Quinone Reduction Research Articles

Association of NQO2 With UDP-Glucuronosyltransferases Reduces Menadione Toxicity in Neuroblastoma Cells.

The balance between detoxification and toxicity is linked to enzymes of the drug metabolism Phase I (cytochrome P450 or oxidoreductases) and phase II conjugating enzymes (such as the UGTs). After the reduction of quinones, the product of the reaction, the quinols—if not conjugated—re-oxidizes spontaneously to form the substrate quinone with the concomitant production of the toxic reactive oxygen species (ROS). Herein, we documented the modulation of the toxicity of the quinone menadione on a genetically modified neuroblastoma model cell line that expresses both the quinone oxidoreductase 2 (NQO2, E.C. 1.10.5.1) alone or together with the conjugation enzyme UDP-glucuronosyltransferase (UGT1A6, E.C. 2.4.1.17), one of the two UGT isoenzymes capable to conjugate menadione. As previously shown, NQO2 enzymatic activity is concomitant to massive ROS production, as previously shown. The quantification of ROS produced by the menadione metabolism was probed by electron-paramagnetic resonance (EPR) on cell homogenates, while the production of superoxide was measured by liquid chromatography coupled to mass spectrometry (LC-MS) on intact cells. In addition, the dysregulation of the redox homeostasis upon the cell exposure to menadione was studied by fluorescence measurements. Both EPR and LCMS studies confirmed a significant increase in the ROS production in the NQO2 overexpressing cells due to the fast reduction of quinone into quinol that can re-oxidize to form superoxide radicals. However, the effect of NQO2 inhibition was drastically different between cells overexpressing only NQO2 vs. both NQO2 and UGT. Whereas NQO2 inhibition decreases the amount of superoxide in the first case by decreasing the amount of quinol formed, it increased the toxicity of menadione in the cells co-expressing both enzymes. Moreover, for the cells co-expressing QR2 and UGT the homeostasis dysregulation was lower in presence of menadione than for the its counterpart expressing only QR2. Those results confirmed that the cooperation of the two enzymes plays a fundamental role during the cells’ detoxification process. The fluorescence measurements of the variation of redox homeostasis of each cell line and the detection of a glucuronide form of menadiol in the cells co-expressing NQO2 and UGT1A6 enzymes further confirmed our findings.

A Quinol Anion as Catalytic Intermediate Coupling Proton Translocation With Electron Transfer in E. coli Respiratory Complex I.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in Escherichia coli complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213GH variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed.

Redox- and protonation-state driven substrate-protein dynamics in respiratory complex I

Respiratory complex I is a key enzyme in the electron transport chains of mitochondria and bacteria. It transfers two electrons to quinone and couples this redox reaction to proton pumping to electrically charge the membrane it is embedded in. The charge and pH gradient across the membrane drives the synthesis of ATP. The redox reaction and proton pumping in complex I are separated in space and time, which raises the question of how the two reactions are coupled so efficiently. Here, we focus on the unique ∼35 Å long tunnel of complex I, which houses the binding site of quinone reduction. We discuss the redox and protonation reactions that occur in this tunnel and how they influence the dynamics of protein and substrate. On the basis of recent structural data and results from molecular simulations, we review how quinone reduction and dynamics may be coupled to proton pumping in complex I.

Architecture of the mycobacterial succinate dehydrogenase with a membrane-embedded Rieske FeS cluster

Complex II, also known as succinate dehydrogenase (SQR) or fumarate reductase (QFR), is an enzyme involved in both the Krebs cycle and oxidative phosphorylation. Mycobacterial Sdh1 has recently been identified as a new class of respiratory complex II (type F) but with an unknown electron transfer mechanism. Here, using cryoelectron microscopy, we have determined the structure of Mycobacterium smegmatis Sdh1 in the presence and absence of the substrate, ubiquinone-1, at 2.53-Å and 2.88-Å resolution, respectively. Sdh1 comprises three subunits, two that are water soluble, SdhA and SdhB, and one that is membrane spanning, SdhC. Within these subunits we identified a quinone-binding site and a rarely observed Rieske-type [2Fe-2S] cluster, the latter being embedded in the transmembrane region. A mutant, where two His ligands of the Rieske-type [2Fe-2S] were changed to alanine, abolished the quinone reduction activity of the Sdh1. Our structures allow the proposal of an electron transfer pathway that connects the substrate-binding and quinone-binding sites. Given the unique features of Sdh1 and its essential role in Mycobacteria, these structures will facilitate antituberculosis drug discovery efforts that specifically target this complex.

