Decrypting cryptochrome: revealing the molecular identity of the photoactivation reaction

Abstract

Migrating birds fly thousands of miles or more, often without visual cues and in treacherous winds, yet keep direction. They employ for this purpose, apparently as a powerful navigational tool, the photoreceptor protein cryptochrome to sense the geomagnetic field. The unique biological function of cryptochrome supposedly arises from a photoactivation reaction involving radical pair formation through electron transfer. Radical pairs, indeed, can act as a magnetic compass; however, the cryptochrome photoreaction pathway is not fully resolved yet. To reveal this pathway and underlying photochemical mechanisms, we carried out a combination of quantum chemical calculations and molecular dynamics simulations on plant ( Arabidopsis thaliana ) cryptochrome. The results demonstrate that after photoexcitation a radical pair forms, becomes stabilized through proton transfer, and decays back to the protein's resting state on time scales allowing the protein, in principle, to act as a radical pair-based magnetic sensor. We briefly relate our findings on A. thaliana cryptochrome to photoreaction pathways in animal cryptochromes.

Figure 1. Electron and proton-transfer reactions in cryptochrome

(a) Flavin cofactor, the tryptophan triad W400, W377, W324 and the D396 residue forming the active site of crypto-chrome-1 from Arabidopsis thaliana. (b) Schematic representation of the photoactivation reaction. Cryptochrome photoactivation is triggered by blue-light photoexcitation of the FAD cofactor (blue arrow) initially present in the oxidized state. Excited flavin, FAD*, receives an electron from one of the nearby tryptophans (red arrows); either W400(H) (RP-W400) or W377(H) (RP-W377). Electron transfer from tryptophan leads to formation of an ionic FAD^•− + W(H)^•+ radical pair, which is then transformed into a stable neutral FADH^• + W^• radical pair state through proton exchange with the nearby D396 (two green arrows). RP-W400 and RP-W377 interconvert through a W400(H)↔W377(H) electron transfer process (red arrows). In contrast to RP-W377, the neutral radical pair RP-W400 recombines back to the initial state through coupled electron-proton transfer (solid purple arrow). The RP-W377 state is stabilized through W377(H)^•+ deprotonation into solution and, therefore, returns to the initial state only on a very long time scale (dashed purple arrow). The reaction cycle B1 and the reaction B2 are primary candidates for establishing magne-toreception;^, the state labeled S is a primary candidate for being the signaling state (for details see text).

Figure 2. Characterization of the cryptochrome photoreaction involving flavin, W400 and D396

(a) Calculated potential energy profiles of the key electronic states describing cryptochrome photoactivation. The energy of oxidized flavin is shown in red, of excited flavin in blue, and of the radical pair state RP-W400 in green. Filled circles represent computed energies while lines show schematic potential energy surfaces. The colored background distinguishes electron transfer step (light green), proton transfer steps (light blue) and coupled electron-proton transfer step (pink). Reaction steps (i-vi) refer to explanations in the text. (b) Change of electron density due to flavin photoexcitation (S0→S1), photoinduced radical pair formation (S1→S2⁽⁰⁾) and recombination (S2→S0). The initial distribution of electron density is shown in blue, while the final distribution is shown in orange. (c) Rearrangement of the COOH- group of the D396(H) residue catalyzing the protonation of flavin by the W400(H)^•+ radical through formation of a D396⁻ intermediate (minimum S2⁽¹⁾ in (a)).

Figure 3. Rearrangement of D396(H) in the cryptochrome active site

(Top) Relative orientations of flavin, D396(H) and W400(H) residues obtained through all-atom MD simulations of Arabidopsis thaliana cryptochrome in water for (a) cryptochrome with oxidized flavin, i.e., FAD + W400(H), and (b) cryptochrome in the radical pair state FAD^•− + W400(H)^•+. (Middle, Bottom) Time dependence of the hydrogen bond lengths d(H^W400-O^D396), labeled d_H−O at top, and d(H^D396-N5^flavin), labeled d_H−O at top, calculated for the two different redox states shown at top.

Figure 4. Electron transfer through tryptophan diad

(a) Calculated potential energy pro-files for the flavin oxidized state (red), flavin excited state (blue), radical pair state RP-W400 (green) and radical pair state RP-W377 (purple). Filled circles represent the computed energies while lines show a schematic profile of the potential energy surfaces. The colored background highlights the results of QM/MM calculations (light blue), and calculations that account for the presence of three water molecules around W377 (pink). (b) Active site model containing three water molecules in the W377 vicinity. (c) Change of electron density due to electron transfer from W400(H) to flavin (S0→S2⁽⁰⁾), from W377(H) to flavin (S0→S3⁽⁰⁾) and from W377(H) to W400(H)^•+ (S2⁽⁰⁾ →S3⁽⁰⁾). The initial distribution of electron density is shown in blue; the final electron density is shown in orange.