HAH1 IS A COPPER-BINDING PROTEIN WITH DISTINCT AMINO-ACID-RESIDUES MEDIATING COPPER HOMEOSTASIS AND ANTIOXIDANT DEFENSE

HAH1 is a 68-amino acid protein originally identified as a human homol ogue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 Del ta yeast, Molecular modeling of HAH1 predicts a protein structure of t wo alpha-helices overlaying a four-stranded antiparallel beta-sheet wi th a potential metal binding site involving two conserved cysteine res idues. Consistent with this model, in vitro studies with recombinant H AH1 directly demonstrated binding of Cu(I), and site-directed mutagene sis identified these cysteine residues as copper ligands. Expression o f wild type and mutant HAH1 in atx1 Delta yeast revealed the essential role of these cysteine residues in copper trafficking to the secretor y compartment in vivo, as expression of a Cys-12/Cys-15 double mutant abrogated copper incorporation into the multicopper oxidase Fet3p. In contrast, mutation of the highly conserved lysine residues in the carb oxyl terminus of HAH1 had no effect on copper trafficking to the secre tory pathway but eliminated the antioxidant function of HAH1 in sod1 D elta yeast, Taken together, these data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biolo gical processes in eucaryotic cells.