorghes GJ. (2015) Inhibition of Fe(2+)-
and Fe(3+)- induced hydroxyl radical production by the iron-chelating drug deferiprone. Free
Radic Biol Med. 78:118-22. doi: 10.1016/j.freeradbiomed.2014.10.513.
Vale RD, Funatsu T, Pierce DW, Romberg L, Harada Y, Yanagida T. (1996). Direct observation
of single kinesin molecules moving along microtubules. Nature. 380(6573):451-3. doi:
10.1038/380451a0.
Valmalette JC, Dombrovsky A, Brat P, Mertz C, Capovilla M, Robichon A. (2012). Light-
induced electron transfer and ATP synthesis in a carotene synthesizing insect. Scientific Reports
2:579.
van der Stel A-X and Wösten MMSM. (2019). Regulation of respiratory pathways in
Campylobacterota: A review. Frontiers in Microbiology 10:1719.
Volkmann N, Hanein D. Actomyosin: law and order in motility. Curr Opin Cell Biol. 2000
Feb;12(1):26-34. doi: 10.1016/s0955-0674(99)00053-8.
Voet, D., & Voet, J. G. (2011). Biochemistry (4th edn.). Wiley.
Wheatley DN (2003) Diffusion, perfusion and the exclusion principle in the structural and
functional organization of the living cell: reappraisal of the properties of the ground substance. J.
Exp. Biol. 206: 1955-1961. Doi: 10.1242/jeb.00238.

Wilks, J. C., & Slonczewski, J. L. (2007). pH of the cytoplasm and periplasm of Escherichia coli:
Rapid measurement by green fluorescent protein fluorimetry. Journal of Bacteriology, 189(15),
5601–5607.
Xu H, Hou R, Tong T, Li H. (2021) Diffusion Biased by a Soft Neck Linker Regulates Kinesin
Stepping. J Phys Chem B. 125(10):2627-2635. doi: 10.1021/acs.jpcb.1c00549.
Yahalomi D, Atkinson SD, Neuhof M, Chang ES, Philippe H, Cartwright P, Bartholomew JL,
Huchon D. (2020). A cnidarians parasite of salmon (Myxozoa: Henneguya) lacks a
mitochondrial genome. Proceedings of the National Academy of Sciences USA 117: 5358-5363.
Zhang L, Pakrasi HB, and Whitmarsh J. (1994). Photoautotrophic growth of the cyanobacterium
Synechocystis sp. PCC 6803 in the absence of cytochrome c553 and plastocyanin. Journal of
Biological Chemistry 269: 5036-5042.