Diffusible Reactive Oxygen Species Research Articles

What Is the Relevance of Murburn Concept in Thalassemia and Respiratory Diseases?

Murburn concept is a novel perspective for understanding cellular function, deeming cells as simple chemical engines (SCE) that are powered by redox reactions initiated by effective charge separation (ECS). The 1-electron active diffusible reactive (oxygen) species, or DR(O)S, equilibriums involved in these processes are also crucial for homeostasis, coherently networking cells, and rendering electromechanical functions of sensing and responding to stimuli. This perspective presents the true physiological function of oxygen, which is to enable ECS and the generation of DR(O)S. Therefore, DR(O)S must now to be seen as the quintessential elixir of life, although they might have undesired effects (i.e., the traditionally perceived oxidative stress) when present in the wrong amounts, places and times. We also elaborated that tetrameric hemoglobin (Hb) is actually an ATP-synthesizing murzyme (an enzyme working via murburn concept) and postulated that several post-translational modifications (such as glycation) on Hb could result from murburn activity. Murburn perspective has also enabled the establishment of a facile rationale explaining the sustenance of erythrocytes for 3–4 months, despite their lacking nucleus or mitochondria (to coordinate their various functions and mass-produce ATP, respectively). Although thalassemia has its roots in genetic causation, the new awareness of the mechanistic roles of oxygen-hemoglobin-erythrocyte trio significantly impacts our approaches to interpreting research data and devising therapies for this malady. These insights are also relevant in other clinical manifestations that involve respiratory distress (such as asthma, lung cancer, COVID-19 and pneumonia) and mitochondrial diseases. Herein, these contexts and developments are briefly discussed.

Structural foundations for explaining the physiological roles of murzymes embedded in diverse phospholipid membranes

The advent of improved structural biology protocols and bioinformatics methodologies have provided paradigm-shifting insights on metabolic or physiological processes catalyzed by homo−/hetero- proteins (super)complexes embedded in phospholipid membranes of cells/organelles. In this panoramic review, we succinctly elucidate the structural features of select redox proteins from four systems: hepatocyte/adrenal cortex endoplasmic reticulum (microsomes), inner mitochondrial membrane (cristae), thylakoid membrane (grana), and in the flattened disks of rod/cone cells (in retina). Besides catalyzing fast/crucial (photo)chemical reactions, these proteins utilize the redox-active diatomic gaseous molecule of oxygen, the elixir of aerobic life. Quite contrary to extant perceptions that invoke primarily deterministic affinity-binding or conformation-change based “proton-pump”/“serial electron-relay” type roles, we advocate murzyme functions for the membrane-embedded proteins in these systems. Murzymes are proteins that generate/stabilize/utilize diffusible reactive (oxygen) species (DRS/DROS) based activities. Herein, we present a brief compendium of the recently revealed wealth of structural information and mechanistic concepts on how the membrane proteins use DRS/DROS to aid ‘effective charge separation’ and facilitate trans-membrane dynamics of diverse species in milieu, thereby enabling the cells to function as ‘simple chemical engines’.

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

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

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

We recently proposed a diffusible reactive (oxygen) species (DRS/DROS) based function for cytochrome b complexes (CBC) and quinones (Q)/quinols (QH2 ) 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 O2 , DRS, Q/QH2 , 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 (QH2 ) in bioenergetic membranes. In the classical model, QH2 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*-, O2 *-, etc., also called diffusible reactive species, DRS) as wasteful or toxic (patho) physiological manifestations. It is highlighted herein that QH2 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.

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

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 O2 /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 (H2 O2 /H2 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.

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

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 pO2 by cancer cells) and approach for cancer therapy.

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

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 b 6 f 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. In toto, the work provides a new orientation and impetus to photosynthesis research. Communicated by Ramaswamy H. Sarma

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

In this first part of our essay on oxygenic photosynthesis, we address the various aspects of classical and murburn explanations. We had recently pointed out that the classical explanations of- trans-membrane potential (TMP)-based chemiosmotic rotary ATP synthesis, quinone-cycle at cytochrome b 6 f, chartered electron transport by plastocyanin, and modality of light’s interaction with chloroplast pigments- are untenable. In lieu, we proposed a diffusible reactive (oxygen) species (DROS)-based murburn model. In continuum, herein, we assess the operational viability of the cyclic oxygenesis and electron transport chain (ETC) explanations for oxygenic photosynthesis, in the light of updated structural/mechanistic information. Further, thermodynamically validated murburn equations of photolysis-photophosphorylation (Pl-Pp) are provided, along with the rationale for organelle-homeostasis. We propose that the photo-excitation of various pigments leads to the formation of aquated electrons (eaq) and DROS in milieu, which are stabilized by a pool of redox-active elements within chloroplasts. Subsequently, the ‘eaq+DR(O)S’ pool is utilized stochastically via disordered/parallel reactions at the stacked thylakoid membrane interface. Effective charge separation by ‘photosystem switches’ enables delocalized oxygenesis, NAD(P) reduction and ADP phosphorylation. Heat/TMP/DROS generation is also an outcome of this process. Finally, we compare the murburn and classical models of Pl-Pp and delineate agendas for their ratification/falsification. Communicated by Ramaswamy H. Sarma 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.

