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. Study holds ProTherm entries: 11161, 11162, 11163, 11164, 11165, 11166, 11167 Extra Details: F->I (F=folded, I=intermediate) homodimer; iron centers; dissociation constant;,intermediate; secondary structure

Submission Details

ID: 3AziCza5

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Apiyo D;Jones K;Guidry J;Wittung-Stafshede P,Biochemistry (2001) Equilibrium unfolding of dimeric desulfoferrodoxin involves a monomeric intermediate: iron cofactors dissociate after polypeptide unfolding. PMID:11305909
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Desulfoferrodoxin P22076 DFX_DESDA