Probing the Signal Transduction Mechanism of the Light-Activated Adenylate Cyclase OaPAC Using Unnatural Amino Acid Mutagenesis.

OaPAC, the photoactivated adenylyl cyclase from Oscillatoria acuminata, is composed of a blue light using FAD (BLUF) domain fused to an adenylate cyclase (AC) domain. Since both the BLUF and AC domains are part of the same protein, OaPAC is a model for understanding how the ultrafast modulation of the chromophore binding pocket caused by photoexcitation results in the activation of the output domain on the μs-s time scale. In the present work, we use unnatural amino acid mutagenesis to identify specific sites in the protein that are involved in transducing the signal from the FAD binding site to the ATP binding site. To provide insight into site-specific structural dynamics, we replaced W90 which is close to the chromophore pocket, F103 which interacts with W90 across the dimer interface, and F180 in the central core of the AC domain, with the infrared probe azido-Phe (AzPhe). Using ultrafast IR, we show that AzPhe at position 90 responds on multiple time scales following photoexcitation. In contrast, the light minus dark IR spectrum of AzPhe103 shows only a minor perturbation in environment between the dark and light states, while replacement of F180 with AzPhe resulted in a protein with no catalytic activity. We also replaced Y125, which hydrogen bonds with N256 across the dimer interface, with fluoro-Tyr residues. All the fluoro-Tyr substituted proteins retained the light-induced red shift in the flavin absorption spectrum; however, only the 3-FY125 OaPAC retained photoinduced catalytic activity. The loss of activity in 3,5-F₂Y125 and 2,3,5-F₃Y125 OaPAC, which potentially increase the acidity of the Y125 phenol by more than 1000-fold, suggests that deprotonation of Y125 disrupts the signal transduction pathway from the BLUF to the AC domain.