Origin of light-induced spin-correlated radical pairs in cryptochrome

Abstract

Blue-light excitation of cryptochromes and homologues uniformly triggers electron transfer (ET) from the protein surface to the flavin adenine dinucleotide (FAD) cofactor. A cascade of three conserved tryptophan residues has been considered to be critically involved in this photoreaction. If the FAD is initially in its fully oxidized (diamagnetic) redox state, light-induced ET via the tryptophan triad generates a series of short-lived spin-correlated radical pairs comprising an FAD radical and a tryptophan radical. Coupled doublet-pair species of this type have been proposed as the basis, for example, of a biological magnetic compass in migratory birds, and were found critical for some cryptochrome functions in vivo. In this contribution, a cryptochrome-like protein (CRYD) derived from Xenopus laevis has been examined as a representative system. The terminal radical-pair state FAD(•)···W324(•) of X. laevis CRYD has been characterized in detail by time-resolved electron-paramagnetic resonance (TREPR) at X-band microwave frequency (9.68 GHz) and magnetic fields around 345 mT, and at Q-band (34.08 GHz) at around 1215 mT. Different precursor states, singlet versus triplet, of radical-pair formation have been considered in spectral simulations of the experimental electron-spin polarized TREPR signals. Conclusively, we present evidence for a singlet-state precursor of FAD(•)···W324(•) radical-pair generation because at both magnetic fields, where radical pairs were studied by TREPR, net-zero electron-spin polarization has been detected. Neither a spin-polarized triplet precursor nor a triplet at thermal equilibrium can explain such an electron-spin polarization. It turns out that a two-microwave-frequency TREPR approach is essential to draw conclusions on the nature of the precursor electronic states in light-induced spin-correlated radical pair formations.

Figure 1

Structural alignment of the conserved tryptophan cascade of members of the photolyase/cryptochrome protein family. Shown are the structures of Escherichia coli CPD photolyase (blue) (PDB entry: 1DNP), Thermus thermophilus CPD photolyase (red) (PDB entry: 1IQR), Drosophila melanogaster (6–4) photolyase (grey) (PDB entry: 3CVU), Arabidopsis thaliana cryptochrome-1 (orange) (PDB entry: 1U3D), Synechocystis sp. PCC 6803 Cry-DASH (yellow) (PDB entry: 1NP7), and A. thaliana cry3 (Cry-DASH, brown) (PDB entry: 2J4D). For X. laevis CRYD, the conserved tryptophans are Trp_A ≡ W400, Trp_B ≡ W377, and Trp_C ≡ W324.

Figure 2

Complete TREPR data sets of X. laevis CRYD showing the signals of the spin-correlated radical pair FAD^•⋯W324^• generated by pulsed laser excitation (460 nm). A, enhanced absorption; E, emission. (a) X-band TREPR. Each time profile is the average of 120 acquisitions recorded at a microwave frequency of 9.68 GHz, and a microwave power of 2 mW at a detection bandwidth of 100 MHz. (b) Q-band TREPR. Each time profile is the average of 50 acquisitions recorded at a microwave frequency of 34.08 GHz, and a microwave power of 3.7 mW at a detection bandwidth of 100 MHz.

Figure 3

Simulations of TREPR spectra at (a) X-band (9.68 GHz), and (b) Q-band (34.08 GHz) microwave frequencies using the magnetic interaction parameters of the radical-pair state FAD^•⋯W324^• of X. laevis CRYD. A, enhanced absorption; E, emission. Only the exchange interaction parameter J was varied in a range from 0 to +1 mT. Other (fixed) simulation parameters: g-tensor of the flavin radical g_FAD = (2.00431, 2.00360, 2.00217); g-tensor of tryptophan radical g_W324 = (2.00370, 2.00285, 2.00246); orientation of the g_W324-tensor principal axes with respect to the g_FAD-tensor principal axes: Ω_W324 = (127°, 77°, 247°); dipolar coupling between FAD^• and W324^•, D_rp = −0.36 mT; orientation of the dipolar coupling tensor D_rp with respect to the g_FAD-tensor principal axes: Ω_Drp = (0°, 110°, 110°); 2nd moment of the FAD^• hyperfine line broadening, 〈H²〉_HFS(FAD^•) = 0.252 mT²; 2nd moment of the of the W324^• radical, 〈H²〉_HFS(W324^•) = 0.841 mT². 2nd moment of (Gaussian) inhomogeneous broadening, 〈H²〉_inh = 0.371 mT².

Figure 4

Simulations of TREPR spectra of FAD^•⋯W324^• of X. laevis CRYD recorded at (a) X-band (9.68 GHz), and (b) Q-band (34.08 GHz) microwave frequencies based on the spin-correlated coupled radical-pair model with a pure singlet-state precursor. The parameters specified in Figure 3 were used in combination with J = +0.073 mT. Lower panel: experimental spectra (red curves) recorded at 1 µs (X-band) and 130 ns (Q-band) after pulsed laser excitation. Spectral simulations are superimposed as black dashed lines. Upper panel: Spectral simulations of the radical pair FAD^•⋯W324^• together with the contributions of the individual radicals to the overall spectrum (black curves), FAD^• (dashed blue curves); W324^• (dashed green curves).

Figure 5

Simulations of TREPR spectra of FAD^•⋯W324^• of X. laevis CRYD recorded at X-band (9.68 GHz, left curves), and Q-band (34.08 GHz, right curves) microwave frequencies, based on a (a) pure singlet-precursor state, (b.1) pure spin-polarized triplet-precursor state, and (b.2) pure (“relaxed”) triplet-precursor state at thermal equilibrium. The simulation parameters for the singlet-precursor radical-pair spectra (a) are given in Figure 3. For the triplet-precursor radical-pair spectra (b.1 and b.2), the following triplet parameters have been used: D = 68.57 mT, and E = −18.75 mT; zero-field spin-state populations, ρ_X = 0.67, ρ_Y = 0.33, and ρ_Z = 0 (case b.1). For the triplet precursor at thermal equilibrium (T = 274 K) (case b.2), the high-field triplet sublevel populations have been calculated using Eqs. (16) and (17). The principal axes of the D_T-tensor of the flavin triplet precursor were assumed collinear with the principal axes of the g_FAD-tensor.