Redox loops in anaerobic respiration - The role of the widespread NrfD protein family and associated dimeric redox module

In prokaryotes, the proton or sodium motive force required for ATP synthesis is produced by respiratory complexes that present an ion-pumping mechanism or are involved in redox loops performed by membrane proteins that usually have substrate and quinone-binding sites on opposite sides of the membrane. Some respiratory complexes include a dimeric redox module composed of a quinone-interacting membrane protein of the NrfD family and an iron‑sulfur protein of the NrfC family. The QrcABCD complex of sulfate reducers, which includes the QrcCD module homologous to NrfCD, was recently shown to perform electrogenic quinone reduction providing the first conclusive evidence for energy conservation among this family. Similar redox modules are present in multiple respiratory complexes, which can be associated with electroneutral, energy-driven or electrogenic reactions. This work discusses the presence of the NrfCD/PsrBC dimeric redox module in different bioenergetics contexts and its role in prokaryotic energy conservation mechanisms.

P<i>K</i><sub>a</sub> Determination of Strongly Acidic C-H Acids Bearing a (Perfluoroalkyl)sulfonyl Group in Acetonitrile by Means of Voltammetric Reduction of Quinone

A voltammetric method has been developed for the determination of the acid dissociation constant (pKa) of (RfSO2)2CHR (Rf = perfluoroalkyl), which is a strongly acidic molecule that serves as an acid catalyst. In acetonitrile solution containing small quantity of the acid, the reduction peak potential of quinone caused by the acid shifted to a more positive side accompanied by an increase in the acidity of the acid. This relationship was in good agreement with an equation derived from the Nernst equation. The present finding has been successfully applied to the determination of the pKa of the acids in acetonitrile with values ranging from 7 to 17.

On the free radical redox chemistry of 5,6-dihydroxyindole

The poly-indolequinone polymers such as poly-dopamine and melanin are significant materials for advanced soft materials in coatings and devices. In order to make progress towards a holistic thermodynamic model, one can consider the thermodynamics of the redox chemistry of a key monomer building block: 5,6-dihydroxyindole. The work presented here indicates a new observation, that the quinone methide tautomer plays a key role in semiquinone free radical production. The modelling includes a new set of pH dependent, reduction potential equations to account for tautomerisation. Furthermore, presented are estimations for various thermodynamic variables. The 1 electron potential for the quinone reduction is between 0.14 and 0.17 V (NHE); Semiquinone reduction is between −0.10 to −0.068 (NHE); Tautomarisation constant is estimated to be 0.99 to 3.9; Comproportionation formation constant is estimated to be between 3.8 × 103 and 50 × 103. The work presented here may form the basis for understanding the charge transport properties for poly-indolequinones.

Catalytic Reactions and Energy Conservation in the Cytochrome bc1 and b6f Complexes of Energy-Transducing Membranes.

This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc1 and b6f (Cytbc1/b6f) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cytsbc1/b6f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cytbc1/b6f, combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc1/b6f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.

