Photochemistry of flavoprotein light sensors

Abstract

Three major classes of flavin photosensors, light oxygen voltage (LOV) domains, blue light sensor using FAD (BLUF) proteins and cryptochromes (CRYs), regulate diverse biological activities in response to blue light. Recent studies of structure, spectroscopy and chemical mechanism have provided unprecedented insight into how each family operates at the molecular level. In general, the photoexcitation of the flavin cofactor leads to changes in redox and protonation states that ultimately remodel protein conformation and molecular interactions. For LOV domains, issues remain regarding early photochemical events, but common themes in conformational propagation have emerged across a diverse family of proteins. For BLUF proteins, photoinduced electron transfer reactions critical to light conversion are defined, but the subsequent rearrangement of hydrogen bonding networks key for signaling remains highly controversial. For CRYs, the relevant photocycles are actively debated, but mechanistic and functional studies are converging. Despite these challenges, our current understanding has enabled the engineering of flavoprotein photosensors for control of signaling processes within cells.

Figure 1

Redox and protonation states of flavin (FAD or FMN). In flavoprotein light sensors photochemistry drives conversions among these states, which are then coupled to changes in protein conformation.

Figure 2

LOV domain structure and reactivity. (a) Conserved LOV domain structure with labeled secondary elements, represented by VVD (PDB: 2PD7). (b) Critical flavin binding site residues, (VVD numbering). (c) Alignments of LOV domain core structures (gray) with helical extensions depicted to show variations in peripheral structure important for signal transduction: VVD (green, 2PD7), EL222 (gold, 3P7N), RsLOV (blue, 4HIA), YtvA (pink, 2PR5). (d) LOV domain photocycle. Photoconversion to the excited triplet state promotes reaction of the C4A position with an active site Cys residue. A neutral bi radical formed by flavin oxidation of the thiol is a likely intermediate. Return to the ground state is relatively slow and rate limited by N5 deprotonation.

Figure 3

Protein responses to LOV cysteinyl-adduct formation. (a) Overlay of VVD dark and light state monomer (N-terminal regions in blue and yellow, respectively). Arrow indicates movement of N-terminal latch on conversion to the light state. Inset shows schematic of N/C-terminal movement associated with adduct formation and dimerization. Below: Light state dimer of VVD, (3RH8) N-terminal region in yellow. (b) Proposed model for quarternary structural rearrangements in YtvA/YF1 based on the YF1 dark-state structure (4GCZ) and the light state model on right from alignment of LOV core with Pseudomonas putida LOV (3SW1). Schematic of YtvA/YF1-based rearrangement upon exposure to light with rotation and super-coiling of Jα helix.

Figure 4

BLUF domain structure and reactivity. (a) BLUF domain architecture shown as a superposition of AppA with the Trp_in (green, 1YRX), full-length AppA with the Trp_out configuration (cyan, 4HH1) and the Ccap of BprP (magenta, 3GFX). Tyr21, Gln63 and Trp104/Met106 interact along the edge of the flavin ring (yellow). β5, the β4-β5 loop and the α1-β2 loop propagate conformational changes from the flavin center to the Ccap. The Trp104 residue is found in different conformations (out and in) in various structures (b) Proposed changes in active site hydrogen bonding that accompany photoinduced electron transfer between flavin and Tyr21. Four different proposed reaction pathways are shown (i-iv) that involve variations of Gln63 rotation and/or tautomerization and Tyr21 transient oxidation. Note that pathways (i) and (ii) have different starting (ground) states.

Figure 5

Cryptochrome structure and reactivity. (a) The flavin pocket in various CRY and PL structures is used to recognize cofactors, substrates, regulatory elements, targets and small molecule inhibitors; thereby providing mechanisms to couple molecular recognition to flavin chemistry. Superposition of dCRY (grey with red CTT and yellow FAD, 4GU5), murine mCRY2 PL domain with FAD (residues 1-512, blue with dark blue FAD, 4I6G), murine mCRY2 PL domain with a small molecule bound (magenta with green small molecule, 4MLP), and murine mCRY2 PL domain with the C-terminus of FBXL3 bound in the FAD pocket (gold with teal FBXL3 residues 400-428, 4I6J). (b) Changes in FAD redox states driven by light in dCRY, AtCRY and aCRY; both the ASQ and a light-excited ASQ may be signaling states of dCRY; similarly the NSQ and light-excited ASQ could be AtCRY signaling states (see text); aCRY signals from a ground NSQ state (c) dCRY and mCRY2 residues that lie near FAD are strongly conserved.