Faustino E. Morán Vieyra

Singlet oxygen quenching by anthocyanin's flavylium cations

The quenching of singlet molecular oxygen (1O2) by the flavylium cation form of six widespread anthocyanin derivatives: cyanidin 3-glucoside (CG), cyanidin 3-rutinoside (CR), cyanidin 3-galactoside (CGL), malvidin (M), malvidin 3-glucoside (MG) and malvidin 3,5-diglucoside (MDG) was studied in 1% HCl methanol solution by time-resolved phosphorescence detection (TRPD) of 1O2 and photostationary actinometry using perinaphthenone and methylene blue as sensitizers, respectively. The average value of the total (k0) and chemical (kc) quenching rate constants were ∼ 4×108 m−1 s−1 and 3×106 m−1 s−1, respectively, indicating the good performance of the studied anthocyanins as catalytic quenchers of 1O2. The quenching efficiency was larger for malvidin than for cyanidin derivatives, probably by the extra electron-donating methoxy group in ring B of the malvidin derivatives; and it was also dependent on the number and type of glycosylated substitution. As observed for other phenolic-like derivatives, the quenching of 1O2 by anthocyanins was mediated by a charge-transfer mechanism, which was modulated by the total number of –OR substituents that increases the electron-donating ability of these compounds.

Singlet oxygen quenching and radical scavenging capacities of structurally-related flavonoids present in Zuccagnia punctata Cav.

The singlet oxygen (1O2) quenching and free radical (DPPH•, ABTS• + and O2• −) scavenging ability of three structurally-related flavonoids (7-hydroxyflavanone HF, 2′,4′-dihydroxychalcone DHC and 3,7-dihydroxyflavone DHF) present in the Argentinean native shrub Zuccagnia punctata Cav. were studied in solution by combining electrochemical and kinetic measurements, mass spectroscopy, end-point antioxidant assays and computational calculations. The results showed that the antioxidant properties of these flavonoids depend on several factors, such as their electron- and hydrogen atom donor capacity, the ionization degree of the more acidic group, solvatation effects and electrostatic interactions with the oxidant species. The theoretical calculations for both the gas and solution phases at the B3LYP level of theory for the Osanger reaction field model agreed with the experimental findings, thus supporting the characterization of the antioxidant mechanism of the Z. punctata flavonoids.

Modulation of optical properties of dissolved humic substances by their molecular complexity.

In this study, we show that several UV–Vis absorbance, steady‐state and time‐resolved fluorescence parameters of a series of dissolved humic substances (DHS) from different sources (e.g. terrestrial fulvic and humic acids, and humic acid‐like molecules extracted from composted and vermicomposted wastes) correlate with the molar absorptivity at 280 nm per mole of organic carbon (ε280), which in turn is proportional to the molecular complexity (e.g. molecular size, aromaticity and oxidation degree) of the DHS. Both absorbance and fluorescence spectral responses were sensitive to the molecular complexity associated with the maturation degree of the DHS. Depending on the DHS, different emitting responses by excitation at the UVA (340 nm) and VIS (460 nm) regions of the absorption spectra were observed. The results were explained in terms of the extent of intramolecular electronic interactions between electron donor groups, such as polyhydroxylated aromatics and indoles, and more oxidized acceptor groups (e.g. quinones or other oxidized aromatics) as the molecular complexity of the DHS increased.

Effect of dye localization and self-interactions on the photosensitized generation of singlet oxygen by rose bengal bound to bovine serum albumin.

The spectroscopic and photophysical properties of rose bengal (RB) encased in bovine serum albumin (BSA) have been examined to evaluate the photosensitized generation of singlet molecular oxygen ((1)O2). The results show that RB photophysical and photosensitizing properties are highly modulated by the average number of dye molecules per protein (n). At n ≪ 1, the dye molecule is tightly located into the hydrophobic nanocavity site I of the BSA molecule with a binding constant Kb = 0.15 ± 0.01 μM(-1). The interaction with surrounding amino acids induces heterogeneous decay of both singlet and triplet excited states of RB and partially reduce its triplet quantum yield as compared with that in buffer solution. However, despite of the diffusive barrier imposed by the protein nanocavity to (3)O2, the quenching of (3)RB(∗):BSA generates (1)O2 with quantum yield ΦΔ = 0.35 ± 0.05. In turns, the intraprotein generated (1)O2 is able to diffuse through the bulk solution, where is dynamically quenched by BSA itself with an overall quenching rate constant of 7.3 × 10(8) M(-1) s(-1). However, at n>1, nonspecific binding of up to ≈ 6RB molecules per BSA is produced, allowing efficient static quenching of excited states of RB preventing photosensitization of (1)O2. These results provide useful information for development of dye-protein adducts suitable for using as potential intracellular photosensitizers.

