Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.
Keywords: kinetic isotope effect; photoreceptors; proton rocking; proton-coupled electron transfer; stretched dynamics.