Contribution of the [FeII(SCys)4] site to the thermostability of rubredoxins

The thermostabilities of Fe(2+) ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH.S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)(4)] site, and substitutions at this location affect the redox properties of the [Fe(SCys)(4)] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V "exchange" mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)(4)] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn(2+), Cd(2+)) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and (1)H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [Fe(II)(SCys)(4)] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [Fe(II)(SCys)(4)] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)(4)] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H(+), Cd(2+)) on the Cys42 sulfur ligand at the [Fe(SCys)(4)] site occurs through the V8 side and not the V44 side of the iron sit

Thermal stability of the [Fe(SCys)4] site in Clostridium pastearianum rubredoxin : contributions of the local environment and Cys ligand protonation

Thermal denaturation of the mesophilic rubredoxin from Clostridium pasteurianum occurs through a number of temperature-dependent steps, the last and irreversible one being release of iron from the [Fe2+ (SCys)4] site. We show here that thermally induced [Fe2+ (SCys)4] site destruction is largely determined by the local environment, and not directly connected to thermostability of the native polypeptide fold of rubredoxin. Hydrophobic residues on the protein surface, V8 and L41, that shield the [Fe(SCys)4] site from solvent and form N-H efS hydrogen bonds to the metal-coordinating sulfurs, were mutated to residues with both uncharged and charged side chains. On these mutated rubredoxins the temperature dependence was measured for: (1) global unfolding of the protein by NMR, (2) loss of Fe2+ at various ionic strengths and pH values, (3) the rates of non-denaturing displacement of Fe2+ by Cd2+ or Zn2+. For reversible temperature-dependent changes in the global protein folding that occur prior to loss of iron, no thermostability differences were found among the wild-type, V8A, V8D, L41R, and L41D rubredoxins. However, for irreversible loss of iron from the [Fe2+ (SCys)4] site, relative to the wild-type protein, L41R was more thermostable, V8A was somewhat less thermostable, and the acidic mutants L41D, V8D and [V8D, L41D] showed dramatically lowered thermostability. Lower pH facilitated - both kinetically and thermodynamically - thermally induced iron release, likely through protonation of ligand cysteines' thiols. For all of the rubredoxins a direct correlation was found between the midpoint temperature for thermally induced Fe2+ loss and the rate of non-denaturing Fe2+ displacement by Cd2+ or Zn2+ at room temperature. A mechanism is proposed involving transient movement of residue-8 and -41 side chains, allowing, and, in the case of negatively charged side chains, also facilitating, attack of a ligand cysteine by the incoming positively charged species (H+, Cd2+, or Zn2+). Thus, localized charge density and solvent accessibility modulate the stability of Fe2+ ligation in rubredoxin. However, the reduced [Fe(SCys)4] site does not control the thermostability of the native polypeptide fold of rubredoxin