UVA Self‐Photosensitized Oxygenation of β‐Ionone

The steady‐state UVA (350 nm) photolysis of (E)‐β‐ionone (1) in aerated toluene solutions was studied by 1H NMR spectroscopy. The formation of the 1,2,4‐trioxane (2) and 5,8‐endoperoxide (5) derivatives in the ratio of 4:1 was observed. Time‐resolved laser induced experiments at 355 nm, such as laser‐flash photolysis, photoacoustic and singlet oxygen 1O2 phosphorescence detection, confirmed the formation of the excited triplet state of 1 with a quantum yield ΦT = 0.50 as the precursor for the generation of singlet oxygen 1O2 (ΦΔ = 0.16) and the isomeric α‐pyran derivative (3), which was a reaction intermediate detected by NMR. In turn, the reaction of 1O2 with 1 and 3 occurred with rate constants of 1.0 × 106 and 2.5 × 108 m−1s−1 to yield the oxygenated products 5 and 2, respectively, indicating the relevance of the fixed s‐cis configuration in the α‐pyran ring in the concerted [2+4] cycloaddition of 1O2.

Physicochemical characterization of water-soluble chitosan derivatives with singlet oxygen quenching and antibacterial capabilities

New water-soluble chitosan derivatives (WSCh) were obtained by Maillard reaction (MR) between glucosamine (GA) with both low and medium molecular weight chitosans (Ch). The WSCh showed larger solubility than the respective Ch, while their deacetylation degree (DD) decreased by approximately 12%. Infrared spectroscopy experiments of WSCh confirmed the formation of imine bonds after MR with intensified pyranose structure, and sugar molecules as polymer branches. However, a 6-times reduction of the molecular weight of WSCh was measured, indicating the breakdown of the polysaccharide chain during the MR. The polysaccharides quenched singlet molecular oxygen (1O2), with rate quenching constants correlating with the DD value of the samples, suggesting the important role of amino groups (-NH2) in the deactivation of 1O2. Additionally, all polysaccharides presented antimicrobial activity against pathogenic bacteria, e.g. Staphylococcus aureus, Escherichia coli, Salmonella sp., Enterococcus faecalis and Listeria ivanovii, as tested by their minimum inhibitory concentration (MIC). This way we obtained new water-soluble polysaccharides, with similar functional properties to those presented by native Ch, enhancing its potential application as carrier material for bioactive compounds.

Enhancement of Photophysical and Photosensitizing Properties of Flavin Adenine Dinucleotide by Mutagenesis of the C‐Terminal Extension of a Bacterial Flavodoxin Reductase

The role of the mobile C-terminal extension present in Rhodobacter capsulatus ferredoxin-NADP(H) reductase (RcFPR) was evaluated using steady-state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C-terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild-type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD-binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge-transfer quenching of (1) FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet- and triplet-state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified.

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×10(4)M(-1). Taken together, the FAD-protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.

Integration of Temperature and Blue-Light Sensing in Acinetobacter baumannii Through the BlsA Sensor†

BlsA is a BLUF photoreceptor present in Acinetobacter baumannii, responsible for modulation of motility, biofilm formation and virulence by light. In this work, we have combined physiological and biophysical evidences to begin to understand the basis of the differential photoregulation observed as a function of temperature. Indeed, we show that blsA expression is reduced at 37°C, which correlates with negligible photoreceptor levels in the cells, likely accounting for absence of photoregulation at this temperature. Another point of control occurs on the functionality of the BlsA photocycle itself at different temperatures, which occurs with an average quantum yield of photoactivation of the signaling state of 0.20 ± 0.03 at 15°C < T < 25°C, but is practically inoperative at T > 30°C, as a result of conformational changes produced in the nanocavity of FAD. This effect would be important when the photoreceptor is already present in the cell to avoid almost instantaneously further signaling process when it is no longer necessary, for example under circumstances of temperature changes possibly faced by the bacteria. This complex interplay between light and temperature would provide the bacteria clues of environmental location and dictate/modulate light photosensing in A. baumannii.

Kinetics and growth mechanism of the photoinduced synthesis of silver nanoparticles stabilized with lysozyme

A fast and single-step procedure is reported for the preparation of stable solutions of spherical-shaped silver nanoparticles (AgNPs) coated with lysozyme (LZ). The preparation of the AgNP@LZ nanocomposites was based on the reduction of Ag+ with ketyl radicals photo-generated by the UVA-photolysis of the benzoin I-2959. Both reaction precursors bind to LZ, modifying its superficial charge and conformational structure. The photo-induced kinetics of formation of the AgNPs as a function of the LZ concentration was monitored in-situ by UV-vis absorption spectroscopy. The multivariate curve resolution-alternating least square (MCR-ALS) method was used for the deconvolution of the kinetic curves for each transient species formed before the growth of the final AgNPs colloids. The Kolmogorov-Johnson-Mehl-Avrami (KJMA) model to describe the formation of the AgNPs was used, and the respective first-order rate constants for the growth of the AgNPs as a function of the lysozyme concentration were calculated and the role of the protein capping in the growth kinetics was evaluated. Despite the protein being partially oxidized by the photo-generated radicals, it was strongly adsorbed onto the silver surface forming a tight coating shell around the AgNPs of approximately 30-60 protein molecules. As a result of the partial denaturation and crowded packing, its intrinsic lytic activity was strongly reduced.