No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricansflavodoxin

Flavodoxins are proteins with an alpha/beta doubly wound topology that medi ate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K-D = 0.1 nM, ox idized FMN). To study the effect of this organic cofactor on the conformati onal stability, we have characterized apo and hole forms of Desulfovibrio d esulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reacti ons for both holo- and apo-flavodoxin are reversible. However, the unfoldin g curves monitored by far-UV circular dichroism and fluorescence spectrosco py do not coincide. For both apo- and holo-flavodoxin, a native-like interm ediate (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 (Delt a G=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively ). A thermodynamic cycle, connecting FMN binding to folded and unfolded fla vodoxin with the unfolding free energies for apo- and holo-flavodoxin, sugg ests that the binding strength of FMN to unfolded flavodoxin must be very h igh (K-D = 0.2 nM). In agreement, we discovered that the FMN remains coordi nated to the polypeptide upon unfolding. (C) 2000 Elsevier Science B.V. All rights reserved.