Binding and Stabilization of a Semiquinone Radical by an Artificial Metalloenzyme Containing a Binuclear Copper (II) Cofactor.

A binuclear Cu(II) cofactor was covalently bound to a lauric acid anchor. The resulting conjugate was characterized then combined with beta-lactoglobulin (ßLG) to generate a new biohybrid following the so-called "Trojan horse" strategy. This biohybrid was examined for its effectiveness in the oxidation of a catechol derivative to the corresponding quinone. The resulting biohybrid did not exhibit the sought after catecholase activity, likely due to its ability to bind and stabilize the semiquinone radical intermediate DTB-SQ. This semi-quinone radical was stabilized only in the presence of the protein and was characterized using optical and magnetic spectroscopic techniques, demonstrating stability for over 16 hours. Molecular docking studies revealed that this stabilization could occur owing to interactions of the semi-quinone with hydrophobic amino acid residues of ßLG.

Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes.

The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.

Electrostatics and water occlusion regulate covalently-bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR.

Proteins like NADH:ubiquinone oxidoreductase (NQR), an essential enzyme and ion pump in the physiology of several pathogenic bacteria, tightly regulate the redox properties of their cofactors. Although flavin mononucleotide (FMN) is fully reduced in aqueous solution, FMN in subunits B and C of NQR exclusively undergo one-electron transitions during its catalytic cycle. Here, we perform ab initio calculations and molecular dynamics simulations to elucidate the mechanisms that regulate the redox state of FMN in NQR. QM/MM calculations show that binding site electrostatics disfavor anionic forms of FMNH2 , but permit a neutral form of the fully reduced flavin. The potential energy surface is unaffected by covalent bonding between FMN and threonine. Molecular dynamics simulations show that the FMN binding sites are inaccessible by water, suggesting that further reductions of the cofactors are limited or prohibited by the availability of water and other proton donors. These findings provide a deeper understanding of the mechanisms used by NQR to regulate electron transfer through the cofactors and perform its physiologic role. They also provide the first, to our knowledge, evidence of the simple concept that proteins regulate flavin redox states via water occlusion.

Stimuli-Responsive Resorcin[4]arene Cavitands: Toward Visible-Light-Activated Molecular Grippers.

Resorcin[4]arene cavitands, equipped with diverse quinone (Q) and [Ru(bpy)2 dppz]2+ (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) photosensitizing walls in different configurations, were synthesized. Upon visible-light irradiation at 420 nm, electron transfer from the [Ru(bpy)2 dppz]2+ to the Q generates the semiquinone (SQ) radical anion, triggering a large conformational switching from a flat kite to a vase with a cavity for the encapsulation of small guests, such as cyclohexane and heteroalicyclic derivatives, in CD3 CN. Depending on the molecular design, the SQ radical anion can live for several minutes (≈10 min) and the vase can be generated in a secondary process without need for addition of a sacrificial electron donor to accumulate the SQ state. Switching can also be triggered by other stimuli, such as changes in solvent, host-guest complexation, and chemical and electrochemical processes. This comprehensive investigation benefits the development of stimuli-responsive nanodevices, such as light-activated molecular grippers.

In situ reactivity of electrochemically generated semiquinone on Emodin and its CuII/MnII complexes with pyrimidine based nucleic acid bases and calf thymus DNA: Insight into free radical induced cytotoxicity of anthracyclines.

There is enough proof to believe that free-radical intermediates of anthracycline based anticancer agents are involved in different stages of drug action. Subtle therapeutic differences observed in the actions of different anthracyclines largely influence the mechanism of action of the drugs that distinguish one member from another. Redox properties control biological responses related either to the one-electron quinone/semiquinone couple or the two-electron quinone/quinone-dianion couple. Comproportionation also leads to generation of semiquinone. Hence, whatever the form of reduction of the quinone moiety, a substantial amount of semiquinone is eventually formed in the system. Immediately after formation, there is competition between natural radical-decay pathways and one-electron transfer reactions that generate the superoxide-radical anion. Prototropic properties control rate of radical decay while redox properties control rate of electron transfer to molecular oxygen. In aerated medium, semiquinone-radical anion and superoxide-radical anion co-exist while in de-aerated medium semiquinone-radical anion predominates. All the radicals are damaging to the biological system. Through this study, attempt was made to detect changes induced by the radicals on pyrimidine based nucleic acid bases and calf thymus DNA in aerated and de-aerated (Ar saturated) medium to know the mechanism by which Emodin, its CuII/MnII complexes might affect DNA. Semiquinone-radical anion was generated electrochemically maintaining a glassy carbon electrode at the first reduction potential of each compound. Since the chosen compound (Emodin), its complexes are analogues of anthracyclines, findings on them can be extrapolated to understand the differences in anticancer activity or of adverse drug reactions reported in an innumerable number of clinical studies related to anthracyclines where the difference in structure of different members is due to differences in the relative positioning of hydroxy groups on the hydroxy-9, 10-anthraquinone moiety of anthracyclines. The study helps to realize action of compounds of this class as anticancer agents.

