A free cysteine residue at the dimer interface decreases conformational stability of Xenopus laevis copper,zinc superoxide dismutase

The two Cu,Zn superoxide dismutases from the amphibian Xenopus laevis (deno ted XSODA and XSODB) display different heat sensitivities, XSODA being more thermolabile than XSODB. In this study, we have investigated the contribut ion of a free cysteine residue located close to the subunit interface of XS ODA to its lower thermal stability. We have found that mutation of residue Cys 150 to Ala in XSODA makes the thermal stability of this enzyme comparab le to that of the wildtype XSODB isoenzyme, while the introduction of a cys teine residue in the same position of XSODB renders this enzyme variant muc h more heat-sensitive. Differential scanning calorimetry experiments showed that XSODA has a melting temperature about 8.5 degrees C lower than that o f XSODB. On the contrary, the melting temperature of XSODACys150Ala is very close to that of XSODB, while the melting temperature of XSODBSer150Cys is even lower than that of wild-type XSODA. These data indicate that the free cysteine residue present in XSODA affects not only the reversibility of un folding of the enzyme but also its conformational stability. We suggest tha t the large effect of the Cys 150 residue on XSODA stability might be due t o incorrect disulfide bond formation or disulfide bond interchange during h eat-induced unfolding rather than to alteration of the interaction between the enzyme subunits. (C) 2000 Academic Press.