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 AppABLUF and BlsA, and replacing the conserved Tyr with fluoro-Tyr analogs that increase the acidity of the phenol OH has a minor effect on AppABLUF 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 AppABLUF. Using TRIR we show that the introduction of a second hydrogen bond to the C2═O in AppABLUF 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 AppABLUF 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.