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

Blood hemoglobin (Hb), known to transport oxygen, is the most abundant globular protein in humans. Erythrocytes have ∼10−3 M concentration of ATP in steady-state and we estimate that this high amounts cannot be formed from 10−4 − 10−7 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

Murburn Precepts for Cytochrome P450 Mediated Drug/Xenobiotic Metabolism and Homeostasis.

We aim to demonstrate why deeming diffusible reactive oxygen species (DROS) as toxic wastes do not afford a comprehensive understanding of cytochrome P450 mediated microsomal xenobiotic metabolism (mXM). Current pharmacokinetic investigations consider reactive oxygen species formed in microsomal reactions as toxic waste products, whereas our works (Manoj et al., 2016) showed that DROS are the reaction mainstay in cytochrome P450 mediated metabolism and that they play significant roles in explaining several unexplained physiologies. Herein, we strive to detail the thermodynamic and kinetic foundations of murburn precepts of cytochrome P450 mediated drug metabolism. Primarily, in silico approaches (using pdb crystal structure files), murburn reaction chemistry logic and thermodynamic calculations to elucidate the new model of CYP-mediated drug metabolism. The theoretical foundations are used to explain experimental observations. We visually elucidate how murburn model better explains- (i) promiscuity of the unique P450-reductase; (ii) prolific activity and inhibitions of CYP3A4; (iii) structure-function correlations of important key CYP2 family isozymes- 2C9, 2D6 and 2E1; and (iv) mutation studies and mechanism-based inactivation of CYPs. Several other miscellaneous aspects of CYP reaction chemistry are also addressed. In the light of our findings that DROS are crucial for explaining reaction outcomes in mXM, approaches for understanding drug-drug interactions and methodologies for lead drug candidates' optimizations should be revisited.

Light's interaction with pigments in chloroplasts: The murburn perspective

The prevailing understanding on the light reaction of oxygenic photosynthesis (photolytic photophosphorylation & NADPH synthesis) considers the vast majority of the diverse pigments, chlorophyll binding proteins (CBPs) and light harvesting complexes (LHCs) as photon-energy relaying facets; only the chlorophyll a couplets at the reaction center of the two photosystems I & II are deemed to serve as photo-excitable electron emitters. Highlighting the historical perspectives involved, we present reasons why this conventional perception is unmet by theoretical foundations, unsupported by molecular awareness on the various pigments and unverified by physiological data available on chloroplasts. Further, we propose a simple diffusible reactive oxygen species (DROS)-based mechanism for correlating the functions of various light harvesting LHCs and CBPs with the reaction centers of Photosystems I & II. The projected hypothesis may also provide a new perspective to photoreception research.

The murburn precepts for photoreception

Murburn concept is a redox mechanistic scheme involving interactive equilibriums of discretized or organized protein/substrate molecules, unbound ions and radicals (or reactive species); which may afford selective/specific electron transfers, particularly at phospholipid interfaces. Earlier, we have applied murburn concept to provide thermodynamically and kinetically viable explana-tions for various physiological/bioenergetic routines like xenobiotic metabolism, unusual dose responses, aerobic respiration, thermogenesis, homeostasis, trans-membrane potential, oxygenic photosynthesis, etc. While proposing the murburn model for photophosphorylation, we had projected that the murburn mechanism could also be relevant for photoreception physiology. Herein, we expand on this aspect and present the basic scheme and evidence in support of the murburn model of photorecep-tion, with retinal/opsin as the salient photon-impingement response-transducing element. In alignment with our earlier murburn schemes, we propose that diffusible reactive oxygen species (DROS, as exemplified by superoxide, which is currently deemed a toxic product of all-trans retinal and NADPH oxidase interactions) is produced in rod/cone cells upon photoactivation and it plays a crucial role in the visual cycle. This is supported by several facts: (i) Layers of photoreceptive neural cells precede the rod/cone cells (with respect to the presentation to oncoming light ray/photon), (ii) There exists high oxygen demand in the retina, (iii) Copious DROS are detected in functional retina, (iv) NAD(P)H/reductase is involved in the cycle, and (v) Events occur at sub-micrometer dimensioned phospholipid disks stacked to stabilize DROS and minimize free protons (quite like the thylakoids that harbor carotenoids in chloroplasts and cyanobacteria). In the new scheme, photo-electric activation leads to charge sepa-ration and hyperpolarization in rod/cone cells due to negative charge build-up. This electro-chemical signaling serves as the front-runner to trigger an action potential relay along a neuron and the superoxide mediated phosphorylation of GDP bound to transducin serves as the initiator of signal transduction cascade. The newly proposed scheme allows a simple electrical connec-tivity of the retina-photoreceptors with the brain via the optic nerve, is anatomically correlated with the structure and resolution of the retina, and is kinetically viable.

