title: No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricans flavodoxin.
abstract: Flavodoxins are proteins with an alpha/beta doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K(D)=0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (DeltaG=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high (K(D)=0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.
authors.affiliation: Chemistry Department, Tulane University, 6823 St. Charles Ave., New Orleans, LA 70118-5698, USA.
authors.forename: D
authors.initials: D
authors.lastname: Apiyo
authors.forename: J
authors.initials: J
authors.lastname: Guidry
authors.forename: P
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D002942:Circular Dichroism; D003901:Desulfovibrio / Q000737:chemistry*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005418:Flavodoxin; D005486:Flavin Mononucleotide