Semiquinone Form Research Articles

Hydrogen bond network around the semiquinone of the secondary quinone acceptor Q(B) in bacterial photosynthetic reaction centers.

By utilizing a combined pulsed EPR and DFT approach, the high-resolution structure of the QB site semiquinone (SQB) was determined. The development of such a technique is crucial toward an understanding of protein-bound semiquinones on the structural level, as (i) membrane protein crystallography typically results in low resolution structures, and (ii) obtaining protein crystals in the semiquinone form is rarely feasible. The SQB hydrogen bond network was investigated with Q- (∼34 GHz) and X-band (∼9.7 GHz) pulsed EPR spectroscopy on fully deuterated reactions centers from Rhodobacter sphaeroides. Simulations in the SQB g-tensor reference frame provided the principal values and directions of the H-bond proton hyperfine tensors. Three protons were detected, one with an anisotropic tensor component, T = 4.6 MHz, assigned to the histidine NδH of His-L190, and two others with similar anisotropic constants T = 3.2 and 3.0 MHz assigned to the peptide NpH of Gly-L225 and Ile-L224, respectively. Despite the strong similarity in the peptide couplings, all hyperfine tensors were resolved in the Q-band ENDOR spectra. The Euler angles describing the series of rotations that bring the hyperfine tensors into the SQB g-tensor reference frame were obtained by least-squares fitting of the spectral simulations to the ENDOR data. These Euler angles show the locations of the hydrogen bonded protons with respect to the semiquinone. Our geometry optimized model of SQB used in previous DFT work is in strong agreement with the angular constraints from the spectral simulations, providing the foundation for future joint pulsed EPR and DFT semiquinone structural determinations in other proteins.

Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen

Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non-covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped-flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence (see P. Müller and M. Ahmad (2011) J. Biol. Chem. 286, 21033–21040 [1]). In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH. Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH−). Reaction of this with dioxygen leads to transient radical (FADH) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer.

A Switch between One- and Two-electron Chemistry of the Human Flavoprotein Iodotyrosine Deiodinase Is Controlled by Substrate

Reductive dehalogenation is not typical of aerobic organisms but plays a significant role in iodide homeostasis and thyroid activity. The flavoprotein iodotyrosine deiodinase (IYD) is responsible for iodide salvage by reductive deiodination of the iodotyrosine derivatives formed as byproducts of thyroid hormone biosynthesis. Heterologous expression of the human enzyme lacking its N-terminal membrane anchor has allowed for physical and biochemical studies to identify the role of substrate in controlling the active site geometry and flavin chemistry. Crystal structures of human IYD and its complex with 3-iodo-l-tyrosine illustrate the ability of the substrate to provide multiple interactions with the isoalloxazine system of FMN that are usually provided by protein side chains. Ligand binding acts to template the active site geometry and significantly stabilize the one-electron-reduced semiquinone form of FMN. The neutral form of this semiquinone is observed during reductive titration of IYD in the presence of the substrate analog 3-fluoro-l-tyrosine. In the absence of an active site ligand, only the oxidized and two-electron-reduced forms of FMN are detected. The pH dependence of IYD binding and turnover also supports the importance of direct coordination between substrate and FMN for productive catalysis.

Aminoacetone oxidase from Streptococcus oligofermentans belongs to a new three-domain family of bacterial flavoproteins.

The aaoSo gene from Streptococcus oligofermentans encodes a 43kDa flavoprotein, aminoacetone oxidase (SoAAO), which was reported to possess a low catalytic activity against several different L-amino acids; accordingly, it was classified as an L-amino acid oxidase. Subsequently, SoAAO was demonstrated to oxidize aminoacetone (a pro-oxidant metabolite), with an activity ~25-fold higher than the activity displayed on L-lysine, thus lending support to the assumption of aminoacetone as the preferred substrate. In the present study, we have characterized the SoAAO structure-function relationship. SoAAO is an FAD-containing enzyme that does not possess the classical properties of the oxidase/dehydrogenase class of flavoproteins (i.e. no flavin semiquinone formation is observed during anaerobic photoreduction as well as no reaction with sulfite) and does not show a true L-amino acid oxidase activity. From a structural point of view, SoAAO belongs to a novel protein family composed of three domains: an α/β domain corresponding to the FAD-binding domain, a β-domain partially modulating accessibility to the coenzyme, and an additional α-domain. Analysis of the reaction products of SoAAO on aminoacetone showed 2,5-dimethylpyrazine as the main product; we propose that condensation of two aminoacetone molecules yields 3,6-dimethyl-2,5-dihydropyrazine that is subsequently oxidized to 2,5-dimethylpyrazine. The ability of SoAAO to bind two molecules of the substrate analogue O-methylglycine ligand is thought to facilitate the condensation reaction. A specialized role for SoAAO in the microbial defence mechanism related to aminoacetone catabolism through a pathway yielding dimethylpyrazine derivatives instead of methylglyoxal can be proposed.

