Enhancing Proton-Coupled\nElectron Transfer in Blue\nLight Using FAD Photoreceptor AppA<sub>BLUF</sub>

The Blue Light Using FAD (BLUF) photoreceptor utilizes\na noncovalently\nbound FAD to absorb light and trigger the initial ultrafast events\nin receptor activation. FAD undergoes 1 and 2 electron reduction as\nan enzyme redox cofactor, and studies on the BLUF photoreceptor PixD\nrevealed the formation of flavin radicals (FAD<sup>•–</sup> and FADH<sup>•</sup>) during the photocycle, supporting a\ngeneral mechanism for BLUF operation that involves PCET from a conserved\nTyr to the oxidized FAD. However, no radical intermediates are observed\nin the closely related BLUF proteins AppA<sub>BLUF</sub> and BlsA,\nand replacing the conserved Tyr with fluoro-Tyr analogs that increase\nthe acidity of the phenol OH has a minor effect on AppA<sub>BLUF</sub> photoactivation in contrast to PixD where the photocycle is halted\nat FAD<sup>•–</sup>. The hydrogen bonding network in\nBLUF proteins contains several strictly conserved residues but differs\nin the identity of amino acids that interact with the flavin C2O.\nIn PixD there are two hydrogen bonds to the C2O, whereas there\nis only one in AppA<sub>BLUF</sub>. Using TRIR we show that the introduction\nof a second hydrogen bond to the C2O in AppA<sub>BLUF</sub> results in the formation of flavin radicals (FAD<sup>•–</sup> and FADH<sup>•</sup>) during the photocycle. Subsequent replacement\nof the conserved Tyr (Y21) in the double mutant with 2,3,5-trifluoroTyr\nprevents radical formation and generation of the light state, indicating\nthat the AppA<sub>BLUF</sub> photocycle is now similar to that of\nPixD. The ability to trigger PCET provides fundamental insight into\nthe role of electron transfer in the mechanism of BLUF photoactivation.