Publications by authors named "Kelath Murali Manoj"

36 Publications

Interaction of membrane-embedded cytochrome b-complexes with quinols: Classical Q-cycle and murburn model.

Cell Biochem Funct 2022 Jan 13. Epub 2022 Jan 13.

Karel de Grote University College, Antwerp University Association, Campus Hoboken, Hoboken, Belgium.

We recently proposed a diffusible reactive (oxygen) species (DRS/DROS) based function for cytochrome b complexes (CBC) and quinones (Q)/quinols (QH ) in the murburn model of bioenergetics. This proposal is in direct conflict with the classical purview of Q-cycle. Via extensive analyses of the structure-function correlations of membrane-quinones/quinols and proteins, we present qualitative and quantitative arguments to infer that the classical model cannot explain the energetics, kinetics, mechanism and probabilistic considerations. Therefore, it is proposed that Q-cycle is neither necessary nor feasible at CBCs. In contrast, we substantiate that the murburn model explains: (a) crucial structural data of CBCs, (b) why quinones/quinols are utilized in bioenergetic membranes, (c) how trans-membrane potential is generated owing to effective charge separation at CBCs, (d) mobility data of O , DRS, Q/QH , and (e) utility of other reaction/membrane components. Further, the murburn model also accommodates the absence of quinones in anaerobic Archaea, wherein methanophenazines are prevalent. The work mandates that the textbooks and research agendas are refreshed to reflect the new perception. SIGNIFICANCE: The current article must be seen as a critical and detailed analysis of the role and working mechanism of quinone (Q) /quinols (QH ) in bioenergetic membranes. In the classical model, QH are perceived as highly mobile electron-transport agents that bind and donate electrons to cytochrome b complexes (CBCs), using sophisticated electronic circuitries, in order to recycle Q and pump protons. The classical perception sees radicals (such as Q*-, O *-, etc., also called diffusible reactive species, DRS) as wasteful or toxic (patho) physiological manifestations. It is highlighted herein that QH has low mobility and matrix has little protons to pump. New insights from the structural analyses of diverse CBCs and quinols, in conjunction with murburn reaction thermodynamics suggest that the electrons from substrates/quinols are effectively utilized via DRS. This perception fits into a much broader analysis of 1 and 2 electron transfers in overall redox metabolism, as recently brought out by the murburn model, wherein DRS are considered obligatory ingredients of physiology. Thus, the findings mandate a reorientation in the pertinent research field.
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http://dx.doi.org/10.1002/cbf.3682DOI Listing
January 2022

Murburn precepts for lactic-acidosis, Cori cycle, and Warburg effect: Interactive dynamics of dehydrogenases, protons, and oxygen.

J Cell Physiol 2021 Dec 20. Epub 2021 Dec 20.

Department of Biochemistry, Satyamjayatu: The Science & Ethics Foundation, Palakkad, Kerala, India.

It is unresolved why lactate is transported to the liver for further utilization within the physiological purview of Cori cycle, when muscles have more lactate dehydrogenase (LDH) than liver. We point out that the answer lies in thermodynamics/equilibriums. While the utilization of NADH for the reduction of pyruvate to lactate can be mediated via the classical mechanism, the oxidation of lactate (with/without the uphill reduction of NAD ) necessitates alternative physiological approaches. The latter pathway occurs via interactive equilibriums involving the enzyme, protons and oxygen or diffusible reactive oxygen species (DROS). Since liver has high DROS, the murburn activity at LDH would enable the cellular system to tide over the unfavorable energy barriers of the forward reaction (~476 kJ/mol; earlier miscalculated as ~26 kJ/mole). Further, the new mechanism does not necessitate any "smart decision-making" or sophisticated control by/of proteins. The DROS-based murburn theory explains the invariant active-site structure of LDH isozymes and their multimeric nature. The theoretical insights, in silico evidence and analyses of literature herein also enrich our understanding of the underpinnings of "lactic acidosis" (lowering of physiological pH accompanied by lactate production), Warburg effect (increased lactate production at high pO by cancer cells) and approach for cancer therapy.
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http://dx.doi.org/10.1002/jcp.30661DOI Listing
December 2021

Why do cells need oxygen? Insights from mitochondrial composition and function.

Cell Biol Int 2021 Dec 17. Epub 2021 Dec 17.

Industrial Sciences and Technology, Karel de Grote-Hogeschool, Association University and High Schools Antwerp, Antwerpen, Belgium.

Mitochondrial membrane-embedded redox proteins are classically perceived as deterministic "electron transport chain" (ETC) arrays cum proton pumps; and oxygen is seen as an "immobile terminal electron acceptor." This is untenable because: (1) there are little free protons to be pumped out of the matrix; (2) proton pumping would be highly endergonic; (3) ETC-chemiosmosis-rotary ATP synthesis proposal is "irreducibly complex"/"non-evolvable" and does not fit with mitochondrial architecture or structural/distribution data of the concerned proteins/components; (4) a plethora of experimental observations do not conform to the postulates/requisites; for example, there is little evidence for viable proton-pumps/pH-gradient in mitochondria, trans-membrane potential (TMP) is non-fluctuating/non-trappable, oxygen is seen to give copious "diffusible reactive (oxygen) species" (DRS/DROS) in milieu, etc. Quite contrarily, the newly proposed murburn model's tenets agree with known principles of energetics/kinetics, and builds on established structural data and reported observations. In this purview, oxygen is needed to make DRS, the principal component of mitochondrial function. Complex V and porins respectively serve as proton-inlet and turgor-based water-exodus portals, thereby achieving organellar homeostasis. Complexes I to IV possess ADP-binding sites and their redox-centers react/interact with O /DRS. At/around these complexes, DRS cross-react or activate/oxidize ADP/Pi via fast thermogenic one-electron reaction(s), condensing to form two-electron stabilized products (H O /H O/ATP). The varied architecture and distribution of components in mitochondria validate DRS as (i) the coupling agent of oxidative reactions and phosphorylations, and (ii) the primary reason for manifestation of TMP in steady-state. Explorations along the new precepts stand to provide greater insights on mitochondrial function and pathophysiology.
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http://dx.doi.org/10.1002/cbin.11746DOI Listing
December 2021