Magnetoreception in birds: II. Behavioural experiments concerning the cryptochrome cycle.

Behavioural tests of the magnetic compass of birds and corresponding immunohistological studies on the activation of retinal cryptochrome 1a, the putative receptor molecule, showed oriented behaviour and activated Cry1a under 373 nm UV, 424 nm blue, 502 nm turquoise and 565 nm green light, although the last wavelength does not allow the first step of photoreduction of cryptochrome to the semiquinone form. The tested birds had been kept under ‘white’ light before, hence we suggested that there was a supply of semiquinone present at the beginning of the exposure to green light that could be further reduced and then re-oxidized. To test the hypothesis in behavioural experiments, we tested robins, Erithacus rubecula, under various wavelengths (1) after 1 h pre-exposure to total darkness and (2) after 1 h pre-exposure to the same light as used in the test. The birds were oriented under blue and turquoise light, where the full cryptochrome cycle can run, but not under green light. This finding is in agreement with the hypothesis. Orientation under green light appears to be a transient phenomenon until the supply of semiquinone is depleted.

Migrating birds use fully reduced FADH− for navigation

![Figure][1] European robin ( Erithacus rubecula ). © Francis C. Franklin/CC-BY-SA-3.0, via Wikimedia Commons. To us it seems miraculous: a migrating bird can embark from its wintering grounds and successfully return to the breeding site that it may have left months before. Guided only by

Semiquinone-induced Maturation of Bacillus anthracis Ribonucleotide Reductase by a Superoxide Intermediate

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides, and represent the only de novo pathway to provide DNA building blocks. Three different classes of RNR are known, denoted I-III. Class I RNRs are heteromeric proteins built up by α and β subunits and are further divided into different subclasses, partly based on the metal content of the β-subunit. In subclass Ib RNR the β-subunit is denoted NrdF, and harbors a manganese-tyrosyl radical cofactor. The generation of this cofactor is dependent on a flavodoxin-like maturase denoted NrdI, responsible for the formation of an active oxygen species suggested to be either a superoxide or a hydroperoxide. Herein we report on the magnetic properties of the manganese-tyrosyl radical cofactor of Bacillus anthracis NrdF and the redox properties of B. anthracis NrdI. The tyrosyl radical in NrdF is stabilized through its interaction with a ferromagnetically coupled manganese dimer. Moreover, we show through a combination of redox titration and protein electrochemistry that in contrast to hitherto characterized NrdIs, the B. anthracis NrdI is stable in its semiquinone form (NrdIsq) with a difference in electrochemical potential of ∼110 mV between the hydroquinone and semiquinone state. The under anaerobic conditions stable NrdIsq is fully capable of generating the oxidized, tyrosyl radical-containing form of Mn-NrdF when exposed to oxygen. This latter observation strongly supports that a superoxide radical is involved in the maturation mechanism, and contradicts the participation of a peroxide species. Additionally, EPR spectra on whole cells revealed that a significant fraction of NrdI resides in its semiquinone form in vivo, underscoring that NrdIsq is catalytically relevant.

