Calorimetric Studies on the FAD Conformational Change of p-Hydroxybenzoate Hydroxylase. Bruce A. Palfey & Marina Kasimova, Department of Biological Chemistry, University of Michigan, & Department of Biophysics, Johns Hopkins University.

p-Hydroxybenzoate hydroxylase catalyzes the conversion of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in a reaction requiring molecular oxygen and NADPH. FAD is the only prosthetic group at the active site of the homodimeric enzyme. A number of crystal structures have been solved for a variety of enzyme-ligand complexes of the wild-type enzyme and some site-directed mutants. In more than half these structures, the isoalloxazine moiety of the flavin is located in a position ideal for oxygen transfer from the flavin C4a-hydroperoxide, the key reaction intermediate, to the aromatic substrate. The flavin is mostly buried in this arrangement (designated "in"), protecting the labile flavin hydroperoxide from solvent-catalyzed decomposition. However, in a number of instances, a different flavin conformation has been observed, with the isoalloxazine moiety swinging out of its interior binding pocket to adopt a more exposed conformation, one that is not capable of substrate hydroxylation. This second conformation (designated "out") is the result of alterations in the structure of the aromatic ligand, the flavin, or the protein. In many of these cases, the altered enzyme system is still effective in the hydroxylation reaction, indicating that the flavin can move during catalysis.

The energetics of the flavin conformational change in PHBH were studied by isothermal titration calorimetry (ITC) at 10deg., pH6.5. In general, the binding of ligands in the "out" conformation is more exothermic than binding in the "in" conformation. In the wild-type PHBH, this is offset by an unfavorable change in entropy, which disfavors the binding of ligands that cause the "out" conformation. A comparison of ITC data obtained for the wild-type enzyme, which adopts either the "in" or "out" conformation depending upon the ligand, and the Tyr222Phe mutant, which adopts the "out" conformation with most ligands, allowed the conformational change to be dissected into energetic components and indicates that the enthalpy change for flavin movement is near zero but is disfavored entropically by the binding of water molecules to the "out" conformer.