Critical analysis of explanations for cellular homeostasis and electrophysiology from murburn perspective.

J Cell Physiol 2021 Sep 13. Epub 2021 Sep 13.

Department of Mechanical Engineering, Gifu University, Yanagido, Gifu City, Japan.

Pursuits in modern cellular electrophysiology are fraught with disagreements at a fundamental level. While the membrane theory of homeostasis deems the cell membrane and proteins embedded therein as the chief players, the association-induction (or sorption/bulk-phase) hypothesis considers the aqueous phase of dissolved proteins (cytoplasm/protoplasm) as the key determinant of cellular composition and ionic fluxes. In the first school of thought, trans-membrane potential (TMP) and selective ion pumps/channels are deemed as key operative principles. In the latter theory, sorption-desorption dynamics and rearrangements of bulk phase determine the outcomes. In both these schools of thought, theorists believe that the macroscopic phase electroneutrality holds, TMP (whether in resting or in activated state) results solely due to ionic concentration differentials across the membrane, and the concerned proteins undergo major conformation changes to affect/effect the noted outcomes. The new entry into the field, murburn concept, builds starting from molecular considerations to macroscopic observations. It moots "effective charge separation" and intricate "molecule-ion-radical" electron transfer equilibriums as a rationale for ionic concentration differentials and TMP variation. After making an unbiased appraisal of the two classical schools of thought, the review makes a point-wise analysis of some hitherto unresolved observations/considerations and suggests the need to rethink the mechanistic perspectives.
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http://dx.doi.org/10.1002/jcp.30578DOI Listing
September 2021

The murburn precepts for cellular ionic homeostasis and electrophysiology.

J Cell Physiol 2021 Aug 11. Epub 2021 Aug 11.

Department of Mechanical Engineering, Gifu University, Yanagido, Gifu, Japan.

Starting from the basic molecular structure and redox properties of its components, we build a macroscopic cellular electrophysiological model. We first present a murburn purview that could explain ion distribution in bulk-milieu/membrane-interface and support the origin of trans-membrane potential (TMP) in cells. In particular, the discussion focuses on how cells achieve disparity in the distribution of monovalent and divalent cations within (K  > Na  > Mg  > Ca ) and outside (Na  > K  > Ca  > Mg ). We explore how TMP could vary for resting/graded/action potentials generation and project a model for impulse conduction in neurons. Outcomes based on murburn bioenergetic equilibriums leading to solubilization of ion-pairs, membrane's permittivity, protein channels' fluxes, and proteins' innate ability to bind/adsorb ions selectively are projected as the integral rationale. We also provide experimental modalities to ratify the projections.
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http://dx.doi.org/10.1002/jcp.30547DOI Listing
August 2021

Validating the predictions of murburn model for oxygenic photosynthesis: Analyses of ligand-binding to protein complexes and cross-system comparisons.

J Biomol Struct Dyn 2021 Jul 30:1-33. Epub 2021 Jul 30.

RedOx Lab, Department of Life Sciences, Satyamjayatu: The Science & Ethics Foundation, Palakkad District, Kerala, India.

In this second half of our treatise on oxygenic photosynthesis, we provide support for the murburn model of the light reaction of photosynthesis and ratify key predictions made in the first part. Molecular docking and visualization of various ligands of quinones/quinols (and their derivatives) with PS II/Cytochrome complexes did not support chartered 2e-transport role of quinols. A broad variety of herbicides did not show any affinity/binding-based rationales for inhibition of photosynthesis. We substantiate the proposal that disubstituted phenolics (perceived as protonophores/uncouplers or affinity-based inhibitors in the classical purview) serve as interfacial modulators of diffusible reactive (oxygen) species or DR(O)S. The DRS-based murburn model is evidenced by the identification of multiple ADP-binding sites on the extra-membraneous projection of protein complexes and structure/distribution of the photo/redox catalysts. With a panoramic comparison of the redox metabolic machinery across diverse organellar/cellular systems, we highlight the ubiquitous one-electron murburn facets (cofactors of porphyrin, flavin, FeS, other metal centers and photo/redox active pigments) that enable a facile harnessing of the utility of DRS. In the summative analyses, it is demonstrated that the murburn model of light reaction explains the structures of membrane supercomplexes recently observed in thylakoids and also accounts for several photodynamic experimental observations and evolutionary considerations. , the work provides a new orientation and impetus to photosynthesis research. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1953607DOI Listing
July 2021

Structure-function correlations and system dynamics in oxygenic photosynthesis: classical perspectives and murburn precepts.