Backbone assignment of E. coli NfsB and the effects of addition of the cofactor analogue nicotinic acid

E. coli nitroreductase NfsB (also called NfnB) has been studied extensively, largely due to its potential for cancer gene therapy. A homodimeric flavoprotein of 216 residues, it catalyses the reduction of nitroaromatics to cytotoxic hydroxylamines by NADH and NADPH and also the reduction of quinones to hydroxyquinones. Its role in vivo is not known but it is postulated to be involved in reducing oxidative stress. The crystal structures of the wild type protein and several homologues have been determined in the absence and presence of ligands, including nicotinate as a mimic of the headpiece of the nicotinamide cofactors. There is little effect on the overall structure of the protein on binding ligands, but, from the B factors, there appears to be a decrease in mobility of 2 helices near the active site. As a first step towards examining the dynamics of the protein in solution with and without ligand, we have assigned the backbone 13C, 15N, and 1HN resonances of NfsB and examined the effect of the binding of nicotinate on the amide 15N, and 1HN shifts.

Metal-free reduction of unsaturated carbonyls, quinones, and pyridinium salts with tetrahydroxydiboron/water.

A series of unsaturated carbonyls, quinones, and pyridinium salts have been effectively reduced to the corresponding saturated carbonyls, dihydroxybenzenes, and hydropyridines in moderate to high yields with tetrahydroxydiboron/water as a mild, convenient, and metal-free reduction system. Deuterium-labeling experiments have revealed this protocol to be an exclusive transfer hydrogenation process from water.

(Keynote) DT-Diaphorase As a Promising Catalytic Label for Rapid and Sensitive Immunosensors

The most common enzyme labels in biosensors are alkaline phosphatase (ALP) and horseradish peroxidase (HRP), which, however, have some limitations for use in electrochemical immunosensors. DT-diaphorase (DT-D) is a redox enzyme that catalyzes a two-electron reduction of quinone in the presence of NADH or NADPH. Importantly, DT-D from Bacillus stearothermophilus (EC 1.6.99.-) has a low molecular weight (30 kDa) and high thermal and long-term stability. Nevertheless, DT-D has never been employed as a catalytic label in biosensors. In this presentation, we report that DT-D can be used as a bifunctional enzyme label for rapid and sensitive electrochemical immunosensors: DT-D can convert an electrochemically inactive substrate into an electrochemically active product and DT-D can participate in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling induced by the product. We found that DT-D has high catalytic activities for nitroso reduction, phosphoester hydrolysis, and carboxyl ester hydrolysis as well as quinone reduction in the presence of NADH. 4-Nitroso-1-naphthol, 1-amino-2-naphthyl phosphate, and 4-aminonaphthalene-1-yl acetate are used as electrochemically inactive substrates for corresponding enzymatic reactions, which are converted into electrochemically active aminohydroxy-naphthalenes. This combination of DT-D and the substrates allows for high electrochemical signal-to-background ratios, because (i) the catalytic reaction by DT-D is fast, (ii) the electrochemical oxidation of the aminohydroxy-naphthalenes is rapid, even at an indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The electrochemical immunosensor using DT-D and 4-nitroso-1-naphthol detects parathyroid hormone with a low detection limit of 2 pg/mL, the immunosensor using DT-D and 1-amino-2-naphthyl phosphate detects outer membrane vesicles from Escherichia coli with a detection limit of 8 ng/mL, and the immunosensor using DT-D and 4-aminonaphthalene-1-yl acetate detects thyroid-stimulating hormone with a detection limit of ~2 pg/mL. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common ALP and HRP.

Template-Free Growth of Vertical Poly(3,4-ethylenedioxythiophene) Fiber Array By Alternating Current-Bipolar Electrolysis

