Dynamic fluorescence evidence for conformational change in the bacterial luciferase intermediates

摘要

Three fluorescent species produced by the reaction of bacterial luciferase from Vibrio harveyi with its substrates, have the same dynamic fluorescence properties, namely a dominant fluorescence decay of lifetime of 10 ns and a rotational correlation time of 100 ns at 2°C. These three species are, the metastable intermediate formed with the two substrates FMNH{sub}2 and O{sub}2, both in its low fluorescence form and high fluorescence form following light irradiation, and the fluorescent transient formed on including the final substrate tetradecanal. For native luciferase the rotational correlation time is 62 or 74 ns (2°C) derived from the decay of the anisotropy of the intrinsic fluorescence at 340 nm or the fluorescence of bound 8-anilino-1-naphthalene sulfonic acid (470 nm), respectively. This correlation time is in the range calculated for luciferase, with M{sub}r = 77000 and assuming a spherical shape. The much larger value found for the fluorescent intermediate states would be explained if they were highly anisotropic rotators. This would imply a considerable axial ratio change for the luciferase when it forms these intermediate states.