Dissecting the Energetics of the Apoflavodoxin-FMN Complex

Anabel Lostao,Mohamed El Harrous,Fatna Daoudi,Antonio Romero,Antonio Parody-Morreale,Javier Sancho

doi:10.1074/jbc.275.13.9518

Abstract

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 PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule. The estimated contribution of the phosphate is greatest, at 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 contribute to the enthalpy of binding. The ionic strength does not have a large effect on the stability of the FMN complex because, although it weakens the phosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the riboflavin complex, which suggests the isoalloxazine and phosphate binding sites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involved in isoalloxazine binding.

Highlights

Many proteins use tightly bound cofactors to perform their biological functions
The calculated molecular mass of apoflavodoxin in a 60 ␮M and in a 120 ␮M solution in 50 mM Mops, pH 7.0, was 18,500 Ϯ 400 and 18,300 Ϯ 300, respectively, in good agreement with the theoretical mass [18,885], and no deviations from the expected distribution for a monomeric species is observed. This indicates that wild type apoflavodoxin in 50 mM Mops, pH 7.0, is a monomer in the concentration range used in this study
Dissociation Constants of Wild Type and Mutant Apoflavodoxin-Flavin Complexes—The dissociation constants at 25.0 Ϯ 0.1 °C of the FMN-apoflavodoxin complexes, in the absence of phosphate, have been determined for wild type flavodoxin and the 10 mutants in 50 mM Mops, pH 7.0 (Table I; see Fig. 4a for the wild type complex)

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis—The mutant flavodoxins W57Y, W57F, W57L, W57A, Y94W, Y94F, Y94L, and Y94A [4] were prepared by oligonucleotide-directed mutagenesis of the Anabaena PCC 7119 flavodoxin gene using the method of Deng and Nickoloff [17] as implemented in the TransformerTM kit (version 2) from CLONTECH. Protein Concentration—The concentration of wild type apoflavodoxin in solution was determined using an extinction coefficient (280 nm) of. 34.1 Ϯ 0.5 mMϪ1 cm Ϫ1 [15] determined by the method of Gill and von Hippel [19] Since this extinction coefficient hardly deviates from the theoretical one that can be calculated from the total amount of tyrosine and tryptophan residues in the protein (33.0 mMϪ1 cmϪ1), we have quantified the concentration of the apoflavodoxin mutants from their theoretical extinction coefficients. Dissociation Constants—The dissociation constants of the complexes between FMN (or the FMN analogues riboflavin and lumiflavin) and apoflavodoxin were determined by titration of the flavin solution with apoflavodoxin at 25 Ϯ 0.1 °C in darkness. F is the observed emission intensity after each addition, Fend the remaining emission intensity at the end of the titration, F␦ the difference in emission intensity between 1 ␮M free flavin and 1 ␮M flavodoxin, CA the total protein concentration after each addition (apo ϩ holo), Kd the dissociation constant of the apo-flavin complex in ␮M units, CF the starting concentration of flavin, and d the dilution factor of this initial

The abbreviations used are

RESULTS

DISCUSSION