Investigation of the interaction between firefly luciferase and oxyluciferin or its analogues 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 D values of luciferase—fluorophore complexes were determined at pH 7.8 (11.3±0.3 μM for LH 2, 1.5±0.5 μM for MeOLH 2 and 13±1 μM 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.