Time-resolved fluorescence of O-acetylserine sulfhydrylase.
Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers 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 lamda > 470 nm, indicates the presence of two components characterized by lifetimes (tau) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function 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 opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved; (v) the increased emission at 490 nm, caused by acetate binding, is likely due to the stabilization of the ketoenamine tautomer induced by an increase in polarity of the active site microenvironment and/or a decrease in proton dissociation in the excited state; (vi) excitation at 330 nm, where the enolimine tautomer absorbs, leads to emission decays typical of the ketoenamine.
Benci
S
S
Institute of Physical Sciences, University of Parma, Italy. benci@ipruniv.cce.unipr.it
Vaccari
S
S
Mozzarelli
A
A
Cook
P F
PF
D000085:Acetates; D001665:Binding Sites; D003547:Cysteine Synthase / Q000737:chemistry*; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D011732:Pyridoxal Phosphate / Q000737:chemistry; D012545:Schiff Bases / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D014633:Valine / Q000737:chemistry