Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis

The hydrogen bonding network that surrounds the flavin in blue light using flavin adenine dinucleotide (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase from Oscillatoria acuminata (OaPAC). Our work elucidates the direct connection between BLUF photoactivation and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate the formation of the neutral flavin radical as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogues to modulate the physical properties of Y6, the TRIR data reveal that a change in the pK(a) and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pK(a) from 9.9 to < 7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FAD(center dot-). The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the adenylyl cyclase (AC) domain. While the 3F-Y6 and 2,3-F(2)Y6 variants undergo the complete photocycle and catalyze the conversion of ATP into cAMP, enzyme activity is abolished in the 3,5-F(2)Y6 and 2,3,5-F(3)Y6 variants where the photocycle is halted at FAD(center dot-). Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in AC activity.