INVESTIGATION OF THE INTERACTION BETWEEN FIREFLY LUCIFERASE AND OXYLUCIFERIN OR ITS ANALOGS BY STEADY-STATE AND SUBNANOSECOND TIME-RESOLVED FLUORESCENCE

Fluorescence excitation and emission spectra and fluorescence decay curves of firefly luciferase complexes with oxyluciferin (LO), luciferin (LH(2)), 6'-methoxyluciferin (MeOLH(2)) and 2-cyano-6-hydroxy-benzothiazole (BT) were obtained in aqueous (pH 2-10), aqueous-ethanolic and ethanolic solutions. The K-m values of luciferase-fluorophore complexes were determined at pH 7.8 (11.3 +/- 0.3 mu M for LH(2), 1.5 +/- 0.5 mu M for MeOLH(2) and 13 +/- 1 mu M for BT) and proved to be similar to the kinetically determined K-m value for LH(2) and K-i values for MeOLH(2) and BT. The free rotation of the excited fluorophore indicates that enzyme-fluorophore complexes dissociate immediately after excitation. The different fluorescence properties of the enzyme-product (EP) complex isolated from the full reaction mixture and the complex of enzyme with synthesized oxyluciferin (E*LO) indicate structural differences in the oxyluciferin-binding site of luciferase at the time of EP and E*LO formation, which may be due to significant conformational changes in luciferase during the reaction. The microenvironment of excited luciferase-bound LO, LH(2) and MeOLH(2) shows more similarity to that in aqueous solution than that in ethanol. The proposed dissociation mechanism of bioluminescence is as follows: as soon as the electronically excited product (LO*) is formed, dissociation of the enzyme-product complex occurs, followed by LO* deactivation in an aqueous microenvironment (formed by the enzyme amino acid residues) and the rebinding of the deactivated product with luciferase.