RESUMO
Iron is an essential micronutrient for living organisms as it is involved in a broad variety of important biological processes. However, free iron inside the cell could be potentially toxic, generating hydroxyl radicals through the Fenton reaction. Dps (DNA-binding protein from starved cells) belongs to a subfamily of ferritins and can store iron atoms inside the dodecamer. The presence of a ferroxidase centre, composed of highly conserved residues, is a signature of this protein family. In this study, we analysed the role of two conserved histidine residues (H25 and H37) located at the ferroxidase centre of the Campylobacter jejuni Dps protein by replacing them with glycine residues. The C. jejuni H25G/H37G substituted variant showed reduced iron binding and ferroxidase activities in comparison with wt Dps, while DNA-binding activity remained unaffected. We also found that both CjDps wt and CjDps H25G/H37G were able to bind manganese atoms. These results indicate that the H25 and H37 residues at the ferroxidase centre of C. jejuni Dps are not strictly required for metal binding and oxidation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/enzimologia , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Histidina/metabolismo , Ferro/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Campylobacter jejuni/química , Campylobacter jejuni/genética , Ceruloplasmina/genética , Sequência Conservada , Histidina/química , Histidina/genética , Cinética , Dados de Sequência Molecular , OxirreduçãoRESUMO
Dps proteins (DNA binding protein from starved cell) form a distinct group within the ferritin superfamily. All Dps members are composed of 12 identical subunits that assemble into a conserved spherical protein shell. Dps oxidize Fe(2+) in a conserved ferroxidase center located at the interface between monomers, the product of the reaction Fe(3+), is then stored inside the protein shell in the form of non-reactive insoluble Fe2O3. The Campylobacter jejuni Dps (CjDps) has been reported to play a plethora of functions, such as DNA binding and protection, iron storage, survival in response to hydrogen peroxide and sulfatide binding. CjDps is also important during biofilm formation and caecal colonization in poultry. In order to facilitate in vitro characterisation of CjDps, it is important to have a simple and reproducible protocol for protein purification. Here we report an observation that CjDps has an unusual high melting temperature. We exploited this property for protein purification by introducing a thermal treatment step which allowed achieving homogeneity by using only two chromatographic steps. Gel filtration chromatography, circular dichroism, mass spectrometry, DNA-binding and iron oxidation analysis confirmed that the CjDps structure and function were unaffected.