EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer

In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q_B, coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q_B. To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q_B site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920–6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g _x  = 2.0057, g _y  = 2.0048, g _z  ~ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.     

Application of a Stopped-Flow EPR Method for the Detection of Short-Lived Flavonoid Semiquinone Radicals Produced by Oxidation Using <Superscript>15</Superscript>N-Labeled Nitrosodisulfonate Radical (Fremy’s Salt)

A stopped-flow-electron paramagnetic resonance (EPR) method was applied for the detection of short-lived radicals of flavonoids bearing a catechol moiety as the B-ring, such as flavonols (quercetin, fisetin, and rutin), flavanones (eriodictyol and taxifolin), flavanols (catechin and epicatechin), and flavone (luteolin). ¹⁵N-labeled sodium salt of nitrosodisulfonate (¹⁵NDS) was employed to obtain the highly resolved EPR hyperfine structure (hfs) of flavonoid-derived semiquinone radicals under stoichiometrically regulated reaction conditions in aqueous media (pH 10). The EPR hfs of these flavonoids radicals, except catechin and epicatechin, were recorded. Based on the g value and the proton hyperfine coupling constants (hfcc), these flavonoid-derived radicals were assigned to be semiquinone radicals of the catechol moiety (B-ring). For example, the observed EPR hyperfine structure (hfs) of the luteolin radical (Lut^?·) was composed of four sets of doublet splitting, which could be ascribed to the three protons of the B-ring (a^2′?=?0.136, a^5′?=?0.102, and a^6′?=?0.272 mT) and a vinyl proton of the C-ring (a³?=?0.099 mT). In addition, the characteristically small doublet splitting resolved for the fisetin anion radical (Fis^?·, 0.028 mT) was assigned to the aromatic proton at the C₅ carbon of the A-ring, indicating that the unpaired electron of the radials was partially delocalized onto the A-ring through the π bonds involved in the vinyl-carbonyl moiety of the C-ring. The hfcc of the methine protons at the C₂ carbon of taxifolin and eriodictyol-derived radicals (Tax^?· and Eri^?·) was, respectively, evaluated to be 0.102 and 0.230 mT. The assignment of the proton hfcc of flavonoid-derived semiquinone radicals will be discussed in relation with the molecular structure of the C-ring.     

DNA strand breaks induced by concerted interaction of H radicals and low-energy electrons

We propose a mechanism of DNA single strand breaks induced by low-energy electrons. Density functional theory calculations have been performed on a neutral, hydrogenated, and/or negatively charged nucleotide of cytosine in the gas phase to identify barriers for the phosphate-sugar O–C bond cleavage. Attachment of the first excess electron induces intermolecular proton transfer to cytosine. The resulting neutral radical of hydrogenated cytosine binds another excess electron, and the excess charge is localized primarily on the C6 atom. A barrier encountered for proton transfer from the C2’ atom of the adjacent sugar unit to the C6 atom of cytosine is 3.6 and 5.0 kcal/mol, based on the MPW1K and B3LYP electronic energies corrected for zero-point vibrations, respectively. The proton transfer is followed by a barrier-free sugar-phosphate C–O bond cleavage. The proton transfer is impossible for the neutral nucleotide, as there is no local minimum for the product. In the case of anionic and hydrogenated nucleotides the same barrier determined at the B3LYP level is as large as 29.3 and 22.4 kcal/mol respectively. This illustrates that the consecutive hydrogenation and electron attachment make the nucleotide of cytosine susceptible to a strand break. The rate of the C–O bond cleavage in the anion of hydrogenated nucleotide of cytosine is estimated to be ca. 10¹⁰  s^-1. The proposed mechanism proceeds through bound anionic states, not through metastable states with finite lifetimes and discrete energy positions with respect to the neutral target. The results suggest that at least for DNA without hydration even very low-energy electrons may cleave the DNA backbone.     

Study of the R meson produced in 13 GeV/cπ+p interactions

The missing mass spectrum opposite the proton in a 750 000 picture exposure, 13 GeV/cπ⁺p bubble chamber experiment, is investigated in two and four pion channels for structures observed or denied by boson spectrometers at the same energy in the reaction π^?p→X^?p.In the four pion final state the R⁺ enhancement is observed clearly and the possibility of multi-component mass structure is considered. Treating the phenomenon as one object, cross sections and branching ratios are derived for intermediate quasi two and three body decay modes (ωπ, ??, A₂π, ?ππ). The signals in the two pion state and for the neutral state produced opposite Δ⁺⁺(1231) are also presented and an elasticity is calculated.     

