Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Abstract

BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather than an isomerizable cofactor. To understand the physicochemical basis of BLUF domain photoactivation, we have applied femtosecond transient absorption spectroscopy to the Slr1694 BLUF domain of Synechocystis PCC6803. We show that photoactivation of BLUF domains proceeds by means of a radical-pair mechanism, driven by electron and proton transfer from the protein to the flavin, resulting in the transient formation of anionic and neutral flavin radical species that finally result in the long-lived signaling state on a 100-ps timescale. A pronounced deuteration effect is observed on the lifetimes of the intermediate radical species, indicating that proton movements underlie their molecular transformations. We propose a photoactivation mechanism that involves a successive rupture of hydrogen bonds between a conserved tyrosine and glutamine by light-induced electron transfer from tyrosine to flavin and between the glutamine and flavin by subsequent protonation at flavin N5. These events allow a reorientation of the conserved glutamine, resulting in a switching of the hydrogen-bond network connecting the chromophore to the protein, followed by radical-pair recombination, which locks the glutamine in place. It is suggested that the redox potential of flavin generally defines the light sensitivity of flavin-binding photoreceptors.

Fig. 1.

X-ray structure of the R. sphaeroides AppA BLUF domain. (A and B) Close-ups of the vicinity of the FAD chromophore in two proposed orientations of the conserved Gln in dark state (A) and light state (B). The coordinates were taken from Protein Data Bank ID code 1YRX (11).

Fig. 2.

EADS and their corresponding lifetimes resulting from global analysis of ultrafast transient absorption experiments on the Synechocystis Slr1694 BLUF domain upon excitation at 400 nm in H₂O buffer (A) and in D₂O buffer (B).

Fig. 3.

Kinetic traces of the Synechocystis Slr1694 BLUF domain in H₂O (open circles) and D₂O (filled circles) upon excitation at 400 nm, with detection at 483 nm (A), 550 nm (B), 610 nm (C), and 710 nm (D). The solid lines denote the result of the target analysis. Note that the time axis is linear from −10 to 20 ps and logarithmic thereafter.

Fig. 4.

Target analysis and photocycle scheme of the Synechocystis Slr1694 BLUF domain. (Upper) SADS of the various molecular species in the kinetic scheme that result from the application of a target analysis to the ultrafast time-resolved data of the Synechocystis Slr1694 BLUF domain in H₂O (solid lines) and D₂O (dotted lines), with FAD* (blue), Q₁ (black), Q₂ (magenta), and the long-lived signaling state Slr_RED (orange). (Lower) The photocycle scheme of Slr1694, with time constants as they follow from the target analysis and assignments of the intermediate states as described in the text.

Fig. 5.

The proposed reaction mechanism for photoinduced hydrogen-bond switching in the Synechocystis Slr1694 BLUF domain. See text for details.