Heterodimer formation between superoxide dismutase and its copper chaperone

Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS). The molecular mechanisms by whic h CCS recognizes and docks with SOD1 for metal ion insertion are not well u nderstood. Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one mon omer of SOD1 and a dimer of dimers involving interactions between the two h omodimers. We have investigated protein-protein complex formation between c opper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD 1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replace d with phenylalanine (H48F-SOD1). According to gel filtration chromatograph y, dynamic light scattering, analytical ultracentrifugation, and chemical c ross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer. No higher order oligomers were d etected. Heterodimer formation is facilitated by the presence of zinc but d oes not depend on copper loading of yCCS. The complex formed with H38F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species. Notably, heterodimer formation between copper-lo aded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc. These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.