Uno Ferro, a de novo Designed Protein, Binds Transition Metals with High Affinity and Stabilizes Semiquinone Radical Anion.

Metalloenzymes often utilize radicals in order to facilitate chemical reactions. Recently, DeGrado and co-workers have discovered that model proteins can efficiently stabilize semiquinone radical anion produced by oxidation of 3,5-di-tert-butylcatechol (DTBC) in the presence of two zinc ions. Here, we show that the number and the nature of metal ions have relatively minor effect on semiquinone stabilization in model proteins, with a single metal ion being sufficient for radical stabilization. The radical is stabilized by both metal ion, hydrophobic sequestration, and interactions with the hydrophilic residues in the protein interior resulting in a remarkable, nearly 500 mV change in the redox potential of the SQ. - /catechol couple compared to bulk aqueous solution. Moreover, we have created 4G-UFsc, a single metal ion-binding protein with pm affinity for zinc that is higher than any other reported model systems and is on par with many natural zinc-containing proteins. We expect that the robust and easy-to-modify DFsc/UFsc family of proteins will become a versatile tool for mechanistic model studies of metalloenzymes.

Intrinsic chemiluminescence production from the degradation of haloaromatic pollutants during environmentally-friendly advanced oxidation processes: Mechanism, structure-activity relationship and potential applications.

The ubiquitous distribution of halogenated aromatic compounds (XAr) coupled with their carcinogenicity has raised public concerns on their potential risks to both human health and the ecosystem. Recently, advanced oxidation processes (AOPs) have been considered as an "environmentally-friendly" technology for the remediation and destruction of such recalcitrant and highly toxic XAr. During our study on the mechanism of metal-independent production of hydroxyl radicals (OH) by halogenated quinones and H2O2, we found, unexpectedly, that an unprecedented OH-dependent two-step intrinsic chemiluminescene (CL) can be produced by H2O2 and tetrachloro-p-benzoquinone, the major carcinogenic metabolite of the widely used wood preservative pentachlorophenol. Further investigations showed that, in all OH-generating systems, CL can also be produced not only by pentachlorophenol and all other halogenated phenols, but also by all XAr tested. A systematic structure-activity relationship study for all 19 chlorophenolic congeners showed that the CL increased with an increasing number of Cl-substitution in general. More importantly, a relatively good correlation was observed between the formation of quinoid/semiquinone radical intermediates and CL generation. Based on these results, we propose that OH-dependent formation of quinoid intermediates and electronically excited carbonyl species is responsible for this unusual CL production; and a rapid, sensitive, simple, and effective CL method was developed not only to detect and quantify trace amount of XAr, but also to provide useful information for predicting the toxicity or monitoring real-time degradation kinetics of XAr. These findings may have broad chemical, environmental and biological implications for future studies on halogenated aromatic persistent organic pollutants.

Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.

In this mini review, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302C in R. capsulatus and Y278C in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra-molecular organization of bacterial and mitochondrial respiratory chains (i.e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Agaricus meleagris pyranose dehydrogenase: influence of covalent FAD linkage on catalysis and stability.

Pyranose dehydrogenase (PDH) is a monomeric flavoprotein belonging to the glucose-methanol-choline (GMC) family of oxidoreductases. It catalyzes the oxidation of free, non-phosphorylated sugars to the corresponding keto sugars. The enzyme harbors an FAD cofactor that is covalently attached to histidine 103 via an 8α-N(3) histidyl linkage. Our previous work showed that variant H103Y was still able to bind FAD (non-covalently) and perform catalysis but steady-state kinetic parameters for several substrates were negatively affected. In order to investigate the impact of the covalent FAD attachment in Agaricus meleagris PDH in more detail, pre-steady-state kinetics, reduction potential and stability of the variant H103Y in comparison to the wild-type enzyme were probed. Stopped-flow analysis revealed that the mutation slowed down the reductive half-reaction by around three orders of magnitude whereas the oxidative half-reaction was affected only to a minor degree. This was reflected by a decrease in the standard reduction potential of variant H103Y compared to the wild-type protein. The existence of an anionic semiquinone radical in the resting state of both the wild-type and variant H103Y was demonstrated using electron paramagnetic resonance (EPR) spectroscopy and suggested a higher mobility of the cofactor in the variant H103Y. Unfolding studies showed significant negative effects of the disruption of the covalent bond on thermal and conformational stability. The results are discussed with respect to the role of covalently bound FAD in catalysis and stability.