Redox behaviour of some asymmetrically substituted viologens and an alkyl bridged bis-viologen in non-aqueous solvents: a voltammetric and spectroscopic investigation

A compound library of seven asymmetrically substituted viologens and, additionally, a bis-viologen is investigated by voltammetric and EPR- and UV–Vis spectroelectrochemical techniques as well as diffusion-ordered nuclear magnetic resonance (NMR) spectroscopy. In acetonitrile-based electrolytes, all compounds show a stepwise two electron chemically and electrochemically reversible reduction of the viologen moieties. In the case of the bis-viologen, peak potential difference data indicate a weak difference in the formal potentials of the two non-conjugated redox systems. Diffusion coefficients from cyclic voltammetric and pulse gradient spin echo NMR experiments are comparable for all mono-viologens while the size and saturation of the side chain in the mono-viologen compounds has only a minor influence on this transport property. The semi-quinone forms of the viologens are highly stable with respect to disproportionation and they are EPR active. For the mono-viologens, coupling constants for all hydrogen and nitrogen nuclei interacting with the unpaired electron were determined. The bis-viologen forms a bis(radical cation) as well as an EPR-silent (but UV-detectable) π-dimer. Furthermore, the effect of weakly coordinating anions on the different redox states is reported.

Pseudomonas aeruginosa MdaB and WrbA are water-soluble two-electron quinone oxidoreductases with the potential to defend against oxidative stress.

Water-soluble quinone oxidoreductases capable of reducing quinone substrates via a concerted two-electron mechanism have been implicated in bacterial antioxidant defence. Twoelectron transfer avoids formation of dangerously reactive semi-quinone intermediates, moreover previous work in Pseudomonas putida indicated a direct protective effect for the quinols generated by an over-expressed oxidoreductase. Here, the Pseudomonas aeruginosa orthologs of five quinone oxidoreductases--MdaB, ChrR, WrbA, NfsB, and NQO1--were tested for their possible role in defending P. aeruginosa against H2O2 challenge. In in vitro assays, each enzyme was shown to reduce quinone substrates with only minimal semiquinone formation. However, when each was individually over-expressed in P. aeruginosa no overt H2O2-protective phenotype was observed. It was shown that this was due to a masking effect of the P. aeruginosa catalase, KatA; in a katA mutant, H2O2 challenged strains over-expressing the WrbA and MdaB orthologs grew significantly better than the empty plasmid control. A growth advantage was also observed for H2O2 challenged P. putida strains over-expressing P. aeruginosa wrbA, mdaB or katA. Despite not conferring a growth advantage to wild type P. aeruginosa, it is possible that these quinone oxidoreductases defend against H2O2 toxicity at lower concentrations.

Computational design of mixed-ligand adducts of Co aminovinyl ketonates with redox-active o-quinones and their derivatives

The adducts of cobalt aminovinyl ketonates with o-benzoquinones, o-benzoquinone imines, and o-benzoquinone diimines were studied by the density functional theory method (B3LYP*/6-311++G(d,p)). For all of the compounds, the ground states are low-spin structures containing a trivalent cobalt atom and a redox-active ligand in the semiquinonate form. The bulky substituents at the bis-chelate and ligand nitrogen atoms favor a decrease in the energy difference between the low-spin and high-spin isomers of the adducts containing LSCoIII and HSCoII atoms, respectively. These compounds can exhibit valence tautomerism in the crystalline state, but the relatively low stabilization energies of the high-spin isomers in solutions may induce a competing process: dissociation into the original bis-chelate and redox-active ligand.

Energy Landscapes and Catalysis in Nitric-oxide Synthase

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.

Folium Sennae protects against hydroxyl radical-induced DNA damage via antioxidant mechanism: an in vitro study.