J Biomol Struct Dyn 2021 Jul 29:1-27. Epub 2021 Jul 29.

Satyamjayatu: The Science & Ethics Foundation, Kulappully, Kerala, India.

Highlights: Contemporary beliefs on oxygenic photosynthesis are critiqued.Murburn model is suggested as an alternative explanation.In the new model, diffusible reactive species are the main protagonists.All pigments are deemed photo-redox active in the new stochastic mechanism.NADPH synthesis occurs via simple electron transfers, not via elaborate ETC.Oxygenesis is delocalized and not just centered at Mn-Complex.Energetics of murburn proposal for photophosphorylation is provided.The proposal ushers in a paradigm shift in photosynthesis research.
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http://dx.doi.org/10.1080/07391102.2021.1953606DOI Listing
July 2021

The murburn precepts for aerobic respiration and redox homeostasis.

Prog Biophys Mol Biol 2021 Dec 10;167:104-120. Epub 2021 Jun 10.

Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, St. Institutskaya 3, 630090, Novosibirsk, Russia. Electronic address:

Murburn concept is a new perspective to metabolism which posits that certain redox enzymes/proteins mediate catalysis outside their active site, via diffusible reactive oxygen species (DROS, usually deemed as toxic wastes). We have recently questioned the proton-centric chemiosmotic rotary ATP synthesis (CRAS) explanation for mitochondrial oxidative phosphorylation (mOxPhos) and proposed an oxygen-centric murburn model in lieu. Herein, the chemical equations and thermodynamic foundations for this new model of mOxPhos are detailed. Standard transformed Gibbs free energy values of respiratory reactions are calculated to address the spontaneity, control, and efficiency of oxidative phosphorylation. Unlike the deterministic/multi-molecular and 'irreducibly complex' CRAS model, the stochastic/bimolecular and parsimonious murburn reactions afford a more viable precept for the variable and non-integral stoichiometry, higher yield for NADH than FADH, and origin/evolution of oxygen-centric cellular life. Also, we present tangible DROS-based explanations for the multiple roles of various reaction components, HCN > HS order of cellular toxicity in aerobes, and explain why oxygen inhibits anaerobes. We highlight the thermodynamic significance of proton deficiency in NADH/mitochondria and link the 'oxygen → DROS → water' metabolic pathway to the macroscopic physiologies of ATP-synthesis, trans-membrane potential, thermogenesis, and homeostasis. We also provide arguments for the extension of the murburn bioenergetics model to life under anoxic and extreme/unique habitats. In the context of mOxPhos, our findings imply that DROS should be seen as an essential requisite for life, and not merely as pathophysiological manifestations.
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http://dx.doi.org/10.1016/j.pbiomolbio.2021.05.010DOI Listing
December 2021

Mechanism of electron transfers mediated by cytochromes and in mitochondria and endoplasmic reticulum: classical and murburn perspectives.

J Biomol Struct Dyn 2021 May 17:1-18. Epub 2021 May 17.

Satyamjayatu: The Science & Ethics Foundation, Palakkad District, Kerala State, India.

We explore the mechanism of electron transfers mediated by cytochrome , a soluble protein involved in mitochondrial oxidative phosphorylation and cytochrome , a microsomal membrane protein acting as a redox aide in xenobiotic metabolism. We found minimal conservation in the sequence and surface amino acid residues of cytochrome proteins among divergent species. Therefore, we question the evolutionary logic for electron transfer (ET) occurring through affinity binding via recognition of specific surface residues/topography. Also, analysis of putative protein-protein interactions in the crystal structures of these proteins and their redox partners did not point to any specific interaction logic. A comparison of the kinetic and thermodynamic constants of wildtype vs. mutants did not provide strong evidence to support the binding-based ET paradigm, but indicated support for diffusible reactive species (DRS)-mediated process. Topographically divergent cytochromes from one species have been substituted for reaction with proteins from other species, implying the involvement of non-specific interactions. We provide a viable alternative (murburn concept) to classical protein-protein binding-based long range ET mechanism. To account for the promiscuity of interactions and solvent-accessible hemes, we propose that the two proteins act as non- specific redox capacitors, mediating one-electron redox equilibriums involving DRS and unbound ions.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1925154DOI Listing
May 2021

Hemoglobin catalyzes ATP-synthesis in human erythrocytes: a murburn model.

J Biomol Struct Dyn 2021 May 17:1-13. Epub 2021 May 17.

Satyamjayatu: The Science & Ethics Foundation, Kulappully, India.

Blood hemoglobin (Hb), known to transport oxygen, is the most abundant globular protein in humans. Erythrocytes have ∼10 M concentration of ATP in steady-state and we estimate that this high amounts cannot be formed from 10 - 10 M levels of precursors via substrate-level phosphorylation of glycolysis. To account for this discrepancy, we propose that Hb serves as a 'murzyme' (a redox enzyme working along the principles of murburn concept), catalyzing the synthesis of the major amounts of ATP found in erythrocytes. This proposal is along the lines of our earlier works demonstrating DROS (diffusible reactive oxygen species) mediated ATP-synthesis as a thermodynamically and kinetically viable mechanism for physiological oxidative phosphorylation. We support the new hypothesis for Hb with theoretical arguments, experimental findings of reputed peers and in silico explorations. Using in silico methods, we demonstrate that adenosine nucleotide and 2,3-bisphosphoglycerate (2,3-BPG) binding sites are located suitably on the monomer/tetramer, thereby availing facile access to the superoxide emanating from the heme center. Our proposal explains earlier reported in situ experimental findings/suggestions of 2,3-BPG and ADP binding at the same locus on Hb. The binding energy is in the order of 2,3-BPG > NADH > ATP > ADP > AMP and agrees with earlier reports, potentially explaining the bioenergetic physiology of erythrocytes. Also, the newly discovered site for 2,3-BPG shows lower affinity in fetal Hb (as compared to adults) explaining oxygen transfer from mother to embryo. The findings pose significant implications in routine physiology and pathologies like sickle cell anemia and thalassemia.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1925592DOI Listing
May 2021

