The blue light using flavin (BLUF) domain proteins are a novel class of photosensors that bind flavin noncovalently in order to sense and respond to high intensity blue (450 nm) light. The transcriptional antirepressor AppA, is a BLUF photosensor that utilizes a non-covalently bound flavin chromophore which is unable to undergo large scale structural change upon light absorption in contrast to most photoreceptors such as rhodopsin, which undergo structural alterations such as trans/cis isomerization upon irradiation. It is thus of great interest to understand how the BLUF protein matrix senses and responds to flavin photoexcitation. In order to probe the mechanism of photoactivation, the excited state photochemistry of wild-type and mutant AppA proteins has been analyzed using ultrafast time resolved infrared (TRIR) spectroscopy. Reconstitution of the protein with isotopically labeled flavin has permitted unambiguous assignment of the ground and excited state modes associated with the flavin C2=O and C4=O groups which participate in a hydrogen bonding network that surrounds the flavin. This approach has allowed us to probe the role of the hydrogen bonding network in AppA activation. Isotope labeling of the Q63E mutant allowed assignment of a protein mode that appears within 100 fs of excitation, demonstrating that the protein matrix responds instantaneously to flavin excitation. These data have led to a detailed understanding of the photoexcitation mechanism, which involves a tautomerization followed by rotation of residue Q63. Additional insight into photoactivation of AppA has also been obtained by replacing a key tyrosine in the hydrogen bonding network with unnatural fluorotyrosine analogs that have altered pKa values. These data have established the acidity of residue Y21 is crucial in stabilizing the light activated form of the protein.