S77C-ΔC7-CueR: a 199mHg PAC study of the protein metal site structure

Abstract

AbstractThe CueR protein regulates the cytosolic concentration of Cu(I) in bacteria such as E. coli. With this work we aimed to remodel the linear two-coordinate metal site with Cys112 and Cys120 as ligands in CueR to a tricoordinate site similar to that observed in the Hg(II) sensor protein MerR. This was done by introducing an additional cysteine near the metal site in the modified S77C-ΔC7-CueR variant, inspired by the fact that Ser77 in CueR is replaced by a cysteine in MerR. 199mHg PAC spectroscopic data indicate that two NQIs are present at pH 8.0, most likely reflecting HgS2 and HgS3 coordination modes, and demonstrating that the design of a pure HgS3 metal site was not achieved. Lowering the pH to 6.0 or the temperature to −196 °C had surprisingly similar effects, giving rise to highly distorted trigonal Hg(II) coordination. Tentatively, this might reflect that the histidine just next to Cys77 (His76) coordinates forming a HgS2N metal site structure. Further redesign beyond the first coordination sphere appears to be required to efficiently stabilize the HgS3 metal site structure at physiological pH.