Are plastocyanin and ferredoxin specific electron carriers or generic redox capacitors? Classical and murburn perspectives on two photosynthetic proteins.

J Biomol Struct Dyn 2020 Oct 19:1-15. Epub 2020 Oct 19.

Department of Biochemistry, Satyamjayatu: The Science & Ethics Foundation, Palakkad, India.

In the light reaction of oxygenic photosynthesis, plastocyanin (PC) and ferredoxins (Fd) are small/diffusible redox-active proteins playing key roles in electron transfer/transport phenomena. In the Z-scheme mechanistic purview, they are considered as specific affinity binding-based electron-relay agents, linking the functions of Cytochrome (Cyt. ), Photosystem I (PS I) and Fd:NADPH oxidoreductase (FNR). The murburn explanation for photolytic photophosphorylation deems PC/Fd as generic 'redox capacitors', temporally accepting and releasing one-electron equivalents in reaction milieu. Herein, we explore the two theories with respect to structural, distributional and functional aspects of PC/Fd. Amino acid residues located on the surface loci of key patches of PC/Fd vary in electrostatic/contour (topography) signatures. Crystal structures of four different complexes each of Cyt.-PC and Fd-FNR show little conservation in the contact-surfaces, thereby discrediting 'affinity binding-based electron transfers (ET)' as an evolutionary logic. Further, thermodynamic and kinetic data of wildtype and mutant proteins interactions do not align with Z-scheme. Furthermore, micromolar physiological concentrations of PC and the non-conducive architecture of chloroplasts render the classical model untenable. In the murburn model, as PC is optional, the observation that plants lacking PC survive and grow is justified. Further, the low physiological concentration/distribution of PC in chloroplast lumen/stroma is supported by murburn equilibriums, as higher concentrations would limit electron transfers. Thus, structural evidence, interactive dynamics with redox partners and physiological distribution/role of PC/Fd support the murburn perspective that these proteins serve as generic redox-capacitors in chloroplasts.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1835715DOI Listing
October 2020

What is the Role of Lipid Membrane-embedded Quinones in Mitochondria and Chloroplasts? Chemiosmotic Q-cycle versus Murburn Reaction Perspective.

Cell Biochem Biophys 2021 Mar 29;79(1):3-10. Epub 2020 Sep 29.

Department of Biotechnology, Vignan's Foundation for Science, Technology & Research, Vadlamudi, Guntur, 522213, India.

Quinones are found in the lipid membranes of prokaryotes like E. coli and cyanobacteria, and are also abundant in eukaryotic mitochondria and chloroplasts. They are intricately involved in the reaction mechanism of redox phosphorylations. In the Mitchellian chemiosmotic school of thought, membrane-lodged quinones are perceived as highly mobile conveyors of two-electron equivalents from the first leg of Electron Transport Chain (ETC) to the 'second pit-stop' of Cytochrome bc or bf complex (CBC), where they undergo a regenerative 'Q-cycle'. In Manoj's murburn mechanism, the membrane-lodged quinones are perceived as relatively slow-moving one- or two- electron donors/acceptors, enabling charge separation and the CBC resets a one-electron paradigm via 'turbo logic'. Herein, we compare various purviews of the two mechanistic schools with respect to: constraints in mobility, protons' availability, binding of quinones with proteins, structural features of the protein complexes, energetics of reaction, overall reaction logic, etc. From various perspectives, the murburn mechanism appeals as a viable alternative explanation well-rooted in thermodynamics/kinetics and one which lends adequate structure-function correlations for the roles of quinones, lipid membrane and associated proteins.
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http://dx.doi.org/10.1007/s12013-020-00945-yDOI Listing
March 2021

Acute toxicity of cyanide in aerobic respiration: Theoretical and experimental support for murburn explanation.

Biomol Concepts 2020 Mar 17;11(1):32-56. Epub 2020 Mar 17.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India-781039.

