Mechanism of Cu,Zn-Superoxide Dismutase Activation by the Human Metallochaperone hCCS

Abstract

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein.Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.Eukaryotic and prokaryotic cells accumulate essential first row transition metals for various cellular functions. Copper for instance, is maintained at a total intracellular concentration in the 10 Ϫ4-10 Ϫ5 M range. Despite this abundance, it has recently been shown that copper ions are typically unavailable in the cytoplasm for direct substitution into metalloenzymes. In fact, the steady state concentration of the free or labile form of copper ion in the cytoplasm of yeast is far less than one ion per cell (1). This finding suggests that the intracellular milieu has a significant overcapacity for metal chelation, and raises the issue of how apo-proteins acquire the correct metal cofactor.A class of metal ion trafficking proteins is emerging that act in distribution of essential transition metal cofactors to specific targets in the cytoplasm, to the cell surface or integral membranes, or to various intracellular compartments. Several newly discovered metal-receptor proteins called metallochaperones are now known to serve as key agents in cellular trafficking of these cofactors (2-4). These cytosolic proteins are characterized by a metal-exchange partnership with one or more specific intracellular target proteins and by a metalspecific binding activity.A prototypical member of the metallochaperone family, Atx1

AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7

“…Arabidopsis plants, the wild-type ecotype Colombia-0 and the mutant lines paa1-3 [12] and paa2-1 [13] were grown under a 16 h light/ 8 h dark cycle on soil at 23°C. For growth on agar media, seeds were surface-sterilized and sown on solid 1/2 strength Murashige and Skoog (1/2 MS) medium including 1% sucrose and 0.4% agargel [21].…”

“…In yeast, Ccs1p/Lys7p delivers copper to the Cu/ZnSOD (Sod1p) by a direct protein-protein interaction [11,12], a function which is required to maintain the activity of reactive oxygen species sensitive enzymes involved in lysine and methionine biosynthesis [13]. Since chloroplasts are a site of oxygen production it is of interest to note that O 2 and the copper chaperone for Cu/Zn superoxide dismutase (CCS) regulate posttranslational activation of Cu/ZnSOD enzymes [14] by mediating correct disulfide formation [15].…”

The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status

“…Whereas the mechanism by which SOD1 acquires Zn(II) is not fully understood, several aspects of the copper insertion by the copper chaperone for SOD1 (CCS) are well established (7)(8)(9)(10)(11)(12). More recently, Furukawa et al (13) have shown that the intrasubunit disulfide bond is correctly introduced in yeast SOD1 by the copper-bound form of yeast CCS.…”

Les bases moléculaires de l’approvisionnement en cuivre

“…L'holo-molécule adopterait alors une conformation protégeant l'ion de cuivre jusqu'à son transfert à la SOD1. La passation du Cu de Ccs1 au site actif de la SOD1 relèverait du domaine III ; son domaine II quant à lui servirait de point d'arrimage avec la SOD1 [19]. Une particularité de la Ccs1 humaine est qu'elle peut être convertie par une simple mutation ponctuelle en une protéine ayant une activité superoxyde dismutase comparable à celle de la SOD1.…”