Energetics of Copper Trafficking between the Atx 1 Metallochaperone and the Intracellular

The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevi-siae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K exchange ‫؍‬ 1.4 ؎ 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor , glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electro-static forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others. Copper is an essential cofactor in hydrolytic, electron transfer , and oxygen utilization enzymes and is also crucial for high affinity iron uptake in yeast (1, 2). Copper uptake in eukaryotes is mediated by the CTR plasma membrane proteins (3– 6). Once inside the cell, a portion of the copper is delivered to P-type ATPases, which pump this metal ion into vesicles for ultimate incorporation into multicopper oxidases. In yeast the P-type ATPase is Ccc2 (7–9) and the multicopper oxidase is Fet3 (10). Until recently, copper was thought to be delivered to target proteins as glutathione complexes; however, the Atx1 metallochaperone protein, a cytosolic Cu(I) receptor, is required downstream of CTR1 and upstream of Ccc2 and related ATPases (11, 12). Copper chaperone proteins are soluble, intracellular receptors that bind and deliver copper to specific partner proteins (12). Initially identified as antioxidant and biosynthetic pathway proteins, the yeast metallochaperone Atx1 and the copper chaperone for superoxide dismutase (CCS) 1 have since been shown to be Cu(I) receptors that interact with specific vesicular and cytoplasmic targets, respectively (12–15). Copper-trafficking pathway proteins including homologues 23) are highly conserved from yeast to humans. …