The inefficiency of cyanide/HCN (CN) binding with heme proteins (under physiological regimes) is demonstrated with an assessment of thermodynamics, kinetics, and inhibition constants. The acute onset of toxicity and CN's mg/Kg LD50 (μM lethal concentration) suggests that the classical hemeFe binding-based inhibition rationale is untenable to account for the toxicity of CN. In vitro mechanistic probing of CN-mediated inhibition of hemeFe reductionist systems was explored as a murburn model for mitochondrial oxidative phosphorylation (mOxPhos). The effect of CN in haloperoxidase catalyzed chlorine moiety transfer to small organics was considered as an analogous probe for phosphate group transfer in mOxPhos. Similarly, inclusion of CN in peroxidase-catalase mediated one-electron oxidation of small organics was used to explore electron transfer outcomes in mOxPhos, leading to water formation. The free energy correlations from a Hammett study and IC50/Hill slopes analyses and comparison with ligands ( CO/ H 2 S/ N 3 - ) $\left( {\text{CO}}/{{{{\text{H}}_{2}}\text{S}}/{\text{N}_{3}^{\text{-}}}\;}\; \right)$ provide insights into the involvement of diffusible radicals and proton-equilibriums, explaining analogous outcomes in mOxPhos chemistry. Further, we demonstrate that superoxide (diffusible reactive oxygen species, DROS) enables in vitro ATP synthesis from ADP+phosphate, and show that this reaction is inhibited by CN. Therefore, practically instantaneous CN ion-radical interactions with DROS in matrix catalytically disrupt mOxPhos, explaining the acute lethal effect of CN.
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http://dx.doi.org/10.1515/bmc-2020-0004DOI Listing
March 2020

Acute toxicity of cyanide in aerobic respiration: Theoretical and experimental support for murburn explanation.

Biomol Concepts 2020 03 17;11(1):32-56. Epub 2020 Mar 17.

Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India-781039.

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http://dx.doi.org/10.1515/bmc-2020-0004DOI Listing
March 2020

Classical and murburn explanations for acute toxicity of cyanide in aerobic respiration: A personal perspective.

Toxicology 2020 02 30;432:152369. Epub 2020 Jan 30.

Satyamjayatu: The Science & Ethics Foundation, Snehatheeram, Kulappully, Shoranur-2 (PO), Kerala, 679122, India.

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http://dx.doi.org/10.1016/j.tox.2020.152369DOI Listing
February 2020

Murburn concept: a paradigm shift in cellular metabolism and physiology.

Biomol Concepts 2020 Jan 22;11(1):7-22. Epub 2020 Jan 22.

Satyamjayatu: The Science & Ethics Foundation,Snehatheeram, Kulappully, Shoranur-2 (PO), Kerala,India-679122.

Two decades of evidence-based exploratory pursuits in heme-flavin enzymology led to the formulation of a new biological electron/moiety transfer paradigm, called murburn concept. Murburn is a novel literary abstraction from "mured burning" or "mild unrestricted burning". This concept was invoked to explain the longstanding conundrum of maverick physiological dose responses and also applied to remodel the prevailing understanding of drug metabolism and cellular respiration. A conglomeration of simple ideas grounded in the known principles of thermodynamics and reaction chemistry, murburn concept invokes catalytic/functional roles for diffusible reactive species or radicals. Hitherto, diffusible reactive species were primarily seen as toxic agents of chaos, non-conducible to the maintenance of life-order. Since the murburn paradigm offers a distinctly different perspective for several biological phenomena, researchers holding conventional views of cellular metabolism pose a direct conflict of interests to the advancement of murburn concept. Murburn schemes are poised to integrate numerous metabolic motifs with holistic physiological outcomes; redefining pursuits in biology and medicine. To advance this agenda, I present a brief account of murburn concept and point out how redundant ideas are still advocated in some prestigious journals.
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http://dx.doi.org/10.1515/bmc-2020-0002DOI Listing
January 2020

Murburn concept: a paradigm shift in cellular metabolism and physiology.

Biomol Concepts 2020 Jan 22;11(1):7-22. Epub 2020 Jan 22.

Satyamjayatu: The Science & Ethics Foundation,Snehatheeram, Kulappully, Shoranur-2 (PO), Kerala, India-679122.

Two decades of evidence-based exploratory pursuits in heme-flavin enzymology led to the formulation of a new biological electron/moiety transfer paradigm, called murburn concept. Murburn is a novel literary abstraction from " ed ing" or " ild n estricted ing". This concept was invoked to explain the longstanding conundrum of maverick physiological dose responses and also applied to remodel the prevailing understanding of drug metabolism and cellular respiration. A conglomeration of simple ideas grounded in the known principles of thermodynamics and reaction chemistry, murburn concept invokes catalytic/functional roles for diffusible reactive species or radicals. Hitherto, diffusible reactive species were primarily seen as toxic agents of chaos, non-conducible to the maintenance of life-order. Since the murburn paradigm offers a distinctly different perspective for several biological phenomena, researchers holding conventional views of cellular metabolism pose a direct conflict of interests to the advancement of murburn concept. Murburn schemes are poised to integrate numerous metabolic motifs with holistic physiological outcomes; redefining pursuits in biology and medicine. To advance this agenda, I present a brief account of murburn concept and point out how redundant ideas are still advocated in some prestigious journals.
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http://dx.doi.org/10.1515/bmc-2020-0002DOI Listing
January 2020

Refutation of the cation-centric torsional ATP synthesis model and advocating murburn scheme for mitochondrial oxidative phosphorylation.

Biophys Chem 2020 02 18;257:106278. Epub 2019 Nov 18.

Satyamjayatu: The Science & Ethics Foundation, Shoranur-2 PO, Palakkad District, Kerala 679122, India. Electronic address:

The acclaimed explanation for mitochondrial oxidative phosphorylation (mOxPhos) is a proton or cation centric scheme. Such ideas were recently disclaimed and in lieu, an evidence-based oxygen-centric explanation, murburn concept, was proposed. The new understanding vouches for catalytic roles of diffusible reactive oxygen species (DROS). The involvement of DROS explains the "non-discoverability of an enzyme-linked high-energy phosphorylating intermediate", a historical predicament, which had fueled several trans-membrane potential (TMP) based mechano-electrical explanations like the Nath model. This communication aims to briefly apprise the readers some lacunae and inadmissible aspects of the Nath model and project the appeal of murburn scheme of mOxPhos.
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http://dx.doi.org/10.1016/j.bpc.2019.106278DOI Listing
February 2020

Chemiosmotic and murburn explanations for aerobic respiration: Predictive capabilities, structure-function correlations and chemico-physical logic.

