On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA_RED. We have applied ultrafast spectroscopy on the photoaccumulated AppA_RED state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA_RED, the FAD singlet excited state decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH^•, which subsequently decays to the original AppA_RED molecular ground state in 60 ps. Thus, is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA_RED that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA_RED, the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA_RED ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA_RED is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.