Conducting polymers (CPs) play an important role in the field of organic device. Electropolymerization is a very common technique for obtaining CPs, which usually gives a CP film with granular or particulate morphology on the electrode. On the other hand, CP fibers are also of great interest due to their unique properties such as efficient anisotropic carrier mobility. The electropolymerization with a template-modified electrode is regarded as one of the most promising strategies to obtain a CP fiber array. However, the attachment and removal of templates require additional working processes, and the use of a template may hinder other types of surface modification on the substrates. Therefore, a facile way to prepare CP fiber arrays without a template remains a challenging problem.The use of an electric field as a guide for polymer formation is deemed to be efficient to grow CP fibers in a specific direction. We have pioneered the electric field-guided growth of poly(3,4-ethylenedioxythiophene) (PEDOT) fibers. In that system, dendritic PEDOT fibers grew from the terminal of Au wires that were used as bipolar electrodes (BPEs) by the electropolymerization of 3,4-ethylenedioxythiophene (EDOT) under an alternating current (AC).1 Here, we introduce the next progress of a successful template-free fabrication of PEDOT fiber arrays from a flat electrode surface using BPEs under an AC condition (Figure 1).2 An acetonitrile solution containing 50 mM EDOT, 5 mM 1,4-benzoquinone (BQ) and 1 mM Bu4NClO4 was used as an electrolyte. A cylindrical setup with driving electrodes was used here, and a conductive plate acted as a BPE. When an iterative potential was applied to the BPE, the circular anodic or cathodic area appeared alternatively underneath the region covered by the cylinder, with a surrounding cathodic or anodic area simultaneously. A potential difference generated on the BPE surface (Delta V BPE) under an external electric field offers redox reactions on the BPE. When Delta V BPE exceeds Delta V min (1.82 V), the oxidation of EDOT proceeds in the anodic area, while the sacrificial reduction of benzoquinone occurs in the cathodic area.Vertical fiber arrays of PEDOT and its derivatives were successfully grown from the central area of various BPE surfaces by the AC-bipolar electrolysis. The growth of fiber array follows a particle-induced growth mechanism. First, anodic oxidation of EDOT gives a PEDOT film on the BPE surface when the voltage between driving electrodes generates a sufficient Delta V BPE. The film growth proceeds with Stranski-Krastanov (S-K) mode as an induction period to give several particles. Once particles are formed, the deposited particles are considered as nodes to initiate subsequent PEDOT fiber growth.Experimental conditions significantly affected the formation of fiber arrays. Higher applied voltages and shorter gap distances between the BPE and the cylinder were beneficial to form fiber arrays, which was supported by the computational simulation using COMSOL Multiphysics. Higher monomer concentration and lower applied frequency were also in favor of fiber array formation. The choice of solvents and electrolytes also influenced morphology of the fiber arrays. In addition, linear fibers were easily fabricated using a cylinder with a smaller diameter, while branching fibers formed with a larger diameter. This new methodology provides an extremely simple and straightforward way to fabricate uniform fiber arrays without the use of template, which are potentially applicable for sensors and electronic devices. References (1) Y. Koizumi, N. Shida, M. Ohira, H. Nishiyama, I. Tomita, S. Inagi, Nat. Commun., 2016, 7, 10404.(2) Y. Zhou, N. Shida, Y. Koizumi, T. Watanabe, H. Nishiyama, I. Tomita, S. Inagi, J. Mater. Chem. C, 2019, 7, 14745. Figure 1

Secrets of a proton pumping machine

Structural Biology Mitochondrial complex I serves as a primary entry point for electrons from the tricarboxylic acid cycle into the mitochondrial electron transport chain. This massive, membrane-embedded protein complex must couple quinone reduction to conformational changes across more than 150 angstroms within four separate proton pumps. Kampjut et al. determined five structures of complex I in states along the catalytic cycle, a deactive conformation, and one with the inhibitor rotenone bound. The resolution of some structures was sufficient to see water molecules and to trace putative paths for proton transfer within the proton-pumping membrane domain. The structures add valuable details that provide a basis for generating mechanistic hypotheses for this crucial complex. Science , this issue p. [eabc4209][1] [1]: /lookup/doi/10.1126/science.abc4209

Investigating the amino acid sequences of membrane bound dihydroorotate:quinone oxidoreductases (DHOQOs): Structural and functional implications

