title: Spectroscopic characterization of the metal-binding sites in the periplasmic metal-sensor domain of CnrX from Cupriavidus metallidurans CH34.
abstract: CnrX, the dimeric metal sensor of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34, contains one metal-binding site per monomer. Both Ni and Co elicit a biological response and bind the protein in a 3N2O1S coordination sphere with a nearly identical octahedral geometry as shown by the X-ray structure of CnrXs, the soluble domain of CnrX. However, in solution CnrXs is titrated by 4 Co-equiv and exhibits an unexpected intense band at 384 nm that was detected neither by single-crystal spectroscopy nor under anaerobiosis. The data from a combination of spectroscopic techniques (spectrophotometry, electron paramagnetic resonance, X-ray absorption spectroscopy) showed that two sites correspond to those identified by crystallography. The two extra binding sites accommodate Co(II) in an octahedral geometry in the absence of oxygen and are occupied in air by a mixture of low-spin Co(II) as well as EPR-silent Co(III). These extra sites, located at the N-terminus of the protein, are believed to participate to the formation of peroxo-bridged dimers. Accordingly, we hypothesize that the intense band at 384 nm relies on the formation of a binuclear μ-peroxo Co(III) complex. These metal binding sites are not physiologically relevant since they are not detected in full-length NccX, the closest homologue of CnrX. X-ray absorption spectroscopy demonstrates that NccX stabilizes Co(II) in two-binding sites similar to those characterized by crystallography in its soluble counterpart. Nevertheless, the original spectroscopic properties of the extra Co-binding sites are of interest because they are susceptible to be detected in other Co-bound proteins.
authors.affiliation: Institut de Biologie Structurale-Jean-Pierre Ebel, UMR 5075, CNRS-CEA-UJF-Grenoble-1, 41, rue Jules Horowitz, 38027 Grenoble Cedex, France.
authors.forename: Juliette
authors.initials: J
authors.lastname: Trepreau
authors.forename: Eve
authors.initials: E
authors.lastname: de Rosny
authors.forename: Carole
authors.initials: C
authors.lastname: Duboc
authors.forename: Géraldine
authors.initials: G
authors.lastname: Sarret
authors.forename: Isabelle
authors.initials: I
authors.lastname: Petit-Hartlein
authors.forename: Antoine P
authors.initials: AP
authors.lastname: Maillard
authors.forename: Anne
authors.initials: A
authors.lastname: Imberty
authors.forename: Olivier
authors.initials: O
authors.lastname: Proux
authors.forename: Jacques
authors.initials: J
authors.lastname: Covès
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D049921:Cupriavidus / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D013052:Spectrometry, X-Ray Emission; D013056:Spectrophotometry, Ultraviolet; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D003035:Cobalt; D003300:Copper