A Solvent-Exposed Cysteine Forms a Peculiar NiII -Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum

Abstract

In Rhodospirillum rubrum, the maturation of carbon monoxide dehydrogenase (CODH) requires three nickel chaperones, namely RrCooC, RrCooT and RrCooJ. Recently, the biophysical characterisation of the RrCooT homodimer and the X-ray structure of its apo form revealed the existence of a solvent-exposed Ni^II -binding site at the dimer interface, involving the strictly conserved Cys2. Here, a multifaceted approach that used NMR and X-ray absorption spectroscopies, complemented with structural bio-modelling methodologies, was used to characterise the binding mode of Ni^II in RrCooT. This study suggests that Ni^II adopts a square-planar geometry through a N₂ S₂ coordinating environment that comprises the two thiolate and amidate groups of both Cys2 residues at the dimer interface. The existence of a diamagnetic mononuclear Ni^II centre with bis-amidate/bis-thiolate ligands, coordinated by a single-cysteine motif, is unprecedented in biology and raises the question of its role in the activation of CODH at the molecular level.

Keywords: CooT; carbon monoxide dehydrogenases; chaperone protein; enzymes; nickel; nickel-binding cysteine.