Time-resolved fluorescence of O-acetylserine sulfhydrylase

Static and time-resolved fluorescence of the internal aldimine of the pyrid oxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS ) and those of free PLP, and the PLP-L-valine Schiff base have been measure d to gain insight into the photophysics of PLP bound to OASS. Exciting at 3 30 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emissi on with a peak at 420 nm and shoulders at 490 and 530 nm. The emission band s at 420 and 490 nm are attributed to the enolimine and ketoenamine tautome rs of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at lambda > 470 nm, indicates the presence of two co mponents characterized by lifetimes (tau) of 0.6 (0.08) and 3.8 (1.55) ns w ith equal fractional intensity (f). In the presence of acetate the slow com ponent dominates OASS emission with f of 0.98. Excitation at 350 nm as a fu nction of emission wavelengths (400-560 nm) shows at least three components . The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposi te way. Results indicate that: (i) the fast component is associated with th e emission at 530 nm; (ii) the slow component is associated with the emissi on at 420 nm; (iii) a fast additive component, characterized by a very shor t lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including th e emission of the band at 490 nm, that could not be resolved; (v) the incre ased emission at 490 nm, caused by acetate binding, is likely due to the st abilization of the ketoenamine tautomer induced by an increase in polarity of the active site microenvironment and/or a decrease in proton dissociatio n in the excited state; (vi) excitation at 330 nm, where the enolimine taut omer absorbs, leads to emission decays typical of the ketoenamine. (C) 1999 Elsevier Science B.V. All rights reserved.