On the Signaling Mechanism and the Absence of Photoreversibility in the AppA BLUF Domain

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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 10nm, a state known as AppARED. We have applied ultrafast spectroscopy on the photoaccumulated AppARED state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppARED, the FAD singlet excited state FADRED∗ decays monoexponentially in 7ps to form the neutral semiquinone radical FADH•, which subsequently decays to the original AppARED molecular ground state in 60ps. Thus, FADRED∗ 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 AppARED that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppARED, the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppARED ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppARED 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.