Effects of semiquinone-rich surface on the behaviors of vascular cells.

Dopamine has been widely used for surface modification of cardiovascular medical devices as it forms films on most substrates that provide functional groups for surface chemical modification. However, under oxidative stress, the phenolic hydroxyl group on dopamine can undergo reversible transformation into phenol-semiquinone-quinone, which can cause cytotoxicity and immunotoxicity. In this study, we measured the effects of semiquinone on the behavior of vascular wall cells and inflammatory cells under oxidative stress via ultraviolet irradiation with a hydrogen peroxide diluent. Na2S2O3 was used as a stabilizer to obtain a semiquinone-rich poly-dopamine film, then phenol-semiquinone-quinone ratio on its surface was evaluated at three irradiation-oxidation time points. We found that the poly-dopamine film with ultraviolet irradiation in hydrogen peroxide solution for 15 min had the highest semiquinone occupancy of 19.18%. In the experimental group irradiated for 15 min, endothelial cells were cultured statically for 3 days and the number of surface adherent endothelial cells in the group with added semiquinone stabilizer was reduced to 73% of that in the group without stabilizer, indicating that semiquinone rich surface inhibits adhesion and proliferation of endothelial cells; Smooth muscle cells were cultured statically for 3 days, and the number of adherent smooth muscle on surfaces without stabilizer was reduced to 75% of that on surfaces with stabilizer added, indicating that semiquinone rich surfaces promote smooth muscle proliferation. These results demonstrate that semiquinone can adversely affect the repair effect after implantation of cardiovascular materials. Therefore, our study provides a reference for the application and optimization of dopamine in cardiovascular implant materials.

Free radical induced activity of an anthracycline analogue and its MnII complex on biological targets through in situ electrochemical generation of semiquinone.

Cytotoxicity by anthracycline antibiotics is attributed to several pathways. Important among them are formation of free-radical intermediates. However, their generation makes anthracyclines cardiotoxic which is a concern on their use as anticancer agents. Hence, any change in redox behavior that address cardiotoxicity is welcome. Modulation of redox behavior raises the fear that cytotoxicity could be compromised. Regarding the generation of free radical intermediates on anthracyclines, a lot depends on the surrounding environment (oxic or anoxic), polarity and pH of the medium. In case of anthracyclines, one-electron reduction to semiquinone or two-electron reduction to quinone-dianion are crucial both for cytotoxicity and for cardiotoxic side effects. The disproportion-comproportionation equilibria at play between quinone-dianion, free quinone and semiquinone control biological activity. Whatever is the form of reduction, semiquinones are generated as a consequence of the presence of anthracyclines and these interact with a biological target. Alizarin, a simpler anthracycline analogue and its MnII complex were subjected to electrochemical reduction to realize what happens when anthracyclines are reduced by compounds present in cells as members of the electron transport chain. Glassy carbon electrode maintained at the pre-determined reduction potential of a compound was used for reduction of the compounds. Nucleobases and calf thymus DNA that were maintained in immediate vicinity of such radical generation were used as biological targets. Changes due to the generated species under aerated/de-aerated conditions on nucleobases and on DNA helps one to realize the process by which alizarin and its MnII complex might affect DNA. The study reveals alizarin was more effective on nucleobases than the complex in the free radical pathway. Difference in damage caused by alizarin and the MnII complex on DNA is comparatively less than that observed on nucleobases; the complex makes up for any inefficacy in the free radical pathway by its other attributes.

The effects of Fe-bearing smectite clays on OH formation and diethyl phthalate degradation with polyphenols and H2O2.

