Conformational stability of Cu,Zn-superoxide dismutase, the apoprotein, and its zinc-substituted derivatives: second-derivative spectroscopy of phenylalanine and tyrosine residues.
The relative stabilities of bovine copper-zinc superoxide dismutase (SOD), its apoprotein form, and zinc-substituted derivatives were investigated by denaturation in guanidine-HCl solutions. Analysis of the kinetics of changes in the second-derivative spectral bands of both phenylalanine and tyrosine residues was simultaneously performed. It was found that reduction of the cupric site increases the stability of the enzyme. The apoprotein appears to be the least stable form, while addition of zinc ions not only increases stability, but appears to induce a native-like conformation from a disordered form at pH 3.8. By perturbing the solvent with up to 20% ethylene glycol, at pH 6.8, it was determined that the only tyrosyl side chain appears to be about 50% solvent-exposed in the apoprotein, 65% exposed in the zinc derivative, and 75% exposed in the native copper-zinc form. In contrast, all four phenylalanine residues appear to be fully buried in all of these species in the mid-pH range. At pH 2.5, as the apoprotein unfolds, the apparent solvent-exposure of the tyrosyl side chain approaches 100%, while the phenylalanyl side chains become only 70% exposed. Substantial differences in the unfolding rate constants of tyrosine and phenylalanine residues of native and zinc-substituted SOD, but not the apoprotein, suggest the presence of metal-stabilized unfolding intermediates. Unfolding as monitored by the exposure of phenylalanine residues follows first-order kinetics, indicating that Phe 48 located at the interface between the two subunits is being exposed to the solvent simultaneously with the remaining three phenylalanine residues buried in the protein core.
Mach
H
H
Department of Molecular Biology, University of Wyoming, Laramie 82071.
Dong
Z
Z
Middaugh
C R
CR
Lewis
R V
RV
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D007700:Kinetics; D010649:Phenylalanine / Q000737:chemistry*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
D001059:Apoproteins; D014443:Tyrosine; D010649:Phenylalanine; D013482:Superoxide Dismutase; D015032:Zinc