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.

Calorimetric studies of flavin binding protein: flavin analog binding.

The effect of flavin structure variation upon the overall binding reaction of flavin to hen egg white riboflavin binding protein (WRBP) was correlated to thermodynamic parameters obtained via titration calorimetry. This effect was measured by determining a reference binding enthalpy (delta Href) for 3-carboxymethylriboflavin and subsequently comparing delta Href to the binding enthalpies determined for all other flavin analogs in this study. The reference enthalpy change was measured in detail under a variety of temperatures (14, 25, and 38 degrees C), pH's (5.5, 6.5, 7.4, 8.5), and buffer types. The reference enthalpy for the binding of N(3)-carboxymethylriboflavin to WRBP was measured as delta H = -20.9 kcal/mol at 25.0 degrees C and pH 7.4. Evaluation of the binding thermodynamic values for the reference flavin indicates that there is no H+ flux concomitant with binding. A small negative change in heat capacity (delta Cp) occurs during the binding process suggesting a contraction of the protein. Finally, the reaction appears to be independent of pH and buffer type indicating little, if any, involvement of charged residues in the binding of flavin to apoWRBP. Thermodynamic values for the binding of an additional eight flavin analogs were then measured at pH 7.4 and 25.0 degrees C. The thermodynamic binding parameters for these analogs were evaluated by comparison to those determined for the reference flavin. The results indicate that the ribityl C2' position is a major influence in the interaction between the ribityl side chain and the protein; the C9 position of the isoalloxazine ring is sterically restricted in a manner similar to positions C7 and C8; and the positions C7 and C8 appear to be unequal with regard to enthalpy release, suggesting that the C8 position is the most restricted region in the aromatic ring. These various findings indicate the unique ability of titration calorimetry to evaluate structural variation of ligands to their corresponding binding site interactions.

An improved purification procedure and apoprotein preparation for the flavodoxin from Azotobacter vinelandii.

An improved method for the purification of the holoflavodoxin from Azotobacter vinelandii was developed, which resulted in improved yields and degree of homogeneity. The purity and homogeneity of this sample were established by polyacrylamide gel electrophoresis. An apoprotein preparation procedure is outlined, which results in a homogeneous preparation of the apoflavodoxin. The homogeneity of the apoflavodoxin sample was established by polyacrylamide gel electrophoresis.

The thermodynamics of nucleotide binding to proteins.

Models describing the interaction of a small molecule with a protein are typically couched in terms of the stoichiometry, cooperativity, and binding free-energy change. These parameters are readily available from equilibrium dialysis experiments (or appropriate variations). With the recent advent of extremely sensitive calorimeters, it is possible to obtain thermal data for the binding reaction and, thus, the entire set of thermodynamic parameters, delta G', delta H', delta S', delta C', become readily available. This review is limited to the binding of nucleotides and nucleotide analogs to proteins for which complete thermal data are available. While the majority of such systems have been characterized by calorimetry, we have not excluded, per se, van't Hoff enthalpy determinations. The systems we have considered include, but are not limited to, thymidylate synthetase, phosphorylase, several dehydrogenases, aldolase, glutamine synthetase, hemoglobin, asparate transcarbamylase, and ribonuclease. A variety of forces contribute to the total free-energy change upon ligand binding. These forces include ionic, van der Waals, hydrogen bond, and hydrophobic. In several cases, properly designed experiments have allowed a partial resolution of the individual contributions of these various forces. Variation of easily accessible conditions such as temperature, pH, ionic strength, or solvent third component produce changes in the set of thermodynamic parameters which lead to the resolution of the forces. The generality of heat effects makes this method very useful for studying the involvement of protons in binding reactions. The variation in the magnitude and direction (release or uptake) of the proton flux is readily studied by determining the apparent heat of reaction at constant pH, ionic strength, and temperature in two or more buffers of differing heat of ionization. This application has been exploited in several cases and is examined in great detail. An overview of the results in these systems to date suggests that several trends observed in the thermodynamic parameters need to be confirmed by further experimentation and, if they hold, an appropriate theoretical basis must be developed to aid in their interpretation.

A calorimetric investigation of the growth of the luminescent bacteria Beneckea harveyi and Photobacterium leiognathi.

Direct calorimetric determinations of the rate of heat production along with simultaneous determinations of the rate of photon emission and the number of viable cells have provided insight into the growth of Beneckea harveyi and Photobacterium leiognathi. These experiments were performed with a Tronac isothermal microcalorimeter modified with a fiber optic light guide to allow in situ detection of light. Escherichia coli and a dark variant of P. leiognathi were also examined to provide points of reference. It is demonstrated that B. harveyi seems to pause in the rate of metabolic heat production at the same point in time that the enzyme luciferase begins to be synthesized. This effect is not removed if B. harveyi is grown in conditioned medium. The thermograms for all species are correlated with cell generation time. The heat production per cell indicates that uncrowded cultures produce more heat than older, more crowded cultures, supporting the original observation of Bayne-Jones and Rhees (1929). These observations reopen for examination the suggestion that living systems tend toward a state of minimum metabolism per unit mass.