Undetectable intracellular free copper: The requirement of a copper chaperone for superoxide dismutase

The topper chaperone for the superoxide dismutase (CCS) gene is necessary f or expression of an active, copper-bound form of superoxide dismutase (SOD1 ) in vivo in spite of the high affinity of SOD1 for copper (dissociation co nstant = 6 fM) and the high intracellular concentrations of both SOD1 (10 m u M in yeast) and copper (70 mu M in yeast), In vitro studies demonstrated that purified Cu(I)-yCCS protein is sufficient for direct copper activation of apo-ySOD1 but is necessary only when the concentration of free copper i ons ([Cu](free)) is strictly limited. Moreover, the physiological requireme nt for yCCS in vivo was readily bypassed by elevated copper concentrations and abrogation of intracellular copper-scavenging systems such as the metal lothioneins, This metallochaperone protein activates the target enzyme thro ugh direct insertion of the copper cofactor and apparently functions to pro tect the metal ion from binding to intracellular copper scavengers. These r esults indicate that intracellular [Cu](free) is Limited to less than one f ree copper ion per cell and suggest that a pool of free copper ions is not used in physiological activation of metalloenzymes.