Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. The human metallo-chaperone protein Atox1 (known also as Hah1) delivers Cu I to the trans-Golgi network (1, 2). Atx1, the version from the yeast Saccharomyces cerevisiae, was the first copper metallo-chaperone to be identified (3). They both feature the classic ferredoxin ␤␣␤␤␣␤-fold with a CXXC motif acting as a high affinity Cu I -binding site ( Fig. 1) (4, 5). Homologues are found in cyanobacteria (Atx1), in Enterococcus hirae (CopZ), in Bacillus subtilis (CopZ), and in many other organisms (6).The human P 1B -type ATPase ATP7A accepts copper from Atox1 and transports it into the lumen of the trans-Golgi network (2). ATP7B performs a related role in liver cells. The inherited disorders Menkes and Wilson diseases are associated with defects in ATP7A and ATP7B, respectively (7). Equivalent metal transporters exist in other organisms such as Ccc2 from S. cerevisiae (3) and heavy metal ATPases 5-8 (HMA5-8) in the simple plant Arabidopsis thaliana (8). Their N termini contain between one and six metal-binding domains (MBDs) 2 that may interact with and receive Cu I directly from Atox1-type metallo-chaperones (6). It appears that, for some Cu I -ATPases at least, metal-binding sites in the transmembrane domain may also independently receive Cu I from copper chaperones (9). The overall molecular structure and binding site of each MBD is similar to that of Atox1 (10).Accurate estimation of affinities for Cu I (as expressed by the dissociation constant K D ) is essential for a quantitative understanding of reactivity and mechanisms of action. Yet reported K D values are scattered widely as highlighted by those of Atox1-type proteins, which differ by more than 10 orders of magnitude (K D ϳ10 Ϫ5 , 10 Ϫ10 , 10 Ϫ14 , and 10 Ϫ18 M) even though the structures and metal-binding sites of these proteins essentially superimpose (11-16). The various values were determined via different experimental approaches with different ligand probes and affinity standards. The affinities of some of the probes and standards remain in dispute. In addition, the intrinsic instability of free Cu ϩ in aqueous solution and the tendency to aerial oxidation of cysteine ligands impose special conditions on these experiments. These aspects are complicated further by reports that thiol ligands such as endogenous glutathione (GSH) may expand the Cu I coordination sphere in these proteins or lead to polymeric forms (14,17,18).In an attempt to resolve these fundamental issues for this iconic set of proteins, this study surveys the literature values for the Cu I affinities of the four probe ligands bicinchoninate (Bca), bathocuproine disulfonate (Bcs), dithiothreitol (Dtt), and glutathione (GSH) (Scheme 1). By direct experimental comparison, their relative affinities are unifi...

Glutaredoxins have been characterised as enzymes regulating the redox status of protein thiols via cofactors GSSG/GSH. However, such a function has not been demonstrated with physiologically relevant protein substrates in in vitro experiments. Their active sites frequently feature a Cys-xx-Cys motif that is predicted not to bind metal ions. Such motifs are also present in copper-transporting proteins such as Atox1, a human cytosolic copper metallo-chaperone. In this work, we present the first demonstration that: (i) human glutaredoxin 1 (hGrx1) efficiently catalyses interchange of the dithiol and disulfide forms of the Cys(12)-xx-Cys(15) fragment in Atox1 but does not act upon the isolated single residue Cys(41); (ii) the direction of catalysis is regulated by the GSSG/2GSH ratio and the availability of Cu(I); (iii) the active site Cys(23)-xx-Cys(26) in hGrx1 can bind Cu(I) tightly with femtomolar affinity (K(D) = 10(-15.5) M) and possesses a reduction potential of E(o)' = -118 mV at pH 7.0. In contrast, the Cys(12)-xx-Cys(15) motif in Atox1 has a higher affinity for Cu(I) (K(D) = 10(-17.4) M) and a more negative potential (E(o)' = -188 mV). These differences may be attributed primarily to the very low pKa of Cys23 in hGrx1 and allow rationalisation of conclusion (ii) above: hGrx1 may catalyse the oxidation of Atox1(dithiol) by GSSG, but not the complementary reduction of the oxidised Atox1(disulfide) by GSH unless Cu(aq)(+) is present at a concentration that allows binding of Cu(I) to reduced Atox1 but not to hGrx1. In fact, in the latter case, the catalytic preferences are reversed. Both Cys residues in the active site of hGrx1 are essential for the high affinity Cu(I) binding but the single Cys(23) residue only is required for the redox catalytic function. The molecular properties of both Atox1 and hGrx1 are consistent with a correlation between copper homeostasis and redox sulfur chemistry, as suggested by recent cell experiments. These proteins appear to have evolved the features necessary to fill multiple roles in redox regulation, Cu(I) buffering and Cu(I) transport.