Laser Desorption Mass Spectrometry of Some Organic Acids

Abstract

Positive and negative ion laser desorption (LD) mass spectra of organic acids are characterized by the emission of the quasimolecular ions (M+H)⁺ and (M-H)^?. Generation of (M+H)⁺ ions is interpreted as evidence for the occurence of high pressure proton transfer reactions in LD. Fragment ions can be rationalized by loss of stable neutral molecules from quasimolecular ions although decomposition may also be occuring prior to ionization. Features unique to LD, including the detection of pyrolysis products along with ions characteristic of the sample, are discussed in terms of the internal energy distribution in the irradiated microvolume. Negative ion mass spectra of acids are dominated by (M-H)^?, while positive ion spectra contain abundant fragment ions, underlining the utility of detecting negative ions for acidic compounds.     

Molecular dynamics simulations of ubiquinone; a survey over torsional potentials and hydrogen bonds

Molecular dynamics simulations, both classical and Car-Parrinello, have been carried out to investigate ubiquinone (UQ), a proton mediator in both oxidative and photo-phosphorylation. The main objectives have been to follow the dynamics of methoxy groups, conformation of the tail with respect to the ring, hydration and hydrogen bond structure around UQ. The methoxy groups are found to be able to rotate fairly freely. The tail in both UQ and UQ^? is approximately perpendicular to the ring plane. Only weak hydrogen bonds are formed between the neutral form of ubiquinone and water molecules in the solvent, while the anionic form shows a distinct solute-solvent hydrogen bond structure. We also conclude that anionic UQ can be accurately modelled by molecular mechanics methods, but the conformation of the methoxy groups in neutral UQ can hardly be properly modelled using a standard force field.     

The influence of neutron and proton irradiation on the magnetization of biotite

This paper reports on the results of investigations into the field dependences of the magnetization for biotite in the initial state, after heat treatment at a temperature of 1000°C for 15 min, and after irradiation with 14-MeV neutrons at a dose of 1.2×10¹³ cm^?2 or with 3-MeV protons at a dose of 2.2×10¹⁴ cm^?2. It is demonstrated that the magnetization of biotite drastically increases after neutron and proton irradiation. This effect can be associated with the formation of oxide melt at radiation-induced thermal peaks and the freezing of high-temperature phase states corresponding to magnetite or magnetite-hematite solid solutions.     

Quasi-elastic electron scattering (e,e′p) and (e,e′d) from 6Li in a coincidence experiment

Coincidence cross sections for the reactions ⁶Li(e, e′p) and ⁶Li(e, e′d) have been measured in the region of quasi-elastic scattering. Using incident electrons of 2.5 and 2.7 GeV, the four-momentum transfers to the proton were 6.6 fm^?2, 10.0 fm^?2 and 11.6 fm^?2. The proton coincidence data agree with shell-model distributions assuming a Woods-Saxon potential and including short-range nucleon-nucleon correlations. The best fit to the deuteron coincidence data is obtained with a cluster wave function for the p-nucleons and a harmonic oscillator wave function for the s-nucleons taking into account the deuteron yield from the s-shell. The ratio of the deuteron cross section from ⁶Li divided by the elastic e-d scattering cross section depends only slightly on the four-momentum transfer and has a value of ≈ 2.     

Bulk magnetization and 1H NMR spectra of magnetically heterogeneous model systems

Bulk magnetization and ¹H static and magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of two magnetically heterogeneous model systems based on laponite (LAP) layered silicate or polystyrene (PS) with low and high proton concentration, respectively, and ferrimagnetic Fe₂O₃ nano- or micro-particles have been studied. In LAP+Fe₂O₃, a major contribution to the NMR signal broadening is due to the dipolar coupling between the magnetic moments of protons and magnetic particles. In PS+Fe₂O₃, due to the higher proton concentration in polystyrene and stronger proton–proton dipolar coupling, an additional broadening is observed, i.e. ¹H MAS NMR spectra of magnetically heterogeneous systems are sensitive to both proton–magnetic particles and proton–proton dipolar couplings. An increase of the volume magnetization by ～1 emu/cm³ affects the ¹H NMR signal width in a way that is similar to an increase of the proton concentration by ～2×10²²/cm³. ¹H MAS NMR spectra, along with bulk magnetization measurements, allow the accurate determination of the hydrogen concentration in magnetically heterogeneous systems.     

