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

Abstract

To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)₄ site (M = Fe^3+/2+, Zn²⁺, or Cd²⁺) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 °C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe²⁺ [Lt ] Fe³⁺ < Zn²⁺ < Cd²⁺. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 °C for Fe²⁺Rd and 83 °C for Fe³⁺Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and ¹H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50–70 °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 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe³⁺Rd, stoichiometric Fe³⁺/cysteine–ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)₄ site.