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Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV–Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mossbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron.

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.

The interaction between gold sub-nanometer clusters composed of ten atoms (Au10) and tetrakis(4-sulfonatophenyl)porphyrin (TPPS) was investigated through various spectroscopic techniques. Under mild acidic conditions, the formation, in aqueous solutions, of nanohybrid assemblies of porphyrin J-aggregates and Au10 cluster nanoparticles was observed. This supramolecular system tends to spontaneously cover glass substrates with a co-deposit of gold nanoclusters and porphyrin nanoaggregates, which exhibit circular dichroism (CD) spectra reflecting the enantiomorphism of histidine used as capping and reducing agent. The morphology of nanohybrid assemblies onto a glass surface was revealed by atomic force microscopy (AFM), and showed the concomitant presence of gold nanoparticles with an average size of 130 nm and porphyrin J-aggregates with lengths spanning from 100 to 1000 nm. Surface-enhanced Raman scattering (SERS) was observed for the nanohybrid assemblies.

Coproheme decarboxylases (ChdC) catalyze the hydrogen peroxide-mediated conversion of coproheme to heme b. This work compares the structure and function of wild-type (WT) coproheme decarboxylase from Listeria monocytogenes and its M149A, Q187A, and M149A/Q187A mutants. The UV–vis, resonance Raman, and electron paramagnetic resonance spectroscopies clearly show that the ferric form of the WT protein is a pentacoordinate quantum mechanically mixed-spin state, which is very unusual in biological systems. Exchange of the Met149 residue to Ala dramatically alters the heme coordination, which becomes a 6-coordinate low spin species with the amide nitrogen atom of the Q187 residue bound to the heme iron. The interaction between M149 and propionyl 2 is found to play an important role in keeping the Q187 residue correctly positioned for closure of the distal cavity. This is confirmed by the observation that in the M149A variant two CO conformers are present corresponding to open (A0) and closed (A1) conformations. The CO of the latter species, the only conformer observed in the WT protein, is H-bonded to Q187. In the absence of the Q187 residue or in the adducts of all the heme b forms of ChdC investigated herein (containing vinyls in positions 2 and 4), only the A0 conformer has been found. Moreover, M149 is shown to be involved in the formation of a covalent bond with a vinyl substituent of heme b at excess of hydrogen peroxide.

The ligand binding characteristics of heme-containing proteins are determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. In this regard, the heme pockets of truncated hemoglobins (TrHbs) constitute an interesting case study as they share many common features, including a number of polar cavity residues. In this review, we will focus on three proteins of group II TrHbs, from Thermobifida fusca (Tf-HbO) and Pseudoalteromonas haloplanktis TAC125 (Ph-HbO). Although the residues in positions G8 (Trp) and B10 (Tyr) are conserved in all three proteins, the CD1 residue is a Tyr in T. fusca and a His in P. haloplanktis. Comparison of the ligand binding characteristics of these proteins, in particular the hydroxo and CO ligands by means of resonance Raman spectroscopy, reveals that this single difference in the key heme cavity residues markedly affects their ligand binding capability and conformation. Furthermore, although the two Ph-HbOs (Ph-HbO-2217 and Ph-HbO-0030) have identical key cavity residues, they display distinct ligand binding properties.

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O2-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O2 species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O2 uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217.

We investigate the effects of antimicrobial (sodium citrate tribasic, E331) and antioxidant (ascorbic acid, E300 and sodium ascorbate, E301) additives on the meat drip from defrosted yellowfin tuna fish loins obtained from the local market and horse heart myoglobin. The effects have been followed by electronic absorption, its second derivative spectra, and resonance Raman spectroscopies. Upon addition of the additives, a final form is reached after about 24 h. It is characterized by a 4 nm red-shifted Soret band compared to that typical of the oxy species (418 nm) but with similar Q bands. Resonance Raman experiments carried out in 16O2 and 18O2 allowed us to identify the presence of the native oxy form coexisting with a second oxygen bound species, characterized by a ν(Fe O2) stretching frequency upshifted 7 cm−1 compared to the native oxy form and with a greater (33 cm−1) isotopic shift in 18O2. These data suggest the presence of a highly bent ligand conformation. The new species induced by the addition of the additives imparts a red colour to the tuna fish meat, a characteristic that is of some concern. In fact, the presence of the new red form can mask the aging of the product that, consequently, might contain histamine. Furthermore, the electronic absorption spectrum is very similar to that of the tuna fish myoglobin carbon monoxide complex, which has important regulatory consequences. Carbon monoxide treatment of tuna is banned in the EU for masking the effects of aging on the appearance of meats.

Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c–CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys→Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein. Graphical abstract: [Figure not available: see fulltext.]

Structurally and phylogenetically closely related chlorite dismutase (Cld) and HemQ differ fundamentally in their enzymatic properties. Reconstruction of the proximal H-bonding network renders Cld HemQ-like, the latter being proposed to bind coprohaeme and release protohaeme., Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from ‘Candidatus Nitrospira defluvii’ (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV–vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure–function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria.