Theoretical studies on the single proton transfer process in adenine base

The effect of metal ions (Mⁿ⁺ = Na⁺, K⁺, Mg²⁺, Ca²⁺, Zn²⁺ and hydrated Mg²⁺ ions) and water molecules on the tautomerism of adenine induced by single intramolecular proton transfer (SPT) have been investigated theoretically. Calculated results show that the single proton transfer process in adenine base is favored and even becomes thermodynamically spontaneous because of the presence of Mⁿ⁺ interacting at the N₃ position of adenine. On the contrary, if Mⁿ⁺ coordinated to N₇ site, the single proton transfer process will become unfavorable than that in the neutral system. The effects of metal ions on the SPT of adenine base are more pronounced if Lewis acidity of metal ion is increased. Water plays a more important role than metal ions during the SPT process. It is found that water can act not only as a solvent but also as a mediator which gives and accepts protons to promote SPT, playing a bridge role. As a result, inclusion of a water molecule drastically reduces the energy barrier for the SPT. Moreover, two water molecules can yield larger assisting effect on the SPTs compared with one water molecule. We can conclude that the tautomerism of DNA adenine base can be modulated by the metal ions and water molecules. Copyright © 2015 John Wiley & Sons, Ltd.     

Continuum shell-model description of baryon decays of light hypernuclei

We consider the particle emission from excited hypernuclear states produced in (K ^?,π ^?) strangeness transfer. In the shell-model framework including the continuum we calculate the ratio between the various neutron, proton and hyperon channels of the hypernucleus_Λ ¹⁴C. Continuum inclusion does not influence branching ratios of important channels substantially.     

Effect of tetrachloroethane on the equilibrium between ionic and molecular complexes in the methylsulfonic acid-butyl acetate system

In concentrated solutions of methylsulfonic acid in butyl acetate (BA), molecular and ionic complexes were found to be in equilibrium:

$AH \cdots B \cdots HA + HA \leftrightarrow AH \cdots BH^ + + (AHA)^ - .$

The base is fully protonated only at a more than 10-fold excess of the acid. In the presence of tetrachloroethane (TCE), a neutral solvent, the IR spectra of the system are indicative of an increase in the concentration of undissociated acid molecules (HA) and a decrease in the concentration of proton solvates (AHA)^?. This means that the equilibrium shifts to the formation of molecular structures. At C _TCE ⁰ :C _BA ⁰ > 2, TCE molecules attenuate the promoting effect of undissociated acid molecules, which are known to facilitate the transfer of a proton to a base molecule with the formation of an (AHA)^? ion with a strong symmetrical H-bond.

     

Evidence for a possible role of 3‐hydroxyanthranilic acid as an antioxidant

The reactions of 3‐hydroxyanthranilic acid (3‐OHAA) with N₃^?, NO₂^?, NO^?, CCl₃O₂^? , and OH^? radicals were examined using a pulse radiolysis technique mainly at pH 7. The bimolecular electron transfer from secondary one‐electron oxidants results in the formation of anilino radical (λ_max ? 380 nm). The rate constant for the reaction of N₃^? radical with 3‐OHAA at pH 7 was found to be 6.3 × 10⁹ dm³ mol^?1 s^?1. It was observed that the 3‐OHAA reacts with oxygen centered radicals. The repair rate constant for the electron transfer reaction from 3‐OHAA to guanosine radical and chlorpromazine cation radical was also examined using a pulse radiolysis technique. Kinetic studies indicate that 3‐OHAA may act as an antioxidant to repair free‐radical damage to above mentioned biologically important compounds. The rate constants of electron transfer from the 3‐OHAA to the guanosine and chlorpromazine radicals were determined. The one‐electron reduction potential for 3‐OHAA radical was found to be 0.53 ± 0.06 V versus NHE. Copyright © 2008 John Wiley & Sons, Ltd.     

Fluorescence properties of the anti-tumour alkaloid luotonin A and new synthetic analogues: pH modulation as an approach to their fluorimetric quantitation in biological samples

Luotonin A is an alkaloid structurally related to the natural anti-tumour agent camptothecin. The fluorescence behaviour of luotonin A and a series of six analogues is described in the present work. The influence of solvent polarity and pH on the native fluorescence properties of these alkaloids was studied, finding that in organic solvents or in aqueous solutions (pH 5.5–7.2) the neutral form of the luotonin derivatives emit in the region of 410–450 nm but, in both media, acidification to pH values below 3.0 causes a new emission band to appear at about 500 nm. An ESPT reaction occurs due to the protonation of the basic nitrogen atoms of the pentacyclic ring. Acid-base titrations of luotonin A and its derivatives in aqueous and acetonitrile media were carried out in order to determine their pK_a^? values which were around 2, showing these compounds to be very weak bases. In aqueous media, the absence of an iso-emissive point in the emission spectra suggests the existence of more than two species in the proton transfer equilibria. The basicity of the luotonin A derivatives is increased in organic media, and a good correlation between the pK_a^? values and the chemical structure was found. The protonation of luotonin A was also studied by ¹H-NMR and ¹³C-NMR experiments, which proved the protonation of the nitrogen atoms at the positions 5 and 6 of the pentacyclic ring. The fluorescence quantum yields were determined in ethanol and in aqueous solutions under neutral and acidic conditions. The fluorescence quantum yields were higher in water for the case of the more polar compounds, and the opposite result was obtained for the more hydrophobic ones. The remarkable and interesting fluorescence properties of luotonin A prompted the development of its fluorimetric analytical quantitation, obtaining very good analytical features.     

