The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (λmax =649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 μs, 390 μs, and 1.5 ms) for the red-to-green conversion, and 1.2 μs, 340 μs, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the green-absorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.

The total structural volume (ΔV str) and enthalpy (ΔH str) changes associated with the formation of the triplet metal-to-ligand charge transfer (3MLCT) state were determined by laser-induced optoacoustic spectroscopy for the complexes Ru(bpy)3 2+, Ru(bpy)2(CN)2, and Ru(bpy)(CN)4 2- in aqueous solutions of 0.1 M monovalent salts. For the cyano complexes (bound to water through hydrogen bonds) the values of ΔV str vs ΔH str exhibited a linear relationship in the salt series, whereas for Ru(bpy)3 2+ the values were independent of the salt. This linear correlation is interpreted as arising from an enthalpy−entropy compensation effect of the water structure perturbed by the 0.1 M salts. Since in all cases the intrinsic energy of the 3MLCT state was unperturbed by the salts (as determined by the constancy of the absorption and emission spectra), the plots ΔH str vs ΔV str (the latter shown to be proportional to ΔS str) yield the free energy for the formation of the 3MLCT state, ΔG MLCT = (123 ± 8) kJ/mol for Ru(bpy)2(CN)2 and (67 ± 25) kJ/mol for Ru(bpy)(CN)4 2-. The higher stability of the 3MLCT state of Ru(bpy)(CN)4 2- is due to the larger entropic factor which originates in the high flexibility photoinduced in its 3MLCT state by the loosening of four CN-bound water molecules, rather than only two in Ru(bpy)2(CN)2. The correlation ΔV str vs ΔS str (a consequence of the correlation between the environment reorganization parameters ΔV sol and ΔS sol) finds support in the proportionality between ΔV str of the cyano complexes and the calculated water-structuring entropy of the salts, ΔS°str(salt). Water structuring salts [negative ΔS°str(salt) values] afforded a larger ΔV str, due to the extension of the water network, opposite to structure breaking salts [positive ΔS°str(salt) values] which yielded smaller ΔV str values. For Ru(bpy)(CN)4 2- the linear dependence had twice as large a slope as for Ru(bpy)2(CN)2, reflecting the difference in the number of hydrogen-bound CN groups.

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In this chapter, we revise some of the most relevant and widely used synthetic routes available for the preparation of metallic silver nanoparticles. Particular emphasis has been focused in the rationale involved in the formation of the nanostructures, from the early metallic silver atoms formation, passing by atoms nucleation and concluding in the growth of silver nanostructures. We hope the reader will find in this chapter a valuable tool to better understand the relevance of the experimental conditions in the resulting silver nanoparticle size, shape and overall properties.

The photodynamic activities of the free-base 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) and their metal complexes with zinc(II) (ZnTMP), copper(II) (CuTMP) and cadmium(II) (CdTMP) have been compared in two systems: reverse micelle of n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate/water bearing photooxidizable substrates and Hep-2 human larynx carcinoma cell line. The quantum yields of singlet molecular oxygen, O2(1 delta g), production (phi delta) of TMP, ZnTMP and CdTMP in tetrahydrofuran, were determined yielding values of 0.65, 0.73 and 0.73, respectively, while O2(1 delta g) formation was not detected for CuTMP. In the reverse micellar system, the amino acid L-tryptophan (Trp) was used as biological substrate to analyze the O2(1 delta g)-mediated photooxidation. The observed rate constants for Trp photooxidation (kobsTrp) were proportional to the sensitizer quantum yield of O2(1 delta g). A value of approximately 2 x 10(7) s-1 M-1 was found for the second-order rate constant of Trp (krTry) in this system. The response of Hep-2 cells to cytotoxicity photoinduced by these agents in a biological medium was studied. The Hep-2 cultures were treated with 1 microM of porphyrin for 24 h at 37 degrees C and the cells exposed to visible light. The cell survival at different light exposure levels was dependent on phi delta. Under these conditions, the cytotoxic effect increases in the order: Cu-TMP << TMP < ZnTMP approximately CdTMP, correlating with the production of O2(1 delta g). A similar behavior was observed in both the chemical and biological media indicating that the O2(1 delta g) mediation appears to be mainly responsible for the cell inactivation.

