Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer.
Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected.
Department of Chemistry, Tulane University, New Orleans, Louisiana 70118, USA.
B K
BK
Muralidhara
Pernilla
P
Wittung-Stafshede
D000838:Anions; D001059:Apoproteins / Q000737:chemistry; D001665:Binding Sites; D002151:Calorimetry; D045285:Desulfovibrio desulfuricans / Q000737:chemistry*; D019281:Dimerization; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005418:Flavodoxin / Q000737:chemistry*; D008024:Ligands; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D012996:Solutions; D013431:Sulfates / Q000737:chemistry; D013816:Thermodynamics
D000838:Anions; D001059:Apoproteins; D005418:Flavodoxin; D008024:Ligands; D010710:Phosphates; D012996:Solutions; D013431:Sulfates; C026932:apoflavodoxin; D005486:Flavin Mononucleotide