Reaction kinetics and mechanism of magnetic field effects in cryptochrome

Abstract

Creatures as varied as mammals, fish, insects, reptiles, and birds have an intriguing sixth sense that allows them to orient themselves in the Earth's magnetic field. Despite decades of study, the physical basis of this magnetic sense remains elusive. A likely mechanism is furnished by magnetically sensitive radical pair reactions occurring in the retina, the light-sensitive part of animal eyes. A photoreceptor, cryptochrome, has been suggested to endow birds with magnetoreceptive abilities as the protein has been shown to exhibit the biophysical properties required for an animal magnetoreceptor to operate properly. Here, we propose a theoretical analysis method for identifying cryptochrome's signaling reactions involving comparison of measured and calculated reaction kinetics in cryptochrome. Application of the method yields an exemplary light-driven reaction cycle, supported through transient absorption and electron-spin-resonance observations together with known facts on avian magnetoreception. The reaction cycle permits one to predict magnetic field effects on cryptochrome activation and deactivation. The suggested analysis method gives insight into structural and dynamic design features required for optimal detection of the geomagnetic field by cryptochrome and suggests further experimental and theoretical studies.

Figure 1. Arabidopsis thaliana cryptochrome in transient absorption experiment

(Top) The structure of cryptochrome-1 from Arabidopsis thaliana is shown together with the highlighted flavin cofactor (FAD) and the tryptophan triad Trp400, Trp377, Trp324. (Bottom) Transient absorption of cryptochrome is probed by means of the pump-probe experiment, as described in experiment. The sample containing cryptochrome is irradiated with a pulsed laser beam (red) that generates a measurable concentration of excited states (FAD*) in the system. FAD* decays then in a series of intermediates back to FAD, while some of the intermediates being probed by the probe beam (white) used to measure the absorption spectrum of transient species. Additionally, magnetic field effects in cryptochrome can be studied if the sample is subjected to an external magnetic field.

Figure 2. Cryptochrome activation and inactivation reactions

Cryptochrome is activated through absorbing a blue-light photon by the flavin cofactor, responsible for protein's signaling. Initially, the flavin cofactor in cryptochrome is present in its fully oxidized FAD state. After absorbing a photon, FAD becomes promoted to an excited FAD* state. FAD* is then protonated and receives an electron from a nearby tryptophan (see Fig. 1), leading to the formation of the [FADH^• + Trp^•] radical pair, which exists in singlet and the triplet overall electron spin states, denoted as ¹[…] and ³[…], respectively. The Trp^• radical may receive an additional electron from a nearby tyrosine,^, or become deprotonated,^, quenching the radical pair and fixing the electron on the FADH^• cofactor. Under aerobic conditions, FADH^• slowly reverts back to the initial inactive FAD state through the also inactive FADH⁻ state of the flavin cofactor. Before the [FADH^• + Trp^•] radical pair in cryptochrome is quenched, the electron of the FADH^• radical can back-transfer to the Trp^• radical, thereby also ending the signaling state of the protein. The electron back-transfer leads to the formation of FADH⁺ and can only occur if the spins of the two unpaired electrons in the radical pair [FADH^•+Trp^•] are in an overall singlet state. While the flavin cofactor is in its FADH^• state, cryptochrome may absorb a second blue-green photon, thereby transferring it into a non-signaling state with the flavin cofactor in the FADH⁻ conformation.

Figure 3. Cryptochrome transient absorption spectra

Time profiles of the change in the transient absorption spectra from the absorbance prior laser excitation of the sample calculated for cryptochrome excited by laser pulses of different intensity (see Fig. 1), for the probe-beam of wavelengths 550-630 nm (plot a), and 490-550 nm (plot b). The laser pulse duration chosen was 5 ns as in experiment, at 355 nm wavelength. The energies of the pulse used in the calculation are indicated in the inset. The wavelengths used in the calculation were chosen consistent with the experimental measurements of the transient absorption spectra in garden warbler cryptochrome. Dots correspond to the experimentally measured data recorded for the pulse energy of 5 mJ.

Figure 4. Population of transient states in cryptochrome

Time evolution of concentration of the transient states in cryptochrome calculated from equations (2)-(10) for cryptochrome photoexcitation by a laser pulse of 5 ns duration, 5 mJ energy and 355 nm wavelength (plot a, consistent with Ref. 55), and a laser pulse of 6 ns duration, 4 mJ energy and 460 nm wavelength (plot b, consistent with Ref. 56). Grayed areas indicate time duration of laser pulse.

Figure 5. Magnetic field effect in cryptochrome

Change of the transient absorption spectra in cryptochrome due to a change in k_sf caused by an external magnetic field, calculated for the 550-630 nm probe-beam. α is defined in the text. Initial photoexcitation is due to a laser pulse of 5 ns duration, 355 nm wavelength and 5 mJ energy (plot a) and 2.5 mJ energy (plot b). The curves in (a) and (b) reflect change in transient absorption due to an increased value of the k_sf relative to the reference value 10⁶ s⁻¹ (see Tab. 1).

Figure 6. Magnetic field effect and probe concentration

The maximal change of the transient absorption spectra in cryptochrome calculated due to change in k_sf at different concentrations of protein, calculated for a 550-630 nm probe-beam. Initial photoexcitation is due to a laser pulse of 5 ns duration, 355 nm wavelength, 5 mJ energy (plot a) or 2.5 mJ energy (plot b). The curves in (a) and (b) show transient absorption due to an increased k_sf relative to the reference value of 10⁶ s⁻¹ (see Tab. 1), α is defined in the text.