Benoît d'Autréaux
Spectroscopic studies of the Fur protein (Ferric Uptake regulation). Interaction with nitric oxide
Published on 25 November 2002
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Thesis presented November 25, 2002
Abstract:
The FUR protein (Ferric Uptake Regulation) regulates the concentration of iron in Gram negative bacteria. The FUR protein from
Escherichia coli is isolated as a 2x17 kDa dimer, which contains one Zn2+ ion per monomer. The regulation process involves the coordination of a Fe2+ ion leading to a conformational change of the FUR protein. Subsequently to the conformational change, the protein binds to the promoter region of genes associated with iron metabolism and down regulates their expression. We have studied the influence of oligomerization, metallation and nitric oxide on the structure and activity of the
E. coli FUR protein. While the FUR protein is capable to form both tetramer and hexamer, it mainly exists as a dimer at physiological concentrations. Another purification product is a monomer with two disulphide bridges. This monomer is not in equilibrium with the dimer and can not be activated by metallic ions. However, an activable dimer can be obtained from the monomer upon reduction of the disulphide bridges by DDT and the addition of metallic ions (Zn2+, Co2+ or Fe2+). Mössbauer and UV-visible spectroscopies had revealed that these ions occupy a similar site to the one described for Zn2+ in the native dimer. Nitric oxide, NO, is produced by eukaryotes as a mechanism of defense against bacterial invasion. NO is known to react with the iron center of numerous proteins. We have studied its action on iron activated FUR protein. We have found that both
in vivo and
in vitro, NO inhibits FUR activity. EPR spectroscopy studies have shown that NO reacts with the iron in the FUR protein to form an iron-nitrosyl S=1/2 adduct, which is stable in anaerobic conditions and which retains its dimeric structure. Further studies using Mössbauer, ENDOR, FTIR and UV-visible spectroscopies suggest that the reaction product is a dinitrosyl Fur-Fe(NO)2 complex. These studies have demonstrated the link between the control of iron metabolism by FUR and the response to NO.
Keywords:
Nitric oxide, Iron, Cobalt, Zinc, Oligomerization, EPR, ENDOR, Mössbauer, FTIR, Metalloprotein, Transcriptional repressor
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