Copper homeostasis in bacteria cells – exploring cellular metal transfer pathways and mechanisms

Copper's ability to accept and donate single electrons makes it an ideal redox cofactor, and thus one of the most essential metal ions to the survival of the cell. However, copper ions are also involved in the Fenton reaction and hence capable of driving the generation of deleterious hydroxyl radicals, which are deleterious to the cell. Moreover, since Cu(II) is at the top of the Irving-Williams series, it can compete with other metals in metalloproteins. Hence, both prokaryotic systems as well as eukaryotic system have developed a considerable regulation mechanism to maintain negligible copper concentration, in the femtomolar concentration. E.coli cells, in common with the vast majority of bacterial cells, require copper for several important enzymes such as ubiquinole oxidases, Cu,Zn-superoxide dismutases, or cytochrome c oxidase. However, as was mentioned above, copper can be deleterious, making protective mechanisms necessary. Understanding this regulation mechanism in bacteria, is tremendously important from two specific reasons: one over 70% of the putative cuproproteins identified in prokaryotes have homologs in eukaryotes, and thus resolving the copper cycle in prokaryotic systems will also shed light on the copper cycle in eukaryotic systems. Second, copper has been used throughout much of the human civilization as an antimicrobial agent. Hence, understanding in detail the copper resistance mechanism in bacteria, is significantly important for understanding the microorganisms' degree of survival in the mammalian cell. In this talk we will shed some light on two important copper regulation systems in E.coli: the copper periplasmic efflux system, CusCFBA, and the Cu(I) metal sensor, gene expression regulation system, CueR. Using Electron Paramagnetic Resonance (EPR) spectroscopy, together with biochemical experiments and computational methods we will show the essentiality of methionine and lysine residues to the interaction between two proteins in the CusCFBA system. We will also present a structural model for the CueR-Cu(I)-DNA complex, shedding light on the transcription mechanism of the CueR protein. This work will show the importance of EPR as a biophysical tool to study cellular metal ion transfer pathways.