Enhancing Proton-Coupled Electron Transfer in Blue Light Using FAD Photoreceptor AppA_BLUF

The Blue Light Using FAD (BLUF) photoreceptor utilizes a noncovalently bound FAD to absorb light and trigger the initial ultrafast events in receptor activation. FAD undergoes 1 and 2 electron reduction as an enzyme redox cofactor, and studies on the BLUF photoreceptor PixD revealed the formation of flavin radicals (FAD^•- and FADH^•) during the photocycle, supporting a general mechanism for BLUF operation that involves PCET from a conserved Tyr to the oxidized FAD. However, no radical intermediates are observed in the closely related BLUF proteins AppA_BLUF and BlsA, and replacing the conserved Tyr with fluoro-Tyr analogs that increase the acidity of the phenol OH has a minor effect on AppA_BLUF photoactivation in contrast to PixD where the photocycle is halted at FAD^•-. The hydrogen bonding network in BLUF proteins contains several strictly conserved residues but differs in the identity of amino acids that interact with the flavin C2═O. In PixD there are two hydrogen bonds to the C2═O, whereas there is only one in AppA_BLUF. Using TRIR we show that the introduction of a second hydrogen bond to the C2═O in AppA_BLUF results in the formation of flavin radicals (FAD^•- and FADH^•) during the photocycle. Subsequent replacement of the conserved Tyr (Y21) in the double mutant with 2,3,5-trifluoroTyr prevents radical formation and generation of the light state, indicating that the AppA_BLUF photocycle is now similar to that of PixD. The ability to trigger PCET provides fundamental insight into the role of electron transfer in the mechanism of BLUF photoactivation.