Equilibrium unfolding of dimeric desulfoferrodoxin involves a monomeric intermediate: Iron cofactors dissociate after polypeptide unfolding

Here we report the conformational stability of homodimeric desulfoferrodoxi n (dfx) from Desulfovibrio desulfuricans (ATCC 27774). The dimer is formed by two dfx monomers Linked through P-strand interactions in two domains; in addition, each monomer contains two different iron centers: one Fe-(S-Cys) (4) center and one Fe-[S-Cys+(N-His)(4)] center. The dissociation constant for dfx was determined to be 1 muM (DeltaG = 34 kJ/mol of dimer) from the c oncentration dependence of aromatic residue emission. Upon addition of the chemical denaturant guanidine hydrochloride (GuHCl) to dfx, a reversible fl uorescence change occurred at 2-3 M GuHCl. This transition was dependent up on protein concentration, in accord with a dimer to monomer reaction [Delta G(H2O) = 46 kJ/mol of dimer]. The secondary structure did not disappear, ac cording to far-UV circular dichroism (CD), until 6 M GuHCl was added; this transition was reversible (for incubation times of <1 h) and independent of dfx concentration [<Delta>G(H2O) = 50 kJ/mol of monomer]. Thus, dfx equili brium unfolding is at least three-state, involving a monomeric intermediate with native-like secondary structure. Only after complete polypeptide unfo lding (and incubation times of >1 h) did the iron centers dissociate, as mo nitored by disappearance of ligand-to-metal charge transfer absorption, flu orescence of an iron indicator, and reactivity of cysteines to Ellman's rea gent. Iron dissociation took place over several hours and resulted in an ir reversibly denatured dfx. It appears as if the presence of the iron centers , the amino acid composition, and, to a lesser extent, the dimeric structur e are factors that aid in facilitating dfx's unusually high thermodynamic s tability for a mesophilic protein.