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Iron storage and regulation at a molecular level
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Iron is an essential element for the proper functioning of the metabolic network in a
living system. However, it is also toxic in physiological conditions. Apart from precipitation it can
damage and compromise cellular macromolecules by Fenton reactions. Thus, ferritins, hollow
spherical proteins, comes to solve this problem by storing iron in its inner cavity. Dps (DNAbinding
protein in starved cells), focused in this study, has a detoxifying function, protecting
DNA from ROS.
The reaction catalyzed by ferritins can be divided in the following stages: iron intake,
oxidation, storage and release. The latter is the least explored and known function of this
protein.
The M. hydrocarbonoclasticus WrbA flavoprotein, present in the same genome, was
used as a P. nauticas Dps redox partner, to reduced and release iron from the iron core.
Mössbauer spectroscopy was used to investigate the kinetics properties of
WrbA(FMN):NADH:Dps in anaerobic conditions. To determine kinetic parameters it was needed
to acquire spectra for different reaction times. The iron release for wild-type, Q14E and Δ15 Dps
variants follow a first-order kinetic, with rate constants very similar.
Was also explored a more inexpensive and faster kinetic assay based on the ophenanthroline
method, monitored by Visible spectroscopy. The result showed that the three
Dps variants have no significant difference regarding the kinetic profile obtained, but rate
constants were significantly lower than those obtained by Mössbauer spectroscopy probed
kinetic measurements. Phenanthroline might cause an inhibitor effect and in order to
understand that effect, the kinetic assays were repeated in the absence of phenanthroline.
Using bioinformatic tools (docking, modeling and others), was possible to conclude that
exist conserved amino acid (G43, L74, P78 e W149) in Dps that appear to participate and are in
the electron transfer pathway.
Descrição
Palavras-chave
Dps iron release Mössbauer UV-Visible spectroscopy o-phenanthroline assay docking