The thermodynamics of flavin binding to the apoflavodoxin from Azotobacter vinelandii.
A thermodynamic study of the binding of flavins (FMN, FAD, 8-carboxylic acid-riboflavin) to the purified apoflavodoxin from Azotobacter vinelandii has been conducted. The binding of FMN was studied at a number of temperatures (10, 15, 20, 25, and 30 degrees C), pH's (6.0, 7.4, and 9.0), and buffer conditions. The binding of FAD was studied at pH 7.4 and 25 degrees C under a number of buffer conditions. The binding of 8-carboxylic acid-riboflavin to the apoflavodoxin and the binding of FMN to the dimeric form of the apoflavodoxin were investigated at pH 7.4 and 25 degrees C. Enthalpies of binding for FMN, FAD, and 8-carboxylic- acid-riboflavin were -28.3, -16.6, and -14.0 kcal mol-1, respectively. The enthalpy of binding of FMN to the dimeric form of the apoflavodoxin was -22.2 kcal mol of binding sites-1. Binding constants of about 10(8), 10(6), and 10(6) were obtained for the binding of FMN, FAD, and 8-carboxylic acid-riboflavin, respectively. Using established thermodynamic relationships free energy and entropy changes were calculated. The entropy data indicate that a large degree of ordering of the system occurs upon flavin binding. The pH data suggest that FMN may bind in both the mono- and dianion forms, and that binding doesn't change the pKa of any functional group in the system. It appears that the phosphate group is probably responsible for approximately half the binding enthalpy observed for the binding of FMN. The temperature-dependence data over the temperature range studied is biphasic, centered at 20 degrees C, indicating that flavin binding occurs to the protein in two thermodynamic states corresponding to the two heat capacities observed. These findings are used to discuss a model for flavin binding.
D001059:Apoproteins / Q000378:metabolism*; D001395:Azotobacter / Q000378:metabolism*; D002151:Calorimetry; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D012256:Riboflavin / Q000031:analogs & derivatives / Q000378:metabolism; D013816:Thermodynamics
D001059:Apoproteins; D005415:Flavins; D005418:Flavodoxin; D005420:Flavoproteins; C026932:apoflavodoxin; C040747:riboflavin 8-carboxylic acid; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D012256:Riboflavin