Arch Biochem Biophys 2019 11 14;676:108128. Epub 2019 Oct 14.

Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, St. Institutskaya 3, 630090, Novosibirsk, Russia. Electronic address:

Since mid-1970s, the proton-centric proposal of 'chemiosmosis' became the acclaimed explanation for aerobic respiration. Recently, significant theoretical and experimental evidence were presented for an oxygen-centric 'murburn' mechanism of mitochondrial ATP-synthesis. Herein, we compare the predictive capabilities of the two models with respect to the available information on mitochondrial reaction chemistry and the membrane proteins' structure-function correlations. Next, fundamental queries are addressed on thermodynamics of mitochondrial oxidative phosphorylation (mOxPhos): (1) Can the energy of oxygen reduction be utilized for proton transport? (2) Is the trans-membrane proton differential harness-able as a potential energy capable of doing useful work? and (3) Whether the movement of miniscule amounts of mitochondrial protons could give rise to a potential of ~200 mV and if such an electrical energy could sponsor ATP-synthesis. Further, we explore critically if rotary ATPsynthase activity of Complex V can account for physiological ATP-turnovers. We also answer the question- "What is the role of protons in the oxygen-centric murburn scheme of aerobic respiration?" Finally, it is demonstrated that the murburn reaction model explains the fast kinetics, non-integral stoichiometry and high yield of mOxPhos. Strategies are charted to further demarcate the two explanations' relevance in the cellular physiology of aerobic respiration.
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http://dx.doi.org/10.1016/j.abb.2019.108128DOI Listing
November 2019

Aerobic Respiration: Criticism of the Proton-centric Explanation Involving Rotary Adenosine Triphosphate Synthesis, Chemiosmosis Principle, Proton Pumps and Electron Transport Chain.

Biochem Insights 2018 25;11:1178626418818442. Epub 2018 Dec 25.

Satyamjayatu: The Science & Ethics Foundation, Kulappully, Shoranur, India.

The acclaimed explanation for mitochondrial oxidative phosphorylation (mOxPhos, or cellular respiration) is a deterministic proton-centric scheme involving four components: Rotary adenosine triphosphate (ATP)-synthesis, Chemiosmosis principle, Proton pumps, and Electron transport chain (abbreviated as RCPE hypothesis). Within this write-up, the RCPE scheme is critically analyzed with respect to mitochondrial architecture, proteins' distribution, structure-function correlations and their interactive dynamics, overall reaction chemistry, kinetics, thermodynamics, evolutionary logic, and so on. It is found that the RCPE proposal fails to explain key physiological aspects of mOxPhos in several specific issues and also in holistic perspectives. Therefore, it is imperative to look for new explanations for mOxPhos.
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http://dx.doi.org/10.1177/1178626418818442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311555PMC
December 2018

Aerobic respiration: proof of concept for the oxygen-centric murburn perspective.

J Biomol Struct Dyn 2019 10 8;37(17):4542-4556. Epub 2019 Jan 8.

Department of Biotechnology, Indian Institute of Technology Guwahati , Guwahati , Assam, India.

The inner mitochondrial membrane protein complexes (I-V) and prokaryotic respiratory machinery are examined for a deeper understanding of their structure-function correlations and dynamics. analysis of the structure of complexes I-IV, docking studies and erstwhile literature confirm that they carry sites which are in close proximity to DROS (diffusible reactive oxygen species) generating redox centers. These findings provide supportive evidence for the newly proposed oxygen-centric chemical-coupling mechanism (murburn concept), wherein DROS catalyzes the esterification of inorganic phosphate to ADP. Further, in a reductionist system, we demonstrate that a DROS (like superoxide) can effectively esterify inorganic phosphate to ADP. The impact of these findings and the interactive dynamics of classical inhibitors (rotenone and cyanide), uncouplers (dinitrophenol and uncoupling protein) and other toxins (atractyloside and oligomycin) are briefly discussed. • Earlier perception: Complexes (I-IV) pump protons and Complex V make ATP (aided by protons) • Herein: Respiratory molecular machinery is probed for new structure-function correlations • Analyses: Quantitative arguments discount proton-centric ATP synthesis in mitochondria and bacteria • data: ADP-binding sites and O/ diffusible reactive oxygen species (DROS)-accessible channels are unveiled in respiratory proteins • data: Using luminometry, ATP synthesis is demonstrated from ADP, Pi and superoxide • Inference: Findings agree with decentralized ADP-Pi activation via oxygen-centric murburn scheme Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2018.1552896DOI Listing
October 2019

Murburn Concept: A Molecular Explanation for Hormetic and Idiosyncratic Dose Responses.

Dose Response 2018 Apr-Jun;16(2):1559325818774421. Epub 2018 May 9.

Satyamjayatu: The Science and Ethics Foundation, Kerala, India.