BackgroundIn the study, Folium Sennae (FS) was firstly extracted by various solvents to obtain five FS extracts. Then, five FS extracts were evaluated for the protective effects against •OH-induced DNA damage, antioxidant abilities in vitro, and chemical contents using various methods. On this basis, the correlation graphs between the pharmacological effects and chemical contents were plotted to obtain the correlation coefficients (R values). Finally, in order to obtain biological evidence, ethyl acetate extract of FS (EAFS) was investigated for the protective effect against •OH-induced MSCs (mesenchymal stem cells) damage using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) assay.ResultsThe pharmacological assays indicated that five FS extracts could effectively protect against •OH-induced DNA damage. The correlation analysis suggested that the average R values of total phenolics, total anthraquinones, aloe-emodin, rhein, and emodin were respectively 0.843, 0.833, 0.753, 0.820, and 0.784, while those of total sugars and total saponins were respectively 0.103 and 0.0068. The mechanistic analysis revealed that five FS extracts could also scavenge •OH, •O2–, DPPH• & ABTS•+ radicals, and reduce Cu2+ to Cu+. MTT assay revealed that the viability of MSCs which were treated with •OH radicals has been effectively protected by EAFS (3 and 30 μg/mL).ConclusionOn this basis, it can be concluded that: (i) Folium Sennae exhibits a protective effect against •OH-induced damages to DNA and MSCs; (ii ) The effects may be attributed to phytophenols (especially aloe-emodin, rhein, and emodin), not sugars or saponins; (iii) They exert the protective action via hydrogen atom transfer (HAT) and/or sequential electron proton transfer (SEPT) mechanisms which make phenolic –OH moiety be oxidized to stable semi-quinone form; (iv) The stability of semi-quinone form can ultimately be responsible for the protective or antioxidant effect of phytophenols.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-16) contains supplementary material, which is available to authorized users.

Amentoflavone Protects against Hydroxyl Radical-induced DNA Damage via Antioxidant Mechanism

Objective: Oxidative DNA damage is involved in mutation, cell death, carcinogenesis, and aging. Since biflavonoid amentoflavone exhibited beneficial effects on DNA, we therefore investigated its protection against •OH-induced DNA oxidative damage then discussed the mechanism. It will enhance the understanding of interaction between biflavonoid vs DNA mediated by free radicals. Methods: The protective effect of amentoflavone against •OH-induced DNA damage was measured using our method. To explore the mechanism, it was further determined by •OH-induced bases damage, deoxyribose damage, and various antioxidant assays. Results: Amentoflavone increased dose-dependently its protective percentages against •OHinduced damage on DNA, bases, and deoxyribose; The IC50 were 31.85±4.75, 198.75±33.53, 147.14±20.95, 75.15±10.52, 93.75±16.36, 167.69±13.90, and 137.95±19.86 μM, respectively for DNA, cytosine, uracil, adenine, thymine, guanine, and deoxyribose damages. Radical-scavenging assays suggested that amentoflavone could effectively scavenge •O2 -, DPPH•, ABTS•+ radicals (IC50 values were respectively 8.98±0.23, 432.25±84.05, 7.25±0.35 μM). Conclusions: Based on the mechanistic analysis, it is concluded that amentoflavone can effectively protect against •OH-induced oxidative damage DNA (including base & deoxyribose moieties), via deoxynucleotide radical repairing, and reactive oxygen species (ROS) scavenging approaches which may be mediated by donating hydrogen atom (H•) and electron (e). Further analysis indicated that both scavenging and repairing approaches can be primarily attributed to its antioxidant mechanism which may ultimately arise from to the stability of its oxidized product semi-quinone form. Its protection against DNA damage may be generally responsible for the radioprotective and anti-inflammation effects.

Structural Snapshots from the Oxidative Half-reaction of a Copper Amine Oxidase: IMPLICATIONS FOR O2 ACTIVATION

The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O2 activation between the aminoquinol and Cu(I).

Semiquinone and Cluster N6 Signals in His-tagged Proton-translocating NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli

NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 μmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.

In Vitro Polyphenolics Erythrocyte Model and in Vivo Chicken Embryo Model Revealed Gallic Acid to Be a Potential Hemorrhage Inducer: Physicochemical Action Mechanisms

The in vivo chicken embryo model (CEM) demonstrated that gallic acid (GA) induced dysvascularization and hypoxia. Inflammatory edema, Zenker's necrosis, hemolysis, and liposis of cervical muscles were the common symptoms. Levels of the gene hif-1α, HIF-1α, TNF-α, IL-6, and NFκB in cervical muscles were all significantly upregulated, while the vascular endothelial growth factor (VEGF) was downregulated in a dose-responsive manner. Consequently, the cervical muscle inflammation and hemolysis could have been stimulated en route to the tissue TNF-α-canonical and the atypical pathways. We hypothesized that GA could deplete the dissolved oxygen (DO) at the expense of semiquinone and quinone formation, favoring the reactive oxygen species (ROS) production to induce RBC disruption and Fe(2+) ion release. To explore this, the in vitro polyphenolics-erythrocyte model (PEM) was established. PEM revealed that the DO was rapidly depleted, leading to the release of a huge amount of Fe (II) ions and hydrogen peroxide (HPO) in a two-phase kinetic pattern. The kinetic coefficients for Fe (II) ion release ranged from 0.347 h(-1) to 0.774 h(-1); and those for Fe (III) ion production were from 6.66 × 10(-3) h(-1) to 8.93 × 10(-3) h(-1). For phase I HPO production, they ranged from 0.236 h(-1) to 0.774 h(-1) and for phase II HPO production from 0.764 h(-1) to 2.560 h(-1) at GA within 6 μM to 14 μM. Thus, evidence obtained from PEM could strongly support the phenomena of CEM. To conclude, GA tends to elicit hypoxia-related inflammation and hemolysis in chicken cervical muscles through its extremely high prooxidant activity.

