Exploring cellular metal transfer mechanisms by EPR spectroscopy

Sharon Ruthstein, Chemistry, Bar Ilan University, Ramat Gan, Israel

Metals are commonly found as natural constituents of proteins; however, many metal ions can be toxic when free in biological fluids. Hence, the eukaryotic and prokaryotic systems have evolved tremendous sophistication regulatory machinery to manage the intracellular and extracellular concentration of metal ions. In this talk we will shed some light on two important copper regulation systems. One in the human cell, and the other in the E. coli cell. In the human cell Cu(II) is accumulated in our body through diet, it is then reduced to Cu(I) in a mechanism that has not been resolved yet. After reduction the copper transporter (CTR1) delivers Cu(I) to the cell. This research utilizes EPR spectroscopy, to explore the cellular copper import and uptake mechanisms by the CTR1. Herein we will show that that the CTR1 extracellular domain consists both Cu(II) as well as Cu(I) binding sites, suggesting that the copper reduction mechanism occurred by the CTR1. Moreover, this research suggests that a competitive metal ion such as Ag(I) might block the Cu(I) binding sites, causing inhibition of the CTR1. Finally, we will show that Cys189 in the CTR1 c-terminal domain is tremendously important for proper interaction with a cytoplasmic target protein.

In the second part of the talk we will concentrate on the prokaryotic system – the E.coli cell. The E.coli cell has three main regulation systems for copper ion. One of them is the CusFCBA periplasma pump system, that transfer Cu(I) and Ag(I) ions from the cytoplasm out of the cell. Herein, we will show that methionine and lysine residues that are essential for the interaction between the two proteins and for copper coordination control the interaction between the CusF metallochaperone and CusB.

This work will show the importance of EPR as a biophysical tool to study cellular metal ion transfer pathways.