Mapping of a copper-binding site an the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis

CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based oil Computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12-Cu2+ complexes then allowed fragment ions specifically binding the copper ion to be deter-mined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp(16)-Asp(24), Asp(38)-Lys(50) and Asp(70)-Glu(76), with the two first regions containing selected residues for mutation. These data confirmed that copper ligands, involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper-might play a role in the regulation of the biological activity of CP12.