Spectroscopic and functional properties of human cytochrome c and its Tyr67 residue mutants (i.e., Tyr67His and Tyr67Arg) have been investigated. In the case of the Tyr67His mutant, we have observed only a very limited structural alteration of the heme pocket and of the Ω-loop involving, among others, the residue Met80 and its bond with the heme iron. Conversely, in the Tyr67Arg mutant the Fe-Met80 bond is cleaved; consequently, a much more extensive structural alteration of the Ω-loop can be envisaged. The structural, and thus the functional modifications, of the Tyr67Arg mutant are present in both the ferric [Fe(III)] and the ferrous [Fe(II)] forms, indicating that the structural changes are independent of the heme iron oxidation state, depending instead on the type of substituting residue. Furthermore, a significant peroxidase activity is evident for the Tyr67Arg mutant, highlighting the role of Arg as a basic, positively charged residue at pH7.0, located in the heme distal pocket, which may act as an acid to cleave the O-O bond in H2O2. As a whole, our results indicate that a delicate equilibrium is associated with the spatial arrangement of the Ω-loop. Clearly, Arg, but not His, is able to stabilize and polarize the negative charge on the Fe(III)-OOH complex during the formation of Compound I, with important consequences on cytochrome peroxidation activity and its role in the apoptotic process, which is somewhat different in yeast and mammals.

Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys2-Cys2 type zinc fingers and a set of four conserved cysteines arranged in a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster.

In this chapter, we will discuss the paradigmatic case of Thermobifida fusca (Tf-trHb) HbO in its ferrous and ferric states and its behaviour towards a battery of possible ligands. This choice was dictated by the fact that it has been one of the most extensively studied truncated haemoglobins, both in terms of spectroscopic and molecular dynamics studies. Tf-trHb typifies the structural properties of group II trHbs, as the active site is characterized by a highly polar distal environment in which TrpG8, TyrCD1, and TyrB10 provide three potential H-bond donors in the distal cavity capable of stabilizing the incoming ligands. The role of these residues in key topological positions, and their interplay with the iron-bound ligands, has been addressed in studies carried out on the CO, F(-), OH(-), CN(-), and HS(-) adducts formed with the wild-type protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. In this context, such a complete analysis provides an excellent benchmark for the investigation of the relationship between protein structure and function, allowing one to translate physicochemical properties of the active site into the observed functional behaviour. Tf-trHb will be compared with other members of the group II trHbs and, more generally, with members of the other trHb subgroups.

The heme-containing peroxidases of the plant peroxidase superfamily have been the subject of extensive studies to gain a complete understanding of their structure and function properties. Spectroscopic techniques have been fundamental to the comprehension of peroxidase function under physiological conditions. Resonance Raman spectroscopy has assumed a particularly important role due to its extreme sensitivity to alterations in the surroundings of the heme, which makes it an ideal tool for studying molecular interactions at the peroxidase active sites. In combination with site-directed mutagenesis and X-ray structural data, it has given insight into the influence exerted on protein architecture in the vicinity of the active site by key amino acids and furnished critical information on subtle structural features important for protein flexibility, functionality and stability. The mutation of key catalytic residues on both the distal and proximal sides of the heme cavity not only revealed their roles in the reaction mechanism of peroxidases, but also demonstrated that in peroxidases there are common structural mechanisms which facilitate communication between the two sides of the heme cavity and highlighted the importance of long range interactions in maintaining the functional properties of the heme. The overwhelming success of this approach using the combination of site-directed mutagenesis together with spectroscopic techniques and X-ray structural data to unveil detailed aspects of the structure–function relationships of heme peroxidases, firstly in the pioneering studies on yeast cytochrome c peroxidase then for other peroxidases, has led to its subsequent widespread general application in the characterization of heme proteins.

Nitrite is an important metabolite in the physiological pathways of NO and other nitrogen oxides in both enzymatic and nonenzymatic reactions. The ferric heme b protein nitrophorin 4 (NP4) is capable of catalyzing nitrite disproportionation at neutral pH, producing NO. Here we attempt to resolve its disproportionation mechanism. Isothermal titration calorimetry of a gallium(III) derivative of NP4 demonstrates that the heme iron coordinates the first substrate nitrite. Contrary to previous low-temperature EPR measurements, which assigned the NP4-nitrite complex electronic configuration solely to a low-spin (S = 1/2) species, electronic absorption and resonance Raman spectroscopy presented here demonstrate that the NP4-NO2(-) cofactor exists in a high-spin/low-spin equilibrium of 7:3 which is in fast exchange in solution. Spin-state interchange is taken as evidence for dynamic NO2(-) coordination, with the high-spin configuration (S = 5/2) representing the reactive species. Subsequent kinetic measurements reveal that the dismutation reaction proceeds in two discrete steps and identify an {FeNO}(7) intermediate species. The first reaction step, generating the {FeNO}(7) intermediate, represents an oxygen atom transfer from the iron bound nitrite to a second nitrite molecule in the protein pocket. In the second step this intermediate reduces a third nitrite substrate yielding two NO molecules. A nearby aspartic acid residue side-chain transiently stores protons required for the reaction, which is crucial for NPs' function as nitrite dismutase.

Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two‐on‐two α‐helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group‐II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph‐2/2HbO), a cold‐adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph‐2/2HbO aquo‐met crystal structure (at 2.21 Å resolution) shows typical features of group‐II truncated hemoglobins, namely the two‐on‐two α‐helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal‐site hydrogen‐bonded network that includes water molecules and several distal‐site residues, including His(58)CD1. Analysis of Ph‐2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph‐2/2HbO can access diverse heme ligation states. Among these, detection of a low‐spin heme hexa‐coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme‐Fe ligand. Altogether, the results show that Ph‐2/2HbO maintains the general structural features of group‐II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment. Fil: Giordano, Daniela. Consiglio Nazionale delle Ricerche; Italia Fil: Pesce, Alessandra. Università degli Studi di Genova; Italia Fil: Boechi, Leonardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bustamante, Juan Pablo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caldelli, Elena. Universita Degli Studi Di Firenze; Italia Fil: Howes, Barry D.. Universita Degli Studi Di Firenze; Italia Fil: Riccio, Alessia. Consiglio Nazionale delle Ricerche; Italia Fil: di Prisco, Guido. Consiglio Nazionale delle Ricerche; Italia Fil: Nardini, Marco. Università degli Studi di Milano; Italia Fil: Estrin, Dario Ariel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Smulevich, Giulietta. Universita Degli Studi Di Firenze; Italia Fil: Bolognesi, Martino. Università degli Studi di Milano; Italia Fil: Verde, Cinzia. Consiglio Nazionale delle Ricerche; Italia. Universita Di Roma; Italia

Protein-bound iron sulfur clusters are prosthetic groups involved in several metabolic pathways. Understanding how they interact with the host protein and which factors influence their stability is therefore an important goal in biology. Here, we have addressed this question by studying the determinants of the 2Fe–2S cluster stability in the IscU/Isu protein scaffold. Through a detailed computational study based on a mixed quantum and classical mechanics approach, we predict that the simultaneous presence of two conserved residues, D39 and H105, has a conflicting role in cluster coordination which results in destabilizing cluster-loaded IscU/Isu according to a ‘tug-of-war’ mechanism. The effect is absent in the D39A mutant already known to host the cluster more stably. Our theoretical conclusions are directly supported by experimental data, also obtained from the H105A mutant, which has properties intermediate between the wild-type and the D39A mutant. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

In this chapter, we will discuss the paradigmatic case of Thermobifida fusca (Tf-trHb) HbO in its ferrous and ferric states and its behaviour towards a battery of possible ligands. This choice was dictated by the fact that it has been one of the most extensively studied truncated haemoglobins, both in terms of spectroscopic and molecular dynamics studies. Tf-trHb typifies the structural properties of group II trHbs, as the active site is characterized by a highly polar distal environment in which TrpG8, TyrCD1, and TyrB10 provide three potential H-bond donors in the distal cavity capable of stabilizing the incoming ligands. The role of these residues in key topological positions, and their interplay with the iron-bound ligands, has been addressed in studies carried out on the CO, F(-), OH(-), CN(-), and HS(-) adducts formed with the wild-type protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. In this context, such a complete analysis provides an excellent benchmark for the investigation of the relationship between protein structure and function, allowing one to translate physicochemical properties of the active site into the observed functional behaviour. Tf-trHb will be compared with other members of the group II trHbs and, more generally, with members of the other trHb subgroups.

The unique architecture of the active site of Thermobifida fusca truncated hemoglobin (Tf-trHb) and other globins belonging to the same family has stimulated extensive studies aimed at understanding the interplay between iron-bound ligands and distal amino acids. The behavior of the heme-bound hydroxyl, in particular, has generated much interest in view of the relationships between the spin-state equilibrium of the ferric iron atom and hydrogen-bonding capabilities (as either acceptor or donor) of the OH− group itself. The present investigation offers a detailed molecular dynamics and spectroscopic picture of the hydroxyl complexes of the WT protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. Each mutant is characterized by a complex interplay of interactions in which the hydroxyl ligand may act both as a H-bond donor or acceptor. The resonance Raman stretching frequencies of the Fe−OH moiety, together with electron paramagnetic resonance spectra and MD simulations on each mutant, have enabled the identification of specific contributions to the unique ligand-inclusive H-bond network typical of this globin family. Fil: Nicoletti, Francesco P.. Universita Degli Studi Di Firenze; Italia Fil: Bustamante, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Droghetti, Enrica. Universita Degli Studi Di Firenze; Italia Fil: Howes, Barry D.. Universita Degli Studi Di Firenze; Italia Fil: Fittipaldi, Maria. Universita Degli Studi Di Firenze; Italia Fil: Bonamore, Alessandra. Universita Di Roma; Italia Fil: Baiocco, Paola. Italian Institute of Technology; Italia Fil: Feis, Alessandro. Universita Degli Studi Di Firenze; Italia Fil: Boffi, Alberto. Universita Di Roma; Italia Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Smulevich, Giulietta. Universita Degli Studi Di Firenze; Italia