Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica
Abstract
Dissertation presented to obtain the Ph.D. degree in BiochemistryReactive oxygen and nitrogen species (ROS and RNS) are produced
by phagocytic cells of the human immune system to attack invading
pathogens due to their ability to damage DNA and the metal centres of
proteins. In order to survive inside the host, bacteria activate genes that
encode detoxifier enzymes, like the Escherichia coli nitric oxide-reductase
flavodiiron protein, also known as flavorubredoxin (FlRd), and repairing
proteins, such as the E. coli YtfE di-iron protein involved in the recovery of
damaged Fe-S centres. Using E. coli and Staphylococcus aureus, the work
presented in this thesis aimed at unravelling: i) the role of E. coli FlRd in
bacteria exposed to a combination of oxidative and nitrosative stresses, ii)
the identification and characterisation of S. aureus YtfE homologue, iii) the
study of E. coli YtfE mechanisms that allow the repair of damaged Fe-S
clusters, and iv) the identification of proteins that interact with E. coli YtfE.
To analyse the role of E. coli FlRd in cells submitted to both
hydrogen peroxide and nitric oxide, the transcription and expression of
norV was explored by means of β-galactosidase and immunoblotting
assays, respectively. Under these conditions, it was observed that the norV
transcription and expression were hindered. To identify if the lack of norV
expression was related to its regulator, the NorR transcription factor, the
gene was cloned and expressed, and the protein was purified and the
binding of nitric oxide to NorR in the presence of hydrogen peroxide was
studied. EPR experiments revealed that upon incubation of NorR with nitric
oxide and hydrogen peroxide the oxidation promoted by H2O2 of the monoiron
centre of NorR impairs the ligation of nitric oxide.(...
