Equilibrium unfolding of dimeric desulfoferrodoxin involves a monomeric intermediate: iron cofactors dissociate after polypeptide unfolding.
Here we report the conformational stability of homodimeric desulfoferrodoxin (dfx) from Desulfovibrio desulfuricans (ATCC 27774). The dimer is formed by two dfx monomers linked through beta-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 microM (DeltaG = 34 kJ/mol of dimer) from the concentration dependence of aromatic residue emission. Upon addition of the chemical denaturant guanidine hydrochloride (GuHCl) to dfx, a reversible fluorescence change occurred at 2-3 M GuHCl. This transition was dependent upon protein concentration, in accord with a dimer to monomer reaction [DeltaG(H(2)O) = 46 kJ/mol of dimer]. The secondary structure did not disappear, according 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 [DeltaG(H(2)O) = 50 kJ/mol of monomer]. Thus, dfx equilibrium unfolding is at least three-state, involving a monomeric intermediate with native-like secondary structure. Only after complete polypeptide unfolding (and incubation times of > 1 h) did the iron centers dissociate, as monitored by disappearance of ligand-to-metal charge transfer absorption, fluorescence of an iron indicator, and reactivity of cysteines to Ellman's reagent. Iron dissociation took place over several hours and resulted in an irreversibly denatured dfx. It appears as if the presence of the iron centers, the amino acid composition, and, to a lesser extent, the dimeric structure are factors that aid in facilitating dfx's unusually high thermodynamic stability for a mesophilic protein.
Apiyo
D
D
Chemistry Department and Molecular and Cellular Biology Graduate Program, Tulane University, New Orleans, Louisiana 70118, USA.
Jones
K
K
Guidry
J
J
Wittung-Stafshede
P
P
D002942:Circular Dichroism; D003901:Desulfovibrio / Q000737:chemistry; D019281:Dimerization; D005288:Ferredoxins / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D007501:Iron / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D005288:Ferredoxins; D010455:Peptides; C066602:desulfoferrodoxin; D007501:Iron; D019791:Guanidine