FLAVIN CONFORMATIONAL-CHANGES IN THE CATALYTIC CYCLE OF P-HYDROXYBENZOATE HYDROXYLASE SUBSTITUTED WITH 6-AZIDO-FLAVIN AND 6-AMINOFLAVIN ADENINE-DINUCLEOTIDE

Crystallographic studies have demonstrated two flavin conformations fo r p-hydroxybenzoate hydroxylase (PHBH) [Gatti, D. L., Palfey, B. A., L ah, M. S., Entsch, B., Massey, V., Ballou, D. P., & Ludwig, M. L. (199 4) 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 syst em of one conformation (the ''out'' conformation) is significantly mor e exposed to solvent and is not in position for necessary catalytic re actions, but when the natural substrate is bound to the enzyme, the is oalloxazine is in the correct position (the ''in'' conformation) for i ts chemical function. In this study, several aspects of the function o f the conformational change in catalysis were explored using the wild- type and Tyr222Phe forms of PHBH substituted with 6-azido FAD. This fl avin served as both a spectral probe and a photolabel. The enzyme cont aining 6-azido FAD was a relatively effective catalyst for the hydroxy lation of p-hydroxybenzoate. However, the intermediate reduced 6-azido enzyme was chemically unstable, and a small fraction converted to 6-a mino PHBH by the elimination of N-2 during each catalytic cycle. The r eduction of 6-azido FAD PHBH by NADPH was almost as fast as the reduct ion of the natural enzyme. The characteristic spectral change caused b y NADPH binding prior to hydride transfer strongly suggests that flavi n 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 labele d protein exchanged substrate and was reduced by NADPH much more slowl y than before photolysis. It is therefore concluded that isoalloxazine movement is required for pyridine nucleotide to gain access to the ac tive site and for the exchange of aromatic ligands.