High stability of a ferredoxin from the hyperthermophilic archaeon A-ambivalens: Involvement of electrostatic interactions and cofactors

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a sma ll monomeric seven-iron protein with a thermal midpoint (T-m) of 122 degree sC (pH 7). To gain insight into the basis of its thermostability, we have c haracterized unfolding reactions induced chemically and thermally at variou s pHs. Thermal unfolding of this ferredoxin, in the presence of Various gua nidine hydrochloride (GuHCl) concentrations, yields a linear correlation be tween unfolding enthalpies (DeltaH[T-m]) and T-m from which an upper limit for the heat capacity of unfolding (DeltaC(p)) was determined to be 3.15 +/ - 0.1 kJ/(mole (.) K), Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20 d egreesC) observed ([GuSCN](1/2) = 3.1 M; DeltaG(U)[H2O] = 79 +/- kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, T-m, is 64 +/- 1 degreesC, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U) [H2O] = 20 +/- 1 kJ/mole). These results support that electrostatic interac tions contribute significantly to the stability. Analysis of the three-dime nsional molecular model of the protein shows that there are several possibl e ion pairs on the surface. In addition, ferredoxin incorporates two iron-s ulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron-sulfur clust ers transiently form linear three-iron species (in pH range 2.5 to 10), whi ch are associated with the unfolded polypeptide, before their complete degr adation.