Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of Synechocystis sp PCC6803

The Sensor of blue-light using FAD (BLUF) domain is the flavin-binding fold categorized to a new class of blue-light sensing domain found in AppA from Rhodobactcr sphaeroides and PAC from Euglena gracilis, but little is known concerning the mechanism of blue-light perception. An open reading frame slr1694 in a cyanobacterium Synechocystis sp. PCC6803 encodes a protein possessing the BLUF domain. Here, a full-length Slr1694 protein retaining FAD was expressed and purified and found to be present as an oligomeric form (trimer or tetramer). Using the purified Slr1694, spectroscopic properties of Slr1694 were characterized. Slr1694 was found to show the same red-shift of flavin absorption and quenching of flavin fluorescence by illumination as those of AppA. These changes reversed in the dark although the rate of dark state regeneration was much faster in Slr1694 than AppA, indicating that Slr1694 is a blue-light receptor based on BLUF with the similar photocycle to that, of AppA. The dark decay in D2O was nearly four times slower than in H2O. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was applied to examine the light-indUced structure change of a chromophore and apo-protein with deuteration and universal C-13 and N-15 isotope labeling. The FTIR results indicate that light excitation induced distinct changes in the amide I modes of peptide backbone but relatively limited changes in flavin chromophore. Light excitation predominantly weakened the C(4) = O and C(2) = O bonding and strengthened the NIC10a and/or C4aN5 bonding, indicating formational changes of the isoalloxazine ring II and III of FAD but little formational change in the isoalloxazine ring I. The photocycle of the BLUF is unique in the sense that light excitation leads to the structural rearrangements of the protein moieties Coupled with a minimum formational change of the chromophore.