Last in, first out

Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I-1 double left right arrow unfolded apoflavodoxin double left right arrow I-2 double left right arrow native apoflavodoxin. I-1 is an off-pathway molten globule-like intermediate that populates during denaturant-induced equilibrium unfolding; I-2 is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I-1 + FMN double left right arrow unfolded apoflavodoxin + FMN double left right arrow I-2 + FMN double left right arrow native apoflavodoxin + FMN double left right arrow holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I-1, I-2, and unfolded apoflavodoxin do not interact to a significant extent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur.