... CM Cordas (&) Á P . Raleiras Á F . Auche`re Á I. Moura Á JJG Moura ... de Quımica, Universidade Nova de Lisboa, 2859-516 Caparica, Portugal e-mail: cristina. cordas @dq.fct ... Present Address: P . Raleiras Department of Photochemistry and Molecular Science, PO Box 523, 75120 ...

A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H2O2 oxidizes Fe2+ ions at much higher reaction rates than O-2. The H2O2 oxidation of two Fe2+ ions was proven by Mossbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mossbauer 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 H2O2 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.

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 similar to 80 angstrom in diameter. The main function of ferritin is to oxidize the cytotoxic Fe2+ ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe2+ (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 Fe2+, and the products were characterized by Mossbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe2+Fe3+ species, and a mononuclear Fe2+ 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 mu-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe2+Fe3+ 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.

The enzymatic (tyrosinase) and chemical (NaIO4, Ag2O or Fremys's salt) oxidation of biologically relevant catecholamines, such as dopamine (DA), N-acetyldopamine (NADA) and the Ecstasy metabolites (alpha-MeDA and N-Me-alpha-MeDA) generates the corresponding o-quinone which can be trapped with nitrogen bionucleophiles such as N-acetyl-histidine and imidazole in a regioselective reaction that takes place predominantly at the 6-position of the catecholamine. (C) 2012 Elsevier Inc. All rights reserved.