Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence

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Abstract
The mechanism of the shifting of the bioluminescence spectrum from the reaction of bacterial luciferase by lumazine protein is investigated by methods of fluorescence dynamics. A metastable intermediate is produced on reaction of Vibrio harveyi luciferase with FMNH2 and O2. It has an absorption maximum at 374 nm and a rotational correlation time (.phi.) derived from the decay of its fluorescence (maximum 500 nm) anisotropy of 90 ns (2.degree. C). Lumazine protein from Photobacterium phosphoreum has an absorption maximum at 417 nm and a fluorescence maximum at 475 nm. Lumazine protein forms a protein-protein complex with luciferase, and the complex has a .vphi. of .apprx. 100 ns. A mixture of lumazine protein and the intermediate would be expected to have an average correlation time (.vphi.av) around 100 ns, but instead, the result is anomalous. The .vphi.av is much lower and is also wavelength dependent. For excitation at 375 nm, which is mainly absorbed in the flavin chromophore of the intermediate, .vphi.av = 25 ns, but at 415 nm, mainly absorbed by the lumazine derivative ligand of lumazine protein, .vphi.av .apprx. 50 ns. It is proposed that protein-protein complexation occurs between lumazine protein and the luciferase intermediate and that in this complex energy transfer from the flavin to the lumazine is the predominant channel of anisotropy loss. A distance of 20 .ANG. between the donor and acceptor is calculated. In the bioluminescence reaction of intermediate with tetradecanal, a fluorescent transient species is produced which is the bioluminescence emitter. If this species were the directly excited product, its emission rate (108 s-1) is too fast for the excitation to be efficiently transferred (.apprx. 2 .times. 107 s-1) to the associated lumazine protein acting as a secondary acceptor. It is further shown that at high concentrations of the fluorescent transient, the bioluminescence spectral shift of lumazine protein is inhibited. The results are consistent with the sensitized chemiluminescence mechanism, wherein all these fluorophores present in the reaction compete for the source of the bioluminescence excitation.