Separation of photo-induced radical pair in cryptochrome to a functionally critical distance

Abstract

Cryptochrome is a blue light receptor that acts as a sensor for the geomagnetic field and assists many animals in long-range navigation. The magnetoreceptor function arises from light-induced formation of a radical pair through electron transfer between a flavin cofactor (FAD) and a triad of tryptophan residues. Here, this electron transfer is investigated by quantum chemical and classical molecular dynamics calculations. The results reveal how sequential electron transfer, assisted by rearrangement of polar side groups in the cryptochrome interior, can yield a FAD-Trp radical pair state with the FAD and Trp partners separated beyond a critical distance. The large radical pair separation reached establishes cryptochrome's sensitivity to the geomagnetic field through weakening of distance-dependent exchange and dipole-dipole interactions. It is estimated that the key secondary electron transfer step can overcome in speed both recombination (electron back-transfer) and proton transfer involving the radical pair reached after primary electron transfer.

Figure 1. Electron and proton transfers in cryptochrome.

Shown is the flavin cofactor, the tryptophan triad (W400, W377, W324) and the D396 residue forming the active site of cryptochrome-1 from Arabidopsis thaliana. Electron (red) and proton (green) transfer processes that likely govern cryptochrome activation are presented by arrows. The three electron transfers W400 → flavin, W377 → W400^·+, and W324 → W377^·+ are labeled I, II, and III, respectively. Electron transfer following flavin photo-excitation leads to a radical pair state of cryptochrome: transfer I to [FAD^·− + W400^·+] with the averaged pair separation of 7.4 Å, sequential transfer I + II to [FAD^·− + W377^·+] with pair separation of 12.2 Å, sequential transfer I + II + III to [FAD^·− + W324^·+] with pair separation of 18.2 Å.

Figure 2. Cryptochrome active site model.

The quantum chemical description of the W377 → W400^·+ electron transfer in cryptochrome (electron transfer II in Fig. 1) includes electronic degrees of freedom of riboflavin and of the side chains of amino acid residues S251, R362, W377, D390, D396, Q401, W400, T404, and T406. Residues donating and accepting the electrons (flavin, W400, W377) are shown in licorice representation, while the atoms of coordinating side chains are shown as balls and sticks. The coloring of cryptochrome secondary structure illustrates the charge distribution of the protein's amino acids: positive (blue), negative (red), polar uncharged (green), and hydrophobic uncharged (white). Sidechain groups of all polar, positively and negatively charged amino acids surrounding flavin, W400, and W377 were included into the active site model to describe environmental effects on the W377 → W400^·+ electron transfer; distant charged sidechains, e.g., E312, R313 (transparent), were not included in the quantum chemical description.

Figure 3. Potential energy profiles of the key electronic states in cryptochrome.

The energy for oxidized flavin is shown in green, for radical pair state [FAD^·− + W400^·+] in red and for radical pair state [FAD^·− + W377^·+] in blue. Solid symbols represent calculated energies, while lines represent schematic potential energy surfaces. The energies were computed using the CASSCF method (a) and the perturbation theory-based XMCQDPT-2 method (b). Open circles denote the energies calculated for the optimized [FAD^·− + W400^·+] radical pair after the rearrangement of the W377 residue shown in Fig. 4. Δ₁ is the energy difference between the RP2 and the RP1 state calculated for the RP1-optimized geometry and Δ₂ is the energy difference between the RP1 and the RP2 state calculated for the RP2-optimized geometry; Δ₁₂ is the energy difference between geometry optimized RP2 state and geometry optimized RP1 state. The values of the energies Δ₁, Δ₂, Δ₁₂, according to the data in Tab. 1, are 1.52 eV, 0.50 eV, 0.36 eV, respectively, for the CASSCF method and 1.32 eV, 0.38 eV, 0.26 eV, respectively, for the XMCQDPT-2 method.

Figure 4. Turn of W377 upon electron transfer.

Shown is a rearrangement of the W377 residue in Arabidopsis thaliana cryptochrome, namely a turn of the side group that is coupled to W377 → W400^·+ electron transfer. The turn comes about in the quantum chemical geometry optimization calculations depicted in Fig. 3 and involves also the positive W400^·+ radical and the polar environment of the W377 residue made up of residues Q401, T404, and T406. A T404^OG1 …W377^HE1 hydrogen bond (right panel, magenta) is formed upon the turn of W377.

Figure 5. Spontaneous rearrangement of W377 seen in classical MD simulation.

(a) Time evolution of the distance d between the OG1 atom of the T404 residue and the HE1 atom of the W377 residue (see Fig. 4) calculated for cryptochrome with oxidized flavin, i.e., in the FAD + W400 + W377 state (green), cryptochrome in the radical pair state [FAD^·− + W400^·+] (red), and cryptochrome in the radical pair state [FAD^·− + W377^·+] (blue). (b) Total energy in Arabidopsis thaliana cryptochrome of the neutral W400…W377 diad (green), the diad with the W400^·+ radical (red), and of the diad with the W377^·+ radical (blue). The distances in (a) and the corresponding energies in (b) for the three redox states of cryptochrome are measured for the same MD trajectories. The energies calculated for each step of the simulation are shown with the shaded colors, while intense colors show energies averaged over 50 steps.

Figure 6. Interaction of W377 radical with environment.

(a) Chlorine anion, Cl⁻, spontaneously binding from the bulk solvent to the positively charged W377^·+ residue, as seen in MD simulation. The binding of Cl⁻ is characterized by the hydrogen bond length d_ion which has been analysed for three redox states of the cryptochrome active site: FAD + W400 + W377 (b), [FAD^·− + W400^·+] (c), and FAD^·− + W377^·+] (d).