Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin

Abstract

Flavodoxin is an α/β protein with a noncovalently bound flavin‐mononucleotide (FMN) cofactor. The apo‐protein adopts a structure identical to that of the holo‐form, although there is more dynamics in the FMN‐binding loops. The equilibrium unfolding processes of Azotobacter vinelandii apo‐flavodoxin, and Desulfovibrio desulfuricans ATCC strain 27774 apo‐ and holo‐flavodoxins involve rather stable intermediates. In contrast, we here show that both holo‐ and apo‐forms of flavodoxin from D. desulfuricans ATCC strain 29577 (75% sequence similarity with the strain 27774 protein) unfold in two‐state equilibrium processes. Moreover, the FMN cofactor remains bound to the unfolded holo‐protein. The folding and unfolding kinetics for holo‐flavodoxin exhibit two‐state behavior, albeit an additional slower phase is present at very low denaturant concentrations. The extrapolated folding time in water for holo‐flavodoxin, ∼280 μsec, is in excellent agreement with that predicted from the protein's native‐state topology. Unlike the holo‐protein behavior, the folding and unfolding reactions for apo‐flavodoxin are best described by two kinetic phases, with rates differing ∼15‐fold, suggesting the presence of a kinetic intermediate. Both folding phases for apo‐flavodoxin are orders of magnitude slower (40‐ and 530‐fold, respectively) than that for the holo‐protein. We conclude that polypeptide–cofactor interactions in the unfolded state of D. desulfuricans strain 29577 flavodoxin alter the kinetic‐folding path towards two‐state and speed up the folding reaction.