Dispersive liquid–liquid microextraction based on solidification of floating organic drop for separation and preconcentration of malachite green before its determination by flow injection spectrophotometry

ABSTRACT

A simple and fast dispersive liquid–liquid microextraction based on solidification of floating organic drop has been developed for the separation and preconcentration of malachite green in water samples prior to its determination by flow injection spectrophotometry. Sodium lauryl sulfate, an anionic surfactant, was used for the ion-pair formation with malachite green. The factors affecting the ion-pair formation and extraction were optimized. Under the optimized conditions (volume of 1-undecanol as the extraction solvent, 40 μL; the volume of ethanol as the disperser solvent, 100 μL; sodium lauryl sulfate concentration, 7.5 × 10^?7 mol L^?1, and the pH of the sample, ～3.0), the calibration graph was linear over the range of 0.8–25 µg L^?1 with the detection limit of 0.3 µg L^?1 and the preconcentration factor of 750. The relative standard deviation at 7 µg L^?1 (n = 6) was found to be 2.1%. The developed method was successfully applied to the determination of malachite green in river water and fish farming water samples.     

Kinetics and mechanism of the reactions of O‐aryl S‐(4‐nitrophenyl) dithiocarbonates with anilines in aqueous ethanol

The reactions of O‐(4‐methylphenyl) S‐(4‐nitrophenyl) dithiocarbonate and O‐(4‐chlorophenyl) S‐(4‐nitrophenyl) dithiocarbonate with a series of anilines are subjected to a kinetic investigation in 44 wt% ethanol–water, at 25.0 °C and an ionic strength of 0.2 M. The reactions are followed spectrophotometrically at 420 nm (appearance of 4‐nitrobenzenethiolate anion). Under excess amine, pseudo‐first‐order rate coefficients (k_obs) are found. For the reactions of both substrates with anilines, plots of k_obs versus free amine concentration at constant pH are nonlinear upwards, according to a second‐order polynomial equation. This kinetic behavior is in agreement with a stepwise mechanism consisting of two tetrahedral intermediates, one zwitterionic (T^±) and the other anionic (T^?), with a kinetically significant proton transfer from T^± to an aniline to yield T^?. The rate equation was derived from the proposed mechanism. By nonlinear least‐squares fitting of the rate equation to the experimental data, values of the rate micro‐coefficients involved in both steps were determined. Copyright © 2009 John Wiley & Sons, Ltd.     

Linear free‐energy relationships in semiquinone species and their Mn(II) and Cu(II) complexes

Linear free‐energy relationships for a series of functionalized semiquinone ligands and their Mn^II‐ and Cu^IIhydro‐tris(3‐cumenyl‐5‐methylpyrazolyl) borate complexes were examined. Quinone–semiquinone cycle half‐wave reduction potentials and semiquinone hydrogen hyperfine coupling constants (a_H) were determined and their correlation with Hammett σ parameters reported. A new σ parameter, σ_aH, has been proposed. Mn^II and Cu^II metal complex metal–ligand charge transfer and n → π* UV transitions were found to be modulated by substituents. Satisfactory Hammett correlations between UV transitions and various σ values have been determined and compared in a number of instances. Copyright © 2011 John Wiley & Sons, Ltd.     

Nuclear magnetic relaxation due to slow molecular reorientation and fast internal rotation interference

The relaxation of the two-proton system longitudinal magnetization M_z(t) is considered theoretically, using the Bloch equations with a diffusion term. The condition dd> τ_c ≥ 1, dd> τ ? 1 is fulfilled here, where dd> is the energy of the proton dipole-dipole interaction, τ is the correlation time of molecular diffusion rotation and τ is the correlation time for the proton rotation relative to the molecule. Unlike the ordinary Bloch equations a two-exponential law for the evolution of M_z(t) is obtained for definite values dd>, τ_c and τ. The results of the present work may be useful for the consideration of the system having mobile proton groups in viscous media.