Dihydroorotate:quinone oxidoreductases (DHOQOs) are membrane bound enzymes responsible for oxidizing dihydroorotate (DHO) to orotate with concomitant reduction of quinone to quinol. They have FMN as prosthetic group and are part of the monotopic quinone reductase superfamily. These enzymes are also members of the dihydroorotate dehydrogenases (DHODHs) family, which besides membrane bound DHOQOs, class 2, includes soluble enzymes which reduce either NAD+ or fumarate, class 1. As key enzymes in both the de novo pyrimidine biosynthetic pathway as well as in the energetic metabolism, inhibitors of DHOQOs have been investigated as leads for therapeutics in cancer, immunological disorders and bacterial/viral infections. This work is a thorough bioinformatic approach on the structural conservation and taxonomic distribution of DHOQOs. We explored previously established structural/functional hallmarks of these enzymes, while searching for uncharacterized common elements. We also discuss the cellular role of DHOQOs and organize the identified protein sequences within six sub-classes 2A to 2F, according to their taxonomic origin and sequence traits. We concluded that DHOQOs are present in Archaea, Eukarya and Bacteria, including the first recognition in Gram-positive organisms. DHOQOs can be the single dihydroorotate dehydrogenase encoded in the genome of a species, or they can coexist with other DHODHs, as the NAD+ or fumarate reducing enzymes. Furthermore, we show that the type of catalytic base present in the active site is not an absolute criterium to distinguish between class 1 and class 2 enzymes. We propose the existence of a quinone binding motif (“ExAH”) adjacent to a hydrophobic cavity present in the membrane interacting N-terminal domain.

The coupling mechanism of mammalian respiratory complex I.

Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo-electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.

Quinone transport in the closed light-harvesting 1 reaction center complex from the thermophilic purple bacterium Thermochromatium tepidum

Redox-active quinones play essential roles in efficient light energy conversion in type-II reaction centers of purple phototrophic bacteria. In the light-harvesting 1 reaction center (LH1-RC) complex of purple bacteria, QB is converted to QBH2 upon light-induced reduction and QBH2 is transported to the quinone pool in the membrane through the LH1 ring. In the purple bacterium Rhodobacter sphaeroides, the C-shaped LH1 ring contains a gap for quinone transport. In contrast, the thermophilic purple bacterium Thermochromatium (Tch.) tepidum has a closed O-shaped LH1 ring that lacks a gap, and hence the mechanism of photosynthetic quinone transport is unclear. Here we detected light-induced Fourier transform infrared (FTIR) signals responsible for changes of QB and its binding site that accompany photosynthetic quinone reduction in Tch. tepidum and characterized QB and QBH2 marker bands based on their 15N- and 13C-isotopic shifts. Quinone exchanges were monitored using reconstituted photosynthetic membranes comprised of solubilized photosynthetic proteins, membrane lipids, and exogenous ubiquinone (UQ) molecules. In combination with 13C-labeling of the LH1-RC and replacement of native UQ8 by ubiquinones of different tail lengths, we demonstrated that quinone exchanges occur efficiently within the hydrophobic environment of the lipid membrane and depend on the side chain length of UQ. These results strongly indicate that unlike the process in Rba. sphaeroides, quinone transport in Tch. tepidum occurs through the size-restricted hydrophobic channels in the closed LH1 ring and are consistent with structural studies that have revealed narrow hydrophobic channels in the Tch. tepidum LH1 transmembrane region.

Molecular Pharmacology of NRH:Quinone Oxidoreductase 2: A Detoxifying Enzyme Acting as an Undercover Toxifying Enzyme.

