The copper chaperone for the superoxide dismutase (CCS) gene is necessary for expression of an active, copper-bound form of superoxide dismutase (SOD1) in vivo in spite of the high affinity of SOD1 for copper (dissociation constant = 6 fM) and the high intracellular concentrations of both SOD1 (10 microM in yeast) and copper (70 microM in yeast). In vitro studies demonstrated that purified Cu(I)-yCCS protein is sufficient for direct copper activation of apo-ySOD1 but is necessary only when the concentration of free copper ions ([Cu]free) is strictly limited. Moreover, the physiological requirement for yCCS in vivo was readily bypassed by elevated copper concentrations and abrogation of intracellular copper-scavenging systems such as the metallothioneins. This metallochaperone protein activates the target enzyme through direct insertion of the copper cofactor and apparently functions to protect the metal ion from binding to intracellular copper scavengers. These results indicate that intracellular [Cu]free is limited to less than one free copper ion per cell and suggest that a pool of free copper ions is not used in physiological activation of metalloenzymes.

The copper chaperone for superoxide dismutase (SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown mechanism. We demonstrate here that this process involves the cooperation of three distinct regions of the copper chaperone for SOD1 (CCS): an amino-terminal Domain I homologous to the Atx1p metallochaperone, a central portion (Domain II) homologous to SOD1, and a short carboxyl-terminal peptide unique to CCS molecules (Domain III). These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies. The biological roles of the yeast CCS domains were examined in yeast cells. Surprisingly, Domain I was found to be necessary only under conditions of strict copper limitation. Domain I and Atx1p were not interchangeable in vivo, underscoring the specificity of the corresponding metallochaperones.

Saccharomyces cerevisiae Atx1p represents a member of the family of metallochaperone molecules that escort copper to distinct intracellular targets. Atx1p specifically delivers copper to the Ccc2p copper transporter in the Golgi. Additionally, when overproduced, Atx1p substitutes for superoxide dismutase 1 in preventing oxidative damage; however the mechanistic overlap between these functions is unresolved. The crystal structure of Atx1p has been solved recently. By examining a surface electrostatic potential distribution, multiple conserved lysines are revealed on one face of Atx1p. An additional conserved lysine (Lys 65 ) lies in close proximity to the metal binding site. Through site-directed mutagenesis, residues in the metal binding region including Lys 65 were found to be necessary for both copper delivery to Ccc2p and for Atx1p antioxidant activity. Copper trafficking to Ccc2p also relied on the lysine-rich face of Atx1p. Surprisingly however, elimination of these lysines did not inhibit the antioxidant activity of Atx1p. We provide evidence that Atx1p does not suppress oxidative damage by a metallochaperone mechanism but may directly consume superoxide. Purified Cu-Atx1p reacts noncatalytically with superoxide anion in vitro. We conclude that the copper-trafficking and antioxidant functions of Atx1p arise from chemically and structurally distinct attributes of this metallochaperone.

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

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme l, is isolated through an approach that combines proteolytic hydrolysis and reverse-phase high performance liquid chromatographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotrypsin) or a peptide-free heme (with proteinase K). Both heme l and heme l-peptide species were investigated by paramagnetic