A novel Zn(II)-phthalocyanine (1). peripherally substituted with four bis(N,N,N-trimethyl)amino-2-propyloxy groups prepared by chemical synthesis is shown to be an efficient photodynamic sensitizer with a quantum yield of 0.6 for singlet oxygen generation in neat water, which is reduced to about 0.3 in phosphate-buffered saline. The physicochemical properties of 1 in both the ground and the electronically excited states strongly depend on the nature of the medium; in particular, aggregation of 1 was favoured by polar media of high ionic strength. Compound 1 exhibited an appreciable affinity for a typical Gram-positive bacterium (Staphylococcus aureus) and a typical Gram-negative bacterium (Escherichia coli). Both bacterial strains were extensively inactivated upon 5 min-irradiation with 675 nm light in the presence of 1 microM photosensitizer, even though the binding of 1 to the two bacterial cells appears to occur according to different pathways. In particular, E. coli cells underwent initial photodamage at the level of specific proteins in the outer wall, thus promoting the penetration of the photosensitizer to the cytoplasmic membrane where some enzymes critical for cell survival were inactivated.

Rose bengal (RB) readily binds to human serum albumin (HSA). At low RB concentrations, 90% of the dye is associated to the protein (5 microM), This association takes place in specific binding sites I and/or II. At higher RB concentrations, unspecific binding takes place with up to 10 RB molecules bound per protein molecule. The behavior of excited RB molecules bound to HSA is widely different to that observed in aqueous solution. Furthermore, the data also show that the behavior of bound RB molecules changes with the average number of dye molecules per protein (n). In particular, when n is large, the fluorescence yield is significantly reduced and no measurable long-lived triples and free singlet oxygen formation from bound dyes is detected. These results are related to self-quenching of the singlet and, most likely, excited triplets. All results point to the relevance of intra-protein generated singlet oxygen. However, when the dye is bound to the protein, at low oxygen concentrations such as those prevailing in vivo, trapping by oxygen of the triplet becomes inefficient and type I processes could contribute to the observed photoprocesses.

The photodecarboxylation reaction of 2-(3-benzoylphenyl)propionate (ketoprofen anion, KP-) was studied in water and in 0.1 M phosphate buffer solutions in the pH range 5.7-11.0 by laser-induced optoacoustic spectroscopy (LIOAS, T range 9.5-31.6 degrees C). Upon exciting KP- with 355 nm laser pulses under anaerobic conditions, two components in the LIOAS signals with well-separated lifetimes were found (tau 1 < 20 ns; 250 < tau 2 < 500 ns) in the whole pH range, whereas a long-lived third component (4 < tau 3 < 10 microseconds) was only detected at pH < or = 6.1. The heat and structural volume changes accompanying the first step did not depend on pH or on the presence of buffer. The carbanion resulting from prompt decarboxylation within the nanosecond pulse (< 10 ns) drastically reduces its molar volume ([-18.9 +/- 2.0] cm3/mol) with respect to KP- and its enthalpy content is (256 +/- 10) kJ/mol. At acid pH (ca 6), a species is formed with a lifetime in the hundreds of ns. The enthalpy and structural volume change for this species with respect to KP- are (181 +/- 15) kJ/mol and (+0.6 +/- 2.0) cm3/mol, respectively. This species is most likely a neutral biradical formed by protonation of the decarboxylated carbanion, and decays to the final product 3-ethylbenzophenone in several microsecond. At basic pH (ca 11), direct formation of 3-ethylbenzophenone occurs in hundreds of ns involving a reaction with the solvent. The global decarboxylation reaction is endothermic ([45 +/- 15] kJ/mol) and shows an expansion of (+14.5 +/- 0.5) cm3/mol with respect to KP-. At low pH, the presence of buffer strongly affects the magnitude of the structural volume changes associated with intermolecular proton-transfer processes of the long-lived species due to reactions of the buffer anion with the decarboxylated ketoprofen anion.