PRO108 IS IMPORTANT FOR FOLDING AND STABILIZATION OF ADRENAL FERREDOXIN, BUT DOES NOT INFLUENCE THE FUNCTIONAL PROPERTIES OF THE PROTEIN

The truncated mutant Met-adrenodoxin-(4-107)-peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and c ould be reconstituted to the holoform by chemical or enzymatic methods . The reconstituted protein had spectroscopic, functional and redox pr operties similar to the Met-adrenodoxin-(4-108)-pedtide of adrenal fer redoxin, into which the cluster was inserted upon expression in the sa me Escherichia coli strain. Rate of in vitro cluster insertion into th e Met-adrenodoxin-(4-107) apoprotein was much lower than for the Met-a drenodoxin-(4-108) apoprotein under identical conditions. Comparative thermodynamic studies with the Met-adrenodoxin-(4-108)-peptide indicat ed that removal of Pro108 resulted in an extensive decrease of the ove rall stability of the protein in either oxidation state. The Met-adren odoxin-(4-107)-peprtide showed a higher sensitivity to urea denaturati on and had a sensibly lower denaturation temperature, 44.8 degrees-C, compared with 51.7 degrees-C for mutant Met-adrenodoxin-(4-108). The s tability of the reduced state of both mutants is slightly lower than t hat of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction.