Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis

The presence of the copper ion at the active site of human wild type copper -zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyz e the disproportionation of superoxide into dioxygen and hydrogen peroxide, Wild type CuZnSOD and several of the mutants associated with familial amyo trophic lateral sclerosis (FALS) (Ala(4) --> Val, Gly(93) --> Ala, and Leu( 38) --> Val) were expressed in Saccharomyces cerevisiae, Purified metal-fre e (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification st udies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in thei r proper locations in vitro, Similar studies of the wild type and FALS muta nt CuZnSOD holoenzymes in the "as isolated" metallation state showed abnorm ally low copper-to-zinc ratios, although all of the copper acquired was loc ated at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS), The loss of met al ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS.