We have employed differential scanning calorimetry (DSC) to investigate the thermally induced unfolding of native Cu,Zn-superoxide dismutase (SOD), the apoprotein derived from native SOD, and the zinc-substituted derivatives of the apoprotein. We observe two overlapping melting transitions for native bovine SOD with heat capacity maxima at temperatures (Tm) of 89 and 96 degrees C when a scanning rate of 0.82 deg/min is employed. By contrast, the dithionite-reduced native SOD (which contains Cu+ rather than Cu2+) exhibits only a single transition at 96 degrees C. Significantly, we find that the concentration of O2 present in native SOD samples influences the relative magnitudes of the 89 and 96 degrees C peaks. Specifically, the lower temperature transition becomes less pronounced as the concentration of O2 in the sample decreases. On the basis of these observations, we propose that the lower temperature peak corresponds to the melting of the oxidized native protein, while the higher temperature peak reflects the melting of the reduced native protein, which forms spontaneously during the heating process. Our interpretation profoundly differs from that of Lepock et al. [Lepock, J.R., Arnold, L.D., Torrie, B.H., Andrews, B., & Kruuv, J. (1985) Arch. Biochem. Biophys. 241, 243-251], who have proposed that the low-temperature transition corresponds to the reduced form of the protein. We present evidence that suggests that their experiments were complicated by the presence of potassium ferrocyanide, which, in addition to reducing the cupric center, also perturbs the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
A spectroscopic investigation of the behavior of bovine erythrocuprein and its four-copper-substituted derivative below pH 4.5 suggests strongly that the metal ion bound to the native zinc site is reversibly released in the pH range 3.0 I pH I 4.5. Published spectra of the copper-cobalt derivative suggest that cobalt is similarly lost from that derivative in the same pH range. Dialysis of the native copper-zinc protein at pH 3.6 resulted in removal of 95% of the Zn but only 5% of the Cu, although the visible, UV, and ESR spectra were only slightly changed from the initial spectra at pH 3.6. The release of free cupric ion from the four-copper derivative at low pH was also determined potentiometrically with use of a copper-selective electrode. The pH dependence of the spectral changes observed for the copper-zinc, four-copper, and copper-cobalt derivatives was virtually identical. The zinc-free derivative of erythrocuprein was also observed to undergo small but distinct changes in its ESR and visible spectra when the pH was changed. These changes occurred with a midpoint pH of 5.0. Below pH 3, copper is also lost from the protein.Bovine erythrocuprein is a relatively small (mol wt 31 200) globular protein consisting of two equivalent subunits, each of which binds one copper2 and one zinc3 in close proximity. X-ray crystallographic studies by the RichadsonsM have shown that the major structural feature of each subunit is an eight-stranded barrel of 6 structure; two loops of irregular structure are also seen which are connected to the ends of the strands forming the barrel. The Cu" and Zn" ions in each subunit are separated by approximately 6 A. The CUI' is five coordinate, with the four histidyl imidazole rings that are bound to it (histidines 44, 46, 61, and 118) forming a distorted square plane, the fifth ligand being water. Three of these histidines (44, 46, and 118) have their a carbons in the @barrel portion of the structure. The fourth, histidine 61, provides an imidazole ring that is deprotonated and bridges the Cu" and Zn" ions. That residue is found in the same single loop of irregular structure that contains those residues that provide the other ligands to Zn", Le., histidines 69 and 78 and aspartate 81. The four ligands to Zn" are arranged in a distorted tetrahedral arrangement.
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