Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

Abstract

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature’s smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the Wout, WinNHin and WinNHout conformations using 19F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key WinNHin conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of WinNHin, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.

Elementary Reactions in the Functional Triads of the Blue-Light Photoreceptor BLUF Domain

“…Taken as a whole, our work points out that the key to resolve the dark-state photocycle is to search for trace evidence of the PR intermediate since it is one important missing piece in the puzzle. Besides the PR intermediate (FMNH • /Glu-COO – /TrpH •+ ) in FMN-Glu-Trp when the forward PT2 proceeds before the forward PT1, we recently have captured the PR intermediates in YnF mutants from the AppA and Sy PixD BLUF domain where the central Tyr is mutated into Phe, retaining the nearby Trp . The PR intermediate in the YnF series is identified to be FMN •– /GlnH + /Trp • , implying that the forward PT1 proceeds before the forward PT2 .…”

“…Besides the PR intermediate (FMNH • /Glu-COO − / TrpH •+ ) in FMN-Glu-Trp when the forward PT2 proceeds before the forward PT1, 63 we recently have captured the PR intermediates in YnF mutants from the AppA and SyPixD BLUF domain where the central Tyr is mutated into Phe, retaining the nearby Trp. 67 The PR intermediate in the YnF series is identified to be FMN •− /GlnH + /Trp • , implying that the forward PT1 proceeds before the forward PT2. 67 Nonetheless, the PR intermediates identified so far are both based on Trp as the electron/proton donor, and the PR intermediate in native FMN-Gln-Tyr has yet to be captured.…”

“…67 The PR intermediate in the YnF series is identified to be FMN •− /GlnH + /Trp • , implying that the forward PT1 proceeds before the forward PT2. 67 Nonetheless, the PR intermediates identified so far are both based on Trp as the electron/proton donor, and the PR intermediate in native FMN-Gln-Tyr has yet to be captured. In our current work, when a direct mutation is applied to substitute the central Tyr or Gln, the H-bond network does not rearrange and the BLUF domain will lose its ability as a photoswitch.…”