Flavin Conformational Changes in the Catalytic Cycle of P-hydroxybenzoate Hydroxylase Substituted with 6-azido- and 6-aminoflavin Adenine Dinucleotide

Crystallographic studies have demonstrated two flavin conformations for p-hydroxybenzoate hydroxylase (PHBH) [Gatti, D. L., Palfey, B. A. , Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., & Ludwig, M. L. (1994) Science 266, 110-114. Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., & van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. The isoalloxazine ring system of one conformation (the "out" conformation) is significantly more exposed to solvent and is not in position for necessary catalytic reactions, but when the natural substrate is bound to the enzyme, the isoalloxazine is in the correct position (the "in" conformation) for its chemical function. In this study, several aspects of the function of the conformational change in catalysis were explored using the wild-type and Tyr222Phe forms of PHBH substituted with 6-azido FAD. This flavin served as both a spectral probe and a photolabel. The enzyme containing 6-azido FAD was a relatively effective catalyst for the hydroxylation of p-hydroxybenzoate. However, the intermediate reduced 6-azido enzyme was chemically unstable, and a small fraction converted to 6-amino PHBH by the elimination of N2 during each catalytic cycle. The reduction of 6-azido FAD PHBH by NADPH was almost as fast as the reduction of the natural enzyme. The characteristic spectral change caused by NADPH binding prior to hydride transfer strongly suggests that flavin movement from the "in" to the "out" conformation precedes flavin reduction. Irradiation of 6-azido PHBH with visible light covalently labeled proline 293, an active site residue, under conditions in which the flavin adopted the "in" conformation, while no protein labeling occurred under conditions in which the flavin was "out". The labeled protein exchanged substrate and was reduced by NADPH much more slowly than before photolysis. It is therefore concluded that isoalloxazine movement is required for pyridine nucleotide to gain access to the active site and for the exchange of aromatic ligands.