Mutational and structural studies of the PixD BLUF output signal that affects light-regulated interactions with PixE.

PixD (Slr1694) is a BLUF (blue-light-using FAD) photoreceptor used by the cyanobacterium Synechocystis sp. PCC6803 to control phototaxis toward blue light. In this study, we probe the involvement of a conserved Tyr8-Gln50-Met93 triad in promoting an output signal upon blue light excitation of the bound flavin. Analysis of acrylamide quenching of Trp91 fluorescence shows that the side chain of this residue remains partially solvent exposed in both the lit and dark states. Mutational analysis demonstrates that substitution mutations at Tyr8 and Gln50 result in the loss of the photocycle while a mutation of Met93 does not appreciably disturb the formation of the light-excited state and only minimally accelerates its decay from 5.7 to 4.5 s. However, mutations of Tyr8, Gln50, and Met93 disrupt the ability of PixD dimers to interact with PixE to form a higher-order PixD(10)-PixE(5) complex, which is indicative of a lit conformational state. Solution nuclear magnetic resonance spectroscopy and X-ray crystallographic analyses confirm that a Tyr8 to Phe mutation is locked in a pseudo-light-excited state revealing flexible areas in PixD that likely constitute part of an output signal upon light excitation of wild-type PixD.