The natural formation of hydroxyl radicals (OH) is important for the attenuation of organic contaminants. In this study, seven model polyphenols were selected to react with four types of smectite clays with varied Fe contents in the presence of H2O2. Diethyl phthalate (DEP) was selected as a model organic contaminant due to its wide distribution in environment. The results show the appearance of Fe-bearing smectite clays can significantly promote ·OH formation with polyphenols and H2O2 under anoxic conditions; clay particle size, the content and location of lattice Fe in smectite clays greatly affect OH formation. Hydrogen bond between phenolic group and smectite surfaces, and cation assisted hydrogen bond between carboxylic group and clay surfaces are important types of complexation. Electrons can be transferred from coordinated polyphenols to structural Fe(III) atoms in tetrahedral layers or at broken edges to form structural Fe(II) and/or semiquinone radicals, both of which can induce H2O2 decomposition to OH. DEP can be degraded by OH attack, and the main products are proposed as phthalic acid, monomethyl phthalate, hydroxyl-diethyl phthalates. Our findings suggest that Fe(III)-bearing smectite clay can be reduced by polyphenol and produce OH in anoxic environments, which can induce organic contaminants transformation.

Evidence for the generation of reactive oxygen species from hydroquinone and benzoquinone: Roles in arsenite oxidation.

Natural organic matter (NOM) significantly affects the fate, bioavailability, and toxicity of arsenic in the environment. In the present study, we investigated the oxidation of As(III) in the presence of hydroquinone (HQ) and benzoquinone (BQ), which were selected as model quinone moieties for NOM. It was found that As(III) was oxidized to As(V) in the presence of HQ or BQ at neutral conditions, and the oxidation efficiency of As(III) increased from 33% to 92% in HQ solutions and from 0 to 80% in BQ solutions with pH increasing from 6.5 to 8.5. The oxidation mechanism was further explored with electron spin resonance (ESR) technique. The results showed that semiquinone radicals (SQ(-)) were generated from the comproportionation reaction between BQ and HQ, which mediated the formation of superoxide anion (O2(-)), hydrogen peroxide (H2O2) and hydroxyl radical (OH). Both the SQ(-), H2O2 and OH contributed to the oxidation of As(III). The increase of pH favored the formation of SQ(-), and thus promoted the generation of reactive oxygen species (ROS) as well as As(III) oxidation. Increasing concentrations of HQ and BQ from 0.1 to 1.0 mM enhanced As(III) oxidation from 65% to 94% and from 10% to 53%, respectively. The findings of this study facilitate our understanding of the fate and transformation of As(III) in organic-rich aquatic environments and highlight quinone moieties as the potential oxidants for As(III) in the remediation of arsenic contaminated sites.

Spin-coupling in dimers of 2,3-dicyano-5,6-dichlorosemiquinone radical anions in the crystalline state.

A crystal engineering approach is used to stabilize a radical anion in the crystalline state and to modulate the separation distance within π-stacks of anion radicals. Alkali metal salts of 2,3-dicyano-5,6-dichlorosemiquinone (C8Cl2N2O2, DDQ∙- radical anions were prepared and their crystal structures determined: LiDDQ·2H2O·(CH3)2CO, RbDDQ·2H2O and CsDDQ·2H2O. In these structures, stacked dimers of radical anions are formed within π-stacked columns. Within the stacked dimers, interplanar separation distances are significantly shorter than the sum of the van der Waals radii for two C atoms; the shortest is 2.812 Šfor the Li salt and the longest is 2.925 Šfor the Cs salt. Diamagnetic character, observed by electron paramagnetic resonance spectroscopy, indicates spin-coupling of the unpaired electrons within the radical anion dimer. The electron-rich cyano substituents on DDQ∙- influence the electron redistribution within the ring skeleton. The crystalline compounds are also characterized by IR spectroscopy, complemented by quantum-chemical calculations based on both isolated and periodic models.

Formation and stabilization of persistent free radicals.

We demonstrate that stable and relatively unreactive "environmentally persistent free radicals (PFRs)" can be readily formed in the post-flame and cool-zone regions of combustion systems and other thermal processes. These resonance-stabilized radicals, including semiquinones, phenoxyls, and cyclopentadienyls, can be formed by the thermal decomposition of molecular precursors including catechols, hydroquinones and phenols. Association with the surfaces of fine particles imparts additional stabilization to these radicals such that they can persist almost indefinitely in the environment. A mechanism of chemisorption and electron transfer from the molecular adsorbate to a redox-active transition metal or other receptor is shown through experiment, and supported by molecular orbital calculations, to result in PFR formation. Both oxygen-centered and carbon-centered PFRs are possible that can significantly affect their environmental and biological reactivity.