F-19 NMR-STUDY ON THE REGIOSPECIFICITY OF HYDROXYLATION OF TETRAFLUORO-4-HYDROXYBENZOATE BY WILD-TYPE AND Y385F P-HYDROXYBENZOATE HYDROXYLASE - EVIDENCE FOR A CONSECUTIVE OXYGENOLYTIC DEHALOGENATION MECHANISM

The regiospecificity of hydroxylation of tetrafluoro-4-hydroxybenzoate (F-4-POHB) by p-hydroxybenzoate hydroxylase (PHBH) and its active sit e mutant Y385F was investigated by F-19 NMR. Evidence is provided that the hydroxylation of F-4-POHB is not restricted to the C3 center of t he aromatic ring but rather involves sequential oxygenation and dehalo genation steps, The catalytic efficiency of PHBH and Y385F with F-4-PO HB was optimal near pH 6.5, Below pH 7.0, substantial substrate inhibi tion occurred. Dianionic F-4-POHB was a competent effector, highly sti mulating upon binding the rate of flavin reduction by NADPH. Hydroxyla tion of F-4-POHB involved the formation of quinone intermediates as pr imary products of oxygenolytic defluorination. Ascorbate competed favo rably with NADPH for the nonenzymatic reduction of these reactive inte rmediates and prevented the accumulation of nonspecific oxidation prod ucts, F-19 NMR showed that the initial aromatic product 2,5,6-trifluor o-3,4-dihydroxybenzoate (F-3-DOHB) was further converted to 5,6-difluo ro-2,3,4-trihydroxybenzoate (5,6-F-2-TOHB). This reaction was most eff icient with Y385F. F-3-DOHB was not bound in a unique regiospecific or ientation as also 2,6-difluoro-3,4,5-trihydroxybenzoate (2,6-F-2-TOHB) was formed, The oxygenolytic dehalogenation of F-3-DOHB by PHBH and Y 385F is consistent with the electrophilic aromatic substitution mechan ism proposed for this class of flavoenzymes. Nucleophilic attack of th e carbon centers of F-3-DOHB onto the distal oxygen of the electrophil ic flavin C(4a)-hydroperoxide occurs when the carbon center has a rela tively high HOMO density and is relatively close to the distal oxygen of the flavin C(4a)-hydroperoxide.