Recently, electron transfers and catalyses in a bevy of redox reactions mediated by hemeproteins were explained by murburn concept. The term "murburn" is abstracted from "" or "" and connotes a novel "" interaction paradigm. Quite unlike the genetic regulations and protein-level affinity-based controls that govern order and specificity/selectivity in conventional treatments, murburn concept is based on stochastic/thermodynamic regulatory principles. The novel insight necessitates a "reactivity outside the active-site" perspective, because select redox enzymatic activity is obligatorily mediated via diffusible radical/species. Herein, reactions employing key hemeproteins (as exemplified by CYP2E1) establish direct experimental connection between "additive-influenced redox catalysis" and "unusual dose responses" in reductionist and physiological milieu. Thus, direct and conclusive molecular-level experimental evidence is presented, supporting the mechanistic relevance of murburn concept in "maverick" concentration-based effects brought about by additives. Therefore, murburn concept could potentially explain several physiological hormetic and idiosyncratic dose responses.
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http://dx.doi.org/10.1177/1559325818774421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946624PMC
May 2018

Functioning of Microsomal Cytochrome P450s: Murburn Concept Explains the Metabolism of Xenobiotics in Hepatocytes.

Front Pharmacol 2016 23;7:161. Epub 2016 Jun 23.

REDOx Lab, PSG Institute of Advanced Studies Coimbatore, India.

Using oxygen and NADPH, the redox enzymes cytochrome P450 (CYP) and its reductase (CPR) work in tandem to carry out the phase I metabolism of a vast majority of drugs and xenobiotics. As per the erstwhile understanding of the catalytic cycle, binding of the substrate to CYP's heme distal pocket allows CPR to pump electrons through a CPR-CYP complex. In turn, this trigger (a thermodynamic push of electrons) leads to the activation of oxygen at CYP's heme-center, to give Compound I, a two-electron deficient enzyme reactive intermediate. The formation of diffusible radicals and reactive oxygen species (DROS, hitherto considered an undesired facet of the system) was attributed to the heme-center. Recently, we had challenged these perceptions and proposed the murburn ("mured burning" or "mild unrestricted burning") concept to explain heme enzymes' catalytic mechanism, electron-transfer phenomena and the regulation of redox equivalents' consumption. Murburn concept incorporates a one-electron paradigm, advocating obligatory roles for DROS. The new understanding does not call for high-affinity substrate-binding at the heme distal pocket of the CYP (the first and the most crucial step of the erstwhile paradigm) or CYP-CPR protein-protein complexations (the operational backbone of the erstwhile cycle). Herein, the dynamics of reduced nicotinamide nucleotides' consumption, peroxide formation and depletion, product(s) formation, etc. was investigated with various controls, by altering reaction variables, environments and through the incorporation of diverse molecular probes. In several CYP systems, control reactions lacking the specific substrate showed comparable or higher peroxide in milieu, thereby discrediting the foundations of the erstwhile hypothesis. The profiles obtained by altering CYP:CPR ratios and the profound inhibitions observed upon the incorporation of catalytic amounts of horseradish peroxidase confirm the obligatory roles of DROS in milieu, ratifying murburn as the operative concept. The mechanism of uncoupling (peroxide/water formation) was found to be dependent on multiple one and two electron equilibriums amongst the reaction components. The investigation explains the evolutionary implications of xenobiotic metabolism, confirms the obligatory role of diffusible reactive species in routine redox metabolism within liver microsomes and establishes that a redox enzyme like CYP enhances reaction rates (achieves catalysis) via a novel (hitherto unknown) modality.
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http://dx.doi.org/10.3389/fphar.2016.00161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918403PMC
July 2016

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 2016 Jun 10;125:91-111. Epub 2016 Mar 10.

CIDR-MCBL, Indian Institute of Science, Bengaluru, Karnataka 560012, India; Hemoproteins Lab, School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632014, India. Electronic address:

Background: Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016].

Methods: A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes.

Results: When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry.

Conclusion: It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations.

Significance: The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems.
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http://dx.doi.org/10.1016/j.biochi.2016.03.003DOI Listing
June 2016

Functioning of drug-metabolizing microsomal cytochrome P450s: In silico probing of proteins suggests that the distal heme 'active site' pocket plays a relatively 'passive role' in some enzyme-substrate interactions.

In Silico Pharmacol 2016 Dec 19;4(1). Epub 2016 Feb 19.

Formerly at PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004, India.

Purpose: The currently held mechanistic understanding of microsomal cytochrome P450s (CYPs) seeks that diverse drug molecules bind within the deep-seated distal heme pocket and subsequently react at the heme centre. To explain a bevy of experimental observations and meta-analyses, we indulge a hypothesis that involves a "diffusible radical mediated" mechanism. This new hypothesis posits that many substrates could also bind at alternate loci on/within the enzyme and be reacted without the pertinent moiety accessing a bonding proximity to the purported catalytic Fe-O enzyme intermediate.

Methods: Through blind and heme-distal pocket centered dockings of various substrates and non-substrates (drug molecules of diverse sizes, classes, topographies etc.) of microsomal CYPs, we explored the possibility of access of substrates via the distal channels, its binding energies, docking orientations, distance of reactive moieties (or molecule per se) to/from the heme centre, etc. We investigated specific cases like- (a) large drug molecules as substrates, (b) classical marker drug substrates, (c) class of drugs as substrates (Sartans, Statins etc.), (d) substrate preferences between related and unrelated CYPs, (e) man-made site-directed mutants' and naturally occurring mutants' reactivity and metabolic disposition, (f) drug-drug interactions, (g) overall affinities of drug substrate versus oxidized product, (h) meta-analysis of in silico versus experimental binding constants and reaction/residence times etc.