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

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.

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

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. In silico 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.Highlights• 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• In silico data: ADP-binding sites and O2/ diffusible reactive oxygen species (DROS)-accessible channels are unveiled in respiratory proteins• In vitro data: Using luminometry, ATP synthesis is demonstrated from ADP, Pi and superoxide• Inference: Findings agree with decentralized ADP–Pi activation via oxygen-centric murburn schemeCommunicated by Ramaswamy H. Sarma

Murburn scheme for mitochondrial thermogenesis

The physiology of thermogenesis in mitochondria (mediated by uncoupling protein, UCP) has traditionally been explained as the dissipation of proton gradient across the inner mitochondrial membrane into heat. However, there are differences of opinion on how thermogenesis is achieved by UCPs and the mechanistic theories have not been correlated sufficiently with UCP`s structure. Recent experimental evidence suggests strong correlation of diffusible reactive oxygen species (DROS) with UCP-induced thermogenesis. Further, the mechanistic explanations of mitochondrial oxidative phosphorylation (mOxPhos) were recently revamped with murburn concept, which considers DROS as an obligatory catalytic agent in mOxPhos. Herein, we propose that UCPs (aided by the large pore and positively charged amino acids of aqueous-phase loops) enable protonation and transport of DROS. Thus, UCP facilitates DROS-reactions amongst themselves, forming water and liberating heat around the inner mitochondrial membrane. Thereby, the simple murburn scheme for biothermogenesis integrates structural information of UCP with its attributed physiological function.

Debunking Chemiosmosis and Proposing Murburn Concept as the Operative Principle for Cellular Respiration

Via a concomitant communication (the first part of my work), I have conclusively debunked the prevailing explanations for mitochondrial oxidative phosphorylation and established the need for a novel rationale to account for the reaction paradigm. Towards the same, murburn concept is hereby floated as a viable explanation (in the second part of my work). It is proposed that the inner mitochondrial membrane (harboring the various metal and flavin enzyme complexes) serves as means to confine and stabilize radical reactions, which effectively couple and bring about ATP synthesis in the proton-deficient microcosm. The proposed scheme is un-ordered and favored by Ockham's razor and evolutionary perspectives. Murburn concept is a paradigm-shift in biochemistry because it advocates that diffusible reactive (oxygen) species are mainstay of routine cellular metabolic process within the mitochondria.

Electron transfer pathways analysis of oxygen tolerant [NiFe]-hydrogenases for hydrogen production: A quantum mechanics/molecular mechanics – statistical coupled analysis

Biotechnological hydrogen production by enzymes is a promising alternative energy source, either by direct generation of biogas or coupled to fuel cells. Nevertheless, most of the hydrogenase enzymes are easily inactivated by traces of O2, generating spectroscopically detectable non-catalytic species, diffusible reactive oxygen species, or directly by oxidation of the protein matrix. To implement technologies based on hydrogenases, the atomic and electronic features that rule the electron transfer process still need to be identified and understood. We studied the electron transfer pathways between the metal cluster and protein matrix of [NiFe]-hydrogenase from Salmonella enterica using multi-level molecular simulation: all-atom molecular dynamics, quantum mechanics/molecular mechanics, and with bioinformatics tools, such as statistical coupled analysis, to identify key residues of the process. The identification of these residues could be a more advantageous alternative compared with random point mutations, in order to improve catalytic performance or operational stability by genetic engineering.

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

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.

DNA damage by histone radicals in nucleosome core particles.

Although DNA binding proteins shield the genetic material from diffusible reactive oxygen species by reacting with them, the resulting protein (peroxyl) radicals can oxidize the bound DNA. To explore this possible DNA damage by protein radicals, histone H4 proteins containing an azoalkane radical precursor at defined sites were prepared. Photolysis of a nucleosome core particle containing the modified protein produces DNA damage that is consistent with selective C4′-oxidation. The nucleotide(s) damaged is highly dependent on proximity to the protein radical. These experiments provide insight into the effects of oxidative stress on protein-bound DNA, revealing an additional layer of complexity concerning nucleic acid damage.

The Curious Case of Benzbromarone: Insight into Super-Inhibition of Cytochrome P450

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.