Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy

O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimu rium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the c oenzyme and lysine 41 generate structured absorption and fluorescence spect ra upon one-photon excitation. We investigated the protein population heter ogeneity by fluorescence correlation spectroscopy and lifetime techniques u pon two-photon excitation. We sampled the fluorescence intensity from a sma ll number of molecules (similar to 10) and analyzed the distribution of pho ton counts to separately determine the number and the fluorescence brightne ss of excited protein molecules. The changes in the average number of molec ules and in the fluorescence brightness with the excitation wavelength indi cate the presence of at least two fluorescent species, with two-photon exci tation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimi ne. Their relative abundance is estimated to be 4:1, whereas the ratio of t heir two-photon cross sections is reversed with respect to the single-photo n excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity . At least two components were detected, with lifetimes of similar to2.5 an d 0.5 ns. The lifetimes are very close to the values measured in bulk solut ions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited sta te.