The role of Di-iron proteins in pathogen resistance

Dissertation presented to obtain the Ph.D. degree in Biochemistry

Reactive 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.(...)