N-ribosyldihydronicotinamide:quinone oxidoreductase 2 (NQO2/QR2, Enzyme Commission number 1.10.99.2) is a cytosolic enzyme, abundant in the liver and variably expressed in mammalian tissues. Cloned 30 years ago, it was characterized as a flavoenzyme catalyzing the reduction of quinones and pseudoquinones. To do so, it uses exclusively N-alkyl nicotinamide derivatives, without being able to recognize NADH, the reference hydrure donor compound, in contrast to its next of a kind, NAD(P)H:quinone oxidoreductase 1 (NQO1). For a long time both enzymes have been considered as key detoxifying enzymes in quinone metabolism, but more recent findings point to a more toxifying function of NQO2, particularly with respect to ortho-quinones. In fact, during the reduction of substrates, NQO2 generates fairly unstable intermediates that reoxidize immediately back to the original quinone, creating a futile cycle, the byproducts of which are deleterious reactive oxygen species. Beside this peculiarity, it is a target for numerous drugs and natural compounds such as melatonin, chloroquine, imiquimod, resveratrol, piceatannol, quercetin, and other flavonoids. Most of these enzyme-ligand interactions have been documented by numerous crystallographic studies, and now NQO2 is one of the best represented proteins in the structural biology database. Despite evidence for a causative role in several important diseases, the functional role of NQO2 remains poorly explored. In the present review, we aimed at detailing the main characteristics of NQO2 from a molecular pharmacology perspective. By drawing a clear border between facts and speculations, we hope to stimulate the future research toward a better understanding of this intriguing drug target. SIGNIFICANCE STATEMENT: Evidence is reviewed on the prevalent toxifying function of N-ribosyldihydronicotinamide:quinone oxidoreductase 2 while catalyzing the reduction of ortho-quinones such as dopamine quinone. The product of this reaction is unstable and generates a futile but harmful cycle (substrate/product/substrate) associated with reactive oxygen species generation.

Exploring the binding pocket of quinone/inhibitors in mitochondrial respiratory complex I by chemical biology approaches.

ATP: adenosine triphosphate; BODIPY: boron dipyrromethene; complex I: proton-translocating NADH-quinone oxidoreductase; DIBO: dibenzocyclooctyne; EM: electron microscopy; FeS: iron-sulfur; FMN: flavin adenine mononucleotide; LDT: ligand-directed tosylate; NADH: nicotinamide adenine dinucleotide; ROS: reactive oxygen species; SMP: submitochondrial particle; TAMRA: 6-carboxy-N,N,N',N'-tetramethylrhodamine; THF: tetrahydrofuran; TMH: transmembrane helix.

Synthesis and Electrochemical Properties of 2,5-Disubstituted Derivatives of 1,4-Bis(4,5-diphenylidimidazol-2-yl)benzene

The data on the synthesis and electrochemical properties of 2,5-disubstituted derivatives of 1,4-bis(4,5-diphenyldimidazol-2-yl)benzene are reported. The effect of substituents on redox properties has been comprehensively investigated. The electrochemical reduction of quinones into the biradical occurs at positive potentials and is a result of two stages of single-electron transfer, through the stage of the monoradical formation. The correlation between the reduction potentials and the salts structure has been found.

Charge polarization imposed by the binding site facilitates enzymatic redox reactions of quinone

Quinone reduction site (Qi) of cytochrome bc1 represents one of the canonical sites used to explore the enzymatic redox reactions involving semiquinone (SQ) states. However, the mechanism by which Qi allows the completion of quinone reduction during the sequential transfers of two electrons from the adjacent heme bH and two protons to C1- and C4-carbonyl remains unclear. Here we established that the SQ coupled to an oxidized heme bH is a dominant intermediate of catalytic forward reaction and, contrary to the long-standing assumption, represents a significant population of SQ detected across pH 5–9. The pH dependence of its redox midpoint potential implicated proton exchange with histidine. Complementary quantum mechanical calculations revealed that the SQ anion formed after the first electron transfer undergoes charge and spin polarization imposed by the electrostatic field generated by histidine and the aspartate/lysine pair interacting with the C4- and C1-carbonyl, respectively. This favors a barrierless proton exchange between histidine and the C4-carbonyl, which continues until the second electron reaches the SQi. Inversion of charge polarization facilitates the uptake of the second proton by the C1-carbonyl. Based on these findings we developed a comprehensive scheme for electron and proton transfers at Qi featuring the equilibration between the anionic and neutral states of SQi as means for a leak-proof stabilization of the radical intermediate. The key catalytic role of the initial charge/spin polarization of the SQ anion at the active site, inherent to the proposed mechanism, may also be applicable to the other quinone oxidoreductases.