Experimental resolution of the free energies of aqueous solvation contributions to ligand-protein binding: quinone-QA site interactions in the photosynthetic reaction center protein.
Equilibrium binding free energies of 14 benzo-, naphtho-, and anthraquinone cofactors have been determined at the QA redox catalytic site of the purified photosynthetic reaction center protein from Rhodobacter sphaeroides solubilized in water (delta G degrees B,w), in hexane solution containing 30 mM water (delta G degrees B,hh), and after partial dehydration (delta G degrees B,dh) with magnesium sulfate. Our aim is to resolve the contributions of aqueous bulk phase solvation and protein hydration contributions to binding in order to characterize in detail the direct interactions between the ligands and protein at the QA site. This is accomplished by comparing the differences between delta G degrees B,w and delta G degrees B,hh (or delta G degrees B,dh) with the water to hexane solvent transfer free energies of the quinones (delta G degrees tr,Q). Values of delta G degrees tr,Q are determined separately in binary solution and range from 0.65 to -5.69 kcal/mol (1 cal = 4.184 J). The results are interpreted in terms of a thermodynamic cycle that links the species involved in the binding and solvent transfer equilibria. Values of delta G degrees B,hh -delta G degrees B,w are linearly correlated with -delta G degrees tr,Q (slope, 0.78 +/- 0.04; ordinate intercept, -0.13 +/- 0.12 kcal/mol). The deviation of the experimental slopes from the predicted value of unity is attributed in part to a systematic decrease of quinone thermodynamic activity in the aqueous binding medium relative to the aqueous phase in the binary partitioning solvent system. The difference between the quinone-QA site binding free energies measured in hydrated hexane and water is therefore related only to the difference in bulk phase quinone solvation, as given by 0.78 delta G degrees tr,Q. The linear relation obtained using delta G degrees B,dh -delta G degrees B,w has the same slope, but the intercept is decreased to -1.48 +/- 0.19 kcal/mol, indicating that quinone binding strengths in the hexane system are uniformly enhanced after partial dehydration. This suggests that the quinones encounter a common opposition to interaction with the site in the hydrated, relative to the partially dehydrated, state. The further utility of the method to directly assess ligand-site binding free energies is demonstrated with examples that address the contributions of molecular size and dipolar or hydrogen bond interactions to the binding of quinones at the QA site.
Warncke
K
K
Johnson Research Foundation, University of Pennsylvania, Philadelphia 19104.
Dutton
P L
PL
D006586:Hexanes; D007700:Kinetics; D008024:Ligands; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D012996:Solutions; D013816:Thermodynamics; D014867:Water
D006586:Hexanes; D008024:Ligands; D045322:Photosynthetic Reaction Center Complex Proteins; D011809:Quinones; D012996:Solutions; D014867:Water