Biphasic reduction of cytochrome b559 by plastoquinol in photosystem II membrane fragments: Evidence for two types of cytochrome b559/plastoquinone redox equilibria

In photosystem II membrane fragments with oxidized cytochrome (Cyt) b559 reduction of Cyt b559 by plastoquinol formed in the membrane pool under illumination and by exogenous decylplastoquinol added in the dark was studied. Reduction of oxidized Cyt b559 by plastoquinols proceeds biphasically comprising a fast component with a rate constant higher than (10s)−1, named phase I, followed by a slower dark reaction with a rate constant of (2.7min)−1 at pH6.5, termed phase II. The extents of both components of Cyt b559 reduction increased with increasing concentrations of the quinols, with that, maximally a half of oxidized Cyt b559 can be photoreduced or chemically reduced in phase I at pH6.5. The photosystem II herbicide dinoseb but not 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) competed with the quinol reductant in phase I. The results reveal that the two components of the Cyt b559 redox reaction reflect two redox equilibria attaining in different time domains. One-electron redox equilibrium between oxidized Cyt b559 and the photosystem II-bound plastoquinol is established in phase I of Cyt b559 reduction. Phase II is attributed to equilibration of Cyt b559 redox forms with the quinone pool. The quinone site involved in phase I of Cyt b559 reduction is considered to be the site regulating the redox potential of Cyt b559 which can accommodate quinone, semiquinone and quinol forms. The properties of this site designated here as QD clearly suggest that it is distinct from the site QC found in the photosystem II crystal structure.

Synthesis and electronic properties of 3,7-dianilino substituted N-hexyl phenothiazines

3,7-Diaminophenothiazine derivatives are readily synthesized via two-fold Buchwald-Hartwig coupling of 10-hexyl 3,7-dibromo-10H-phenothiazine with a series of primary and secondary anilines and amines. All derivatives possess two reversible oxidations at low potentials with remarkable semiquinone formation constants. The electronic structure of this novel class of phenothiazinyl-oligoanilines is additionally studied and rationalized by DFT computations and correlation studies between selected experimental and computational electronic data.

A novel F420‐dependent anti‐oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents

Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420-dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F420-deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F420− mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2-dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F420H2-specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F420 biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents.

Redox-active silica nanoparticles. Part 7. Redox behavior of core/shell structured viologen modified silica particles immobilized at paraffin impregnated graphite electrodes

The redox behavior of viologen modified spherical and nonporous Stöber silica particles with diameters of 125nm immobilized at paraffin impregnated graphite electrodes in 0.1M KCl/H2O is described. A permanent particle coverage was obtained by mechanical attachment to the graphite electrodes. The particles preferentially form clusters on the electrode surface as indicated by SEM. The presence of the viologen salt was shown by EDS of a modified electrode. Cyclic and differential pulse voltammetry were used to study the redox behavior in the immobilized state. Cyclic voltammograms of the modified particles exhibit two chemically reversible reduction waves corresponding to the first and second single electron reduction of the viologen dication. The mid-point potentials of the viologen units in the immobilized state are close to those of the reference compound methyl viologen. However, values of the semi-quinone formation constant Ksem of the modified silica material are increased compared to those of dissolved methyl viologen. This indicates a stabilizing effect of the solid supporting matrix/electrode with respect to disproportionation of the anchored viologen mono radical cations compared to methyl viologen.