Immature copper-zinc superoxide dismutase (Sod1) is activated by its copper chaperone (Ccs1). Ccs1 delivers a single copper ion and catalyzes oxidation of an intra-subunit disulfide bond within each Sod1 monomer through a mechanistically ambiguous process. Here, we use residue specific fluorescent labeling of immature Sod1 to quantitate the thermodynamics of the Sod1•Ccs1 interaction while determining a more complete view of Ccs1 function. Ccs1 preferentially binds a completely immature form of Sod1 that is metal deficient and disulfide reduced (E, E-Sod1SH). However, binding induces structural changes that promote high-affinity zinc binding by the Ccs1-bound Sod1 molecule. This adds further support to the notion that Ccs1 likely plays dual chaperoning roles during the Sod1 maturation process. Further analysis reveals that in addition to the copper-dependent roles during Sod1 activation, the N- and C-terminal domains of Ccs1 also have synergistic roles in securing both Sod1 recognition and its own active conformation. These results provide new and measurable analyses of the molecular determinants guiding Ccs1-mediated Sod1 activation.
Keywords: Copper; Metallo-chaperones; Metallo-enzymes; Sod1.