Dynamic fluorescence properties of bacterial luciferase intermediates.
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 degrees C. These three species are the metastable intermediate formed with the two substrates FMNH2 and O2, both in its low-fluorescence form and in its 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 degrees C) derived from the decay of the anisotropy of the intrinsic fluorescence at 340 nm or the fluorescence of bound 8-anilino-1-naphthalenesulfonic acid (470 nm), respectively. The steady-state anisotropy of the fluorescent intermediates is 0.34, and the fundamental anisotropy from a Perrin plot is 0.385. The high-fluorescence intermediate has a fluorescence maximum at 500 nm, and its emission spectrum is distinct from the bioluminescence spectrum. The fluorescence quantum yield is 0.3 but decreases on dilution with a quadratic dependence on protein concentration. This, and the large value of the rotational correlation time, would be explained by protein complex formation in the fluorescent intermediate states, but no increase in protein molecular weight is observed by gel filtration or ultracentrifugation. The results instead favor a proposal that, in these intermediate states, the luciferase undergoes a conformational change in which its axial ratio increases by 50%.
Lee
J
J
Department of Biochemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
O'Kane
D J
DJ
Gibson
B G
BG
D005486:Flavin Mononucleotide; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014733:Vibrio / Q000201:enzymology*
D005486:Flavin Mononucleotide; D008156:Luciferases