INVESTIGATION OF THE INTERACTION BETWEEN FIREFLY LUCIFERASE AND OXYLUCIFERIN OR ITS ANALOGS BY STEADY-STATE AND SUBNANOSECOND TIME-RESOLVEDFLUORESCENCE
Oa. Gandelman et al., INVESTIGATION OF THE INTERACTION BETWEEN FIREFLY LUCIFERASE AND OXYLUCIFERIN OR ITS ANALOGS BY STEADY-STATE AND SUBNANOSECOND TIME-RESOLVEDFLUORESCENCE, Journal of photochemistry and photobiology.B, Biology, 22(3), 1994, pp. 203-209
Fluorescence excitation and emission spectra and fluorescence decay cu
rves of firefly luciferase complexes with oxyluciferin (LO), luciferin
(LH(2)), 6'-methoxyluciferin (MeOLH(2)) and 2-cyano-6-hydroxy-benzoth
iazole (BT) were obtained in aqueous (pH 2-10), aqueous-ethanolic and
ethanolic solutions. The K-m values of luciferase-fluorophore complexe
s 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 t
o the kinetically determined K-m value for LH(2) and K-i values for Me
OLH(2) and BT. The free rotation of the excited fluorophore indicates
that enzyme-fluorophore complexes dissociate immediately after excitat
ion. The different fluorescence properties of the enzyme-product (EP)
complex isolated from the full reaction mixture and the complex of enz
yme with synthesized oxyluciferin (ELO) indicate structural differenc
es in the oxyluciferin-binding site of luciferase at the time of EP an
d ELO formation, which may be due to significant conformational chang
es 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 me
chanism of bioluminescence is as follows: as soon as the electronicall
y excited product (LO) is formed, dissociation of the enzyme-product
complex occurs, followed by LO deactivation in an aqueous microenviro
nment (formed by the enzyme amino acid residues) and the rebinding of
the deactivated product with luciferase.