Stability and folding of the ferredoxin from the hyperthermophilic archaeon Acidianus ambivalens

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a sma ll monomeric protein containing two iron-sulfur centres, one [3Fe-4S] (1 /0) and one [4Fe-4S] (2 + /1 +). It is an intrinsically hyperstable protein , being expressed at the organism's extreme optimal growth temperature: 80 degrees C. Using spectroscopic methods we have investigated the unfolding r eaction of the Acidianus ambivalens ferredoxin. No unfolding of the oxidise d ferredoxin was observed at pH 7.0, even in the presence of 8 M GuHCl. Upo n increasing the pH to 10.0, the unfolding transition showed a midpoint at 6.3 M GuHCl and an unfolding-free energy of 70 kJ mol(-1) in buffer (pH 10) was estimated. Kinetic-unfolding experiments showed that the polypeptide u nfolding correlated with rearrangement of the iron-sulfur centres to new on es which had strong absorption maxima at 520 and 610 nm. These new, possibl y linear three-iron, clusters were coordinated to the unfolded protein but degraded slowly. From thermal experiments in the presence of GuHCl we estim ated the melting temperature for the Acidianus ambivalens ferredoxin in buf fer (at pH 7) to be 122 degrees C. Possible structural properties that cont ribute to the large thermal stability of the Acidianus ambivalens ferredoxi n are discussed using a three-dimensional protein model. (C) 2000 Elsevier Science Inc. All rights reserved.