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The carbon monoxide dehydrogenase accessory protein CooJ is a histidine-rich multidomain dimer containing an unexpected Ni(II)-binding site.
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Edité par CCSD ; American Society for Biochemistry and Molecular Biology -
International audience. Activation of nickel enzymes requires specific accessory proteins organized in multiprotein complexes controlling metal transfer to the active site. Histidine-rich clusters are generally present in at least one of the metallo-chaperones involved in Ni delivery. The maturation of carbon monoxide dehydrogenase (CODH) in the proteobacterium Rhodospirillum rubrum requires three accessory proteins CooC, CooT and CooJ dedicated to Ni insertion into the active site, a distorted [NiFe3S4] cluster coordinated to an Fe site. Previously, CooJ from R. rubrum (RrCooJ) has been described as a Ni chaperone with 16 histidines and 2 cysteines at its C-terminus. Here, the X-ray structure of a truncated version of RrCooJ, combined with small-angle X-ray scattering (SAXS) data and a modelling study of the full-length protein, revealed a homodimer comprising a coiled-coil with two independent and highly flexible His tails. Using isothermal calorimetry, we characterized several metal-binding sites (four per dimer), involving the His-rich motifs and having similar metal affinity (KD = 1.6 µM). Remarkably, biophysical approaches, site-directed mutagenesis and X-ray crystallography uncovered an additional Ni-binding site at the dimer interface, that binds Ni(II) with an affinity of 380 nM. Although RrCooJ was initially thought to be a unique protein, a proteome database search identified at least 46 bacterial CooJ homologs. These homologs all possess two spatially separated nickel-binding motifs: a variable C-terminal histidine tail and a strictly conserved "H-(W/F)-X2-H-X3-H" motif, identified in this study, suggesting a dual function for CooJ both as a Ni chaperone and as a Ni storage protein.
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