Results: It was found that heme-centered dockings of the substrate/modulator drug molecules with the available CYP crystal structures gave poor docking geometries and distances from Fe-heme centre. In conjunction with several other arguments, the findings discount the relevance of erstwhile hypothesis in many CYP systems. Consequently, the newly proposed hypothesis is deemed a viable alternate, as it satisfies Occam's razor.

Conclusions: The new proposal affords expanded scope for explaining the mechanism, kinetics and overall phenomenology of CYP mediated drug metabolism. It is now understood that the heme-iron and the hydrophobic distal pocket of CYPs serve primarily to stabilize the reactive intermediate (diffusible radical) and the surface or crypts of the apoprotein bind to the xenobiotic substrate (and in some cases, the heme distal pocket could also serve the latter function). Thus, CYPs enhance reaction rates and selectivity/specificity via a hitherto unrecognized modality.
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http://dx.doi.org/10.1186/s40203-016-0016-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760962PMC
December 2016

Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

Biochem Biophys Res Commun 2014 Dec 6;455(3-4):190-3. Epub 2014 Nov 6.

REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004, India. Electronic address:

The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.
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http://dx.doi.org/10.1016/j.bbrc.2014.10.137DOI Listing
December 2014

The curious case of benzbromarone: insight into super-inhibition of cytochrome P450.

PLoS One 2014 3;9(3):e89967. Epub 2014 Mar 3.

REDOx Lab, Biotechnology and Healthcare Division, PSG Institute of Advanced Studies, Peelamedu, Tamil Nadu, India.

Cytochrome P450 (CYP) family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr) and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing "super-inhibition" phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS) effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents) is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089967PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940698PMC
January 2015

Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay.

Biochem Biophys Res Commun 2012 Mar 9;419(2):211-4. Epub 2012 Feb 9.

Heme & Flavo Proteins Laboratory, #204, Center for Biomedical Research, VIT University, Vellore, Tamil Nadu 632014, India.

We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding - (1) the promiscuous role of cytochrome b(5) in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.
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http://dx.doi.org/10.1016/j.bbrc.2012.01.149DOI Listing
March 2012

What is the Functional Role of N-terminal Transmembrane Helices in the Metabolism Mediated by Liver Microsomal Cytochrome P450 and its Reductase?

Cell Biochem Biophys 2012 Feb 1. Epub 2012 Feb 1.

Heme & Flavo Proteins Laboratory, Center for Biomedical Research, VIT University, #204, Vellore, 632014, Tamil Nadu, India.

We sought to clarify on the hitherto unresolved role of N-terminal transmembrane segments (TMS) of cytochrome P450 (CYP) and its' reductase (CPR) in protein interaction/catalysis. TMS analyses show little evolutionary conservation in CYPs. The conserved CPR's TMS poses limited scope for predictable/consistent hetero-recognition with the wide bevy of CYPs' TMS, as evident from preliminary analyses and TMhit server predictions for inter-helical binding. Further, experimentations with four different CPR preparations (preps) and two liver microsomal CYPs (2C9 and 2E1) shows that the hydroxylated product formation rate is not quantitatively correlated to the extent of integrity of the CPR N-terms. Incorporation of cytochrome b (5) in some reactions afforded similar rates while employing either fully intact or partially intact CPR. A survey of literature shows that liver microsomal CYPs function quite well even without the TMS or with significantly altered TMS. These observations negate the hypothesis that N-term TMS of CPR or CYP is obligatory for CYP-CPR interaction and catalysis. Also, in CYP2E1-mediated hydroxylation of para-nitrophenol, the extent of intactness or truncation did not significantly affect the CPR preps' catalytic role at very low or high substrate concentrations. To interpret these results, we draw support from recently published research on reduced nicotinamide adenide dinucleotide phosphate oxidase (Takac et al., J Biol Chem, 286:13304-13313, 2011) and from our pertinent earlier works. We infer that CPR' free TMS segment could alter the diffusible reactive oxygen species' dynamics in the microenvironment, thereby altering the reaction outcome. Based on the evidence, we conclude that TMS merely facilitates "interaction/catalysis" by anchoring the CYP and CPR in the lipid interface.
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http://dx.doi.org/10.1007/s12013-012-9339-0DOI Listing
February 2012

Traces of certain drug molecules can enhance heme-enzyme catalytic outcomes.

Biochem Biophys Res Commun 2012 Jan 26;417(3):1041-5. Epub 2011 Dec 26.

Heme & Flavo Proteins Laboratory, Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, India.

We report that trace amounts (nano- to picomolar concentrations) of dapsone and amiodarone in reaction mixtures of structurally and functionally distinct heme enzymes gave increased product formation rates from diverse substrates. These enhancements are found to be lowered by mild radical quenchers; a lowering well differentiated from heme active site inhibitions. Based on the non-specific nature of the activations in the diverse heme enzyme reactions studied, a generic mechanism is proposed to explain additive-based activity enhancements in heme enzymology.
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http://dx.doi.org/10.1016/j.bbrc.2011.12.090DOI Listing
January 2012
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