Filipe Folgosa

Spectroscopic evidence for and characterization of a trinuclear ferroxidase center in bacterial ferritin from Desulfovibrio vulgaris Hildenborough.

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 Å in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe(2+), and the products were characterized by Mössbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a μ-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe(2+)Fe(3+) state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.

Desulfovibrio vulgaris bacterioferritin uses H2O2 as a co-substrate for iron oxidation and reveals DPS-like DNA protection and binding activities

A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H(2)O(2) oxidizes Fe(2+) ions at much higher reaction rates than O(2). The H(2)O(2) oxidation of two Fe(2+) ions was proven by Mössbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mössbauer parameters of the intermediate species we propose that D. vulgaris Bfr follows a mineralization mechanism similar to the one reported for vertebrate H-type ferritins subunits, in which a diferrous centre at the ferroxidase site is oxidized to diferric intermediate species, that are subsequently translocated into the inner nanocavity. D. vulgaris recombinant Bfr oxidizes and stores up to 600 iron atoms per protein. This Bfr is able to bind DNA and protect it against hydroxyl radical and DNase deleterious effects. The use of H(2)O(2) as an oxidant, combined with the DNA binding and protection activities, seems to indicate a DPS (DNA-binding protein from starved cells)-like role for D. vulgaris Bfr.

New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway

SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 × 10⁷ M⁻¹ · s⁻¹ and 1.3 × 10⁶ M⁻¹ · s⁻¹ for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 × 10⁶ M⁻¹ · s⁻¹ for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.

Two coupled photochromic systems of 3′,4′-(methylenedioxy)flavylium: kinetic and thermodynamic characterization

The chemistry and photochemistry of 3′,4′-(methylenedioxy)flavylium was studied by means of UV–Vis spectrophotometry, 1H NMR, stopped flow, and continuous irradiation, in acidic and basic aqueous solutions. Six species were identified: the flavylium cation, AH+; the hemiketal, B; the cis- and the trans-chalcones, Cc and Ct, and their ionized forms, Cc− and Ct−. These species define a multiequilibria network whose kinetics and thermodynamics were completely characterized. The two pairs of chalcones define two coupled photochromic systems, respectively in acidic and basic media, both allowing cycles capable of writing, reading and erasing to be defined.

Iron management and production of electricity by microorganisms

The increasing dependency on fossil fuels has driven researchers to seek for alternative energy sources. Renewable energy sources such as sunlight, wind, or water are the most common. However, since the 1990s, other sources for energy production have been studied. The use of microorganisms such as bacteria or archaea to produce energy is currently in great progress. These present several advantages even when compared with other renewable energy sources. Besides the energy production, they are also involved in bioremediation such as the removal of heavy metal contaminants from soils or wastewaters. Several research groups have demonstrated that these organisms are able to interact with electrodes via heme and non-heme iron proteins. Therefore, the role of iron as well as iron metabolism in these species must be of enormous relevance. Recently, the influence of cellular iron regulation by Fur in the Geobacter sulfurreducens growth and ability to produce energy was demonstrated. In this review, we aim to briefly describe the most relevant proteins involved in the iron metabolism of bacteria and archaea and relate them and their biological function with the ability of selected organisms to produce energy.

UV radiation effects on a DNA repair enzyme: conversion of a [4Fe–4S]2+ cluster into a [2Fe–2S]2+

Organisms are often exposed to different types of ionizing radiation that, directly or not, will promote damage to DNA molecules and/or other cellular structures. Because of that, organisms developed a wide range of response mechanisms to deal with these threats. Endonuclease III is one of the enzymes responsible to detect and repair oxidized pyrimidine base lesions. However, the effect of radiation on the structure/function of these enzymes is not clear yet. Here, we demonstrate the effect of UV-C radiation on E. coli endonuclease III through several techniques, namely UV–visible, fluorescence and Mössbauer spectroscopies, as well as SDS-PAGE and electrophoretic mobility shift assay. We demonstrate that irradiation with a UV-C source has dramatic consequences on the absorption, fluorescence, structure and functionality of the protein, affecting its [4Fe–4S] cluster and its DNA-binding ability, which results in its inactivation. An UV-C radiation-induced conversion of the [4Fe–4S]2+ into a [2Fe–2S]2+ was observed for the first time and proven by Mössbauer and UV–visible analysis. This work also shows that the DNA-binding capability of endonuclease III is highly dependent of the nuclearity of the endogenous iron–sulfur cluster. Thus, from our point of view, in a cellular context, these results strengthen the argument that cellular sensitivity to radiation can also be due to loss of radiation-induced damage repair ability.

Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14+/-1 subunits of 15254.3+/-7.6 Da. Mössbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.

Imine Ligands Based on Ferrocene: Synthesis, Structural and Mössbauer Characterization and Evaluation as Chromogenic and Electrochemical Sensors for Hg+2

The compounds 1-naphthyliminoferrocene, 1, and 2-methyl-1-naphthyliminoferrocene, 2, were prepared by the reaction of ferrocenecarboxaldehyde and the corresponding amine, 1-naphthylamine and 2-methyl-1-naphthylamine with one drop of pyrrolidine as the catalyst, in CH2Cl2, under an inert atmosphere. The compounds were fully characterized by elemental analyses, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF), FT-IR, 1H and 13C NMR as well as two-dimensional, NOESY, COSY and HSQC, NMR spectroscopy. Mossbauer spectroscopic studies were performed to characterize iron species in both compounds. The solid-state molecular structures of compounds 1 and 2 were determined by single crystal X-ray diffraction. Analysis of the electrochemical behaviour of complexes 1 and 2 was performed by cyclic and square wave voltammetry. Compounds 1 and 2 were tested as chromogenic and electrochemical sensors for Hg2+.