Thermal stability of Clostridium pasteurianum rubredoxin: Deconvoluting the contributions of the metal site and the protein

To provide a framework for understanding the hyperthermostability of some r ubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was underta ken. Rds with three different metals in its M(SCys)(4) site (M = Fe3+/2+, Z n2+. or Cd2+) were examined. Kinetics of metal ion release were monitored a naerobically at several fixed temperatures between 40 and 100 degreesC, and during progressive heating of the iron-containing protein. Both methods ga ve a thermal stability of metal binding in the order Fe2+ << Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the prote in in temperature ramp experiments was 69 degreesC for Fe2+-Rd and 83 degre esC for Fe3+ Rd. Temperature-dependent changes in the protein structure wer e monitored by differential scanning calorimetry, tryptophan fluorescence. binding of a fluorescent hydrophobic probe, and H-1 NMR. Major but reversib le structural changes, consisting of swilling of the hydrophobic core and o pening of a loop region. were found to occur at temperatures (50-70 degrees C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 si te, whereas the final, irreversible loss of metal ion is dependent on the i nherent M-SCys bond strength. In the case of Fe(3+)Rd, stoichiometric Fe3+/ cysteine-ligand redox chemistry also occurs during metal ion loss, The resu lts indicate that thermally induced unfolding of the native Cp Rd must surm ount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)(4) site.