Dissecting the energetics of the apoflavodoxin-FMN complex

Many flavoproteins are non-covalent complexes between FMN and an apoprotein . To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena P CC 7119 by a combination of site-directed mutagenesis, titration calorimetr y, equilibrium binding constant determinations, and x-ray crystallography, Comparison of the strength of the wild type and mutant apoflavodoxin-FMN co mplexes and that of the complexes between wild type apoflavodoxin and short ened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of t he FMN molecule. The estimated contribution of the phosphate is greatest, a t 7 kcal mol(-1); that of the isoalloxazine is of around 5-6 kcal mol(-1) ( mainly due to interaction with Trp-57 and Tyr-94 in the apoprotein) and the ribityl contributes least: around 1 kcal mol(-1). The stabilization of the complex is both enthalpic and entropic although the enthalpy contribution is dominant. Both the phosphate and the isoalloxazine significantly contrib ute to the enthalpy of binding. The ionic strength does not have a large ef fect on the stability of the FMN complex because, although it weakens the p hosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the r iboflavin complex, which suggests the isoalloxazine and phosphate binding s ites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involv ed in isoalloxazine binding.