DIRECT MEASUREMENT OF EXCITATION TRANSFER IN THE PROTEIN COMPLEX OF BACTERIAL LUCIFERASE HYDROXYFLAVIN AND THE ASSOCIATED YELLOW FLUORESCENCE PROTEINS FROM VIBRIO-FISCHERI Y1

Time-resolved fluorescence was used to directly measure the energy tra nsfer rate constant in the protein-protein complex involved in the yel low bioluminescence of Vibrio fischeri, strain Y1. In this reaction th e putative donor is the fluorescent transient intermediate, luciferase hydroxyflavin, which exhibits a major fluorescence lifetime of the bo und flavin of 10 ns. On addition of the acceptor, the V. fischeri yell ow fluorescence protein containing either FMN or riboflavin as ligand, a rapid decay time, 0.25 ns, becomes predominant. The same results ar e observed using rec-luciferase from Photobacterium leiognathi to prod uce the donor. Because of favorable spectral separation in this system , this rapid decay rate of 4 ns(-1), can be directly equated to the en ergy transfer rate. This rate is ten times higher than the rate previo usly observed in the Photobacterium luciferase hydroxyflavin-lumazine protein, donor-acceptor system, derived from emission anisotropy measu rements. This ten-times ratio is close to the ratio of spectral overla ps of the donor fluorescence with the acceptor absorption, between the se two systems, so it is concluded that the topology of the protein co mplexes in both cases, must be very similar. Energy transfer is also m onitored by the loss of steady-state fluorescence intensity at 460 nm of the donor, on addition of the acceptor protein. A fluorescence titr ation indicates that luciferase hydroxyflavin and the yellow protein c omplex with a 1:1 stoichiometry with a K-d of 0.7 mu M (0 degrees C). These parameters account for the bioluminescence spectral shifting eff ects observed in these reactions.