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Night-migratory songbirds are remarkably proficient navigators 1 . Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass 2,3 . The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds 4-7 . Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin-tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds.

The fact that many animals, including migratory birds, use the Earth's magnetic field for orientation and compass-navigation is fascinating and puzzling in equal measure. The physical origin of these phenomena has not yet been fully understood, but arguably the most likely hypothesis is based on the radical pair mechanism (RPM). Whilst the theoretical framework of the RPM is well-established, most experimental investigations have been conducted at fields several orders of magnitude stronger than the Earth's. Here we use transient absorption spectroscopy to demonstrate a pronounced orientation-dependence of the magnetic field response of a molecular triad system in the field region relevant to avian magnetoreception. The chemical compass response exhibits the properties of an inclination compass as found in migratory birds. The results underline the feasibility of a radical pair based avian compass and also provide further guidelines for the design and operation of exploitable chemical compass systems.

Drosophila have been used as model organisms to explore both the biophysical mechanisms of animal magnetoreception and the possibility that weak, low-frequency anthropogenic electromagnetic fields may have biological consequences. In both cases, the presumed receptor is cryptochrome, a protein thought to be responsible for magnetic compass sensing in migratory birds and a variety of magnetic behavioural responses in insects. Here, we demonstrate that photo-induced electron transfer reactions in Drosophila melanogaster cryptochrome are indeed influenced by magnetic fields of a few millitesla. The form of the protein containing flavin and tryptophan radicals shows kinetics that differ markedly from those of closely related members of the cryptochrome-photolyase family. These differences and the magnetic sensitivity of Drosophila cryptochrome are interpreted in terms of the radical pair mechanism and a photocycle involving the recently discovered fourth tryptophan electron donor., Competing Interests: The authors declare no competing financial interests.

Migratory birds use the Earth's magnetic field as a source of navigational information. This light-dependent magnetic compass is thought to be mediated by cryptochrome proteins in the retina. Upon light activation, electron transfer between the flavin adenine dinucleotide cofactor and tryptophan residues leads to the formation of a spin-correlated radical pair, whose subsequent fate is sensitive to external magnetic fields. To learn more about the functional requirements of this complex chemical compass, we have created a family of simplified, adaptable proteins-maquettes-that contain a single tryptophan residue at different distances from a covalently bound flavin. Despite the complete absence of structural resemblance to the native cryptochrome fold or sequence, the maquettes exhibit a strong magnetic field effect that rivals those observed in the natural proteins in vitro. These novel maquette designs offer unprecedented flexibility to explore the basic requirements for magnetic sensing in a protein environment.

Even though the interaction of a <1 mT magnetic field with an electron spin is less than a millionth of the thermal energy at room temperature (kBT), it still can have a profound effect on the quantum yields of radical pair reactions. We present a study of the effects of sub-millitesla magnetic fields on the photoreaction of flavin mononucleotide with ascorbic acid. Direct control of the reaction pathway is achieved by varying the rate of electron transfer from ascorbic acid to the photo-excited flavin. At pH 7.0, we verify the theoretical prediction that, apart from a sign change, the form of the magnetic field effect is independent of the initial spin configuration of the radical pair. The data agree well with model calculations based on a Green's function approach that allows multinuclear spin systems to be treated including the diffusive motion of the radicals, their spin-selective recombination reactions, and the effects of the inter-radical exchange interaction. The protonation states of the radicals are uniquely determined from the form of the magnetic field-dependence. At pH 3.0, the effects of two chemically distinct radical pair complexes combine to produce a pronounced response to ∼500 μT magnetic fields. These findings are relevant to the magnetic responses of cryptochromes (flavin-containing proteins proposed as magnetoreceptors in birds) and may aid the evaluation of effects of weak magnetic fields on other biologically relevant electron transfer processes.

Magnetic fields as weak as the Earth's can change the yields of radical pair reactions even though the energies involved are orders of magnitude smaller than the thermal energy, kBT, at room temperature. Proposed as the source of the light-dependent magnetic compass in migratory birds, the radical pair mechanism is thought to operate in cryptochrome flavoproteins in the retina. Here we demonstrate that the primary magnetic field effect on flavin photoreactions can be amplified chemically by slow radical termination reactions under conditions of continuous photoexcitation. The nature and origin of the amplification are revealed by studies of the intermolecular flavin-tryptophan and flavin-ascorbic acid photocycles and the closely related intramolecular flavin-tryptophan radical pair in cryptochrome. Amplification factors of up to 5.6 were observed for magnetic fields weaker than 1 mT. Substantial chemical amplification could have a significant impact on the viability of a cryptochrome-based magnetic compass sensor.

Cavity enhanced absorption spectroscopy (CEAS) combined with phase-sensitive detection is employed to study the effects of static magnetic fields on radical recombination reactions. The chemical system comprises the photochemically generated thionine semiquinone radical and a 1,4-diazabicyclo[2.2.2]octane (DABCO) cationic radical in a micellar solution of sodium dodecyl sulphate. Data obtained using the modulated CEAS technique, describing the magnetic field effect (MFE) on reaction yields, are shown to be superior to those obtained using conventional transient absorption (TA) flash photolysis methods typically employed for these measurements. The high sensitivity afforded by modulated CEAS detection is discussed in terms of the new possibilities it offers such as the measurement of magnetic field effects in real biological systems which have hitherto been largely beyond the detection capabilities of existing techniques. [ABSTRACT FROM AUTHOR]

Broadbandcavity-enhanced absorption spectroscopy (BBCEAS) is shownto be a sensitive method for the detection of magnetic field effects(MFEs) in two flavin-based chemical reactions which are simple modelsfor cryptochrome magnetoreceptors. The advantages of optical cavity-baseddetection and (pseudo-white-light) supercontinuum radiation have beencombined to provide full spectral coverage across the whole of thevisible spectrum (425 < λ < 700 nm). This region coversthe absorbance spectra of flavin mononucleotide (FMN) and flavin adeninedinucleotide (FAD) as well as their photogenerated radicals. To illustratethe power of this technique, BBCEAS has been used to record the spectraldependence of MFEs for photoinduced radical pairs formed in the intermolecularreaction of FMN with lysozyme and the intramolecular photochemistryof FAD. These reactions have been chosen for their photochemical similaritiesto cryptochrome proteins which have been proposed as key to the magneticcompass sense of many animals including birds. In experiments performedusing low protein concentrations (10 μM) and 1 mm optical path-lengths,absorbance changes as small as 1 × 10–7(representing<0.1% MFEs) have been detected with good signal-to-noise offeringthe prospect of sensitive MFE detection in cryptochrome. [ABSTRACT FROM AUTHOR]

The study of radical pair intermediates in biological systems has been hampered by the low sensitivity of the optical techniques usually employed to investigate these highly reactive species. Understanding the physical principles governing the spin-selective and magneto-sensitive yields and kinetics of their reactions is essential in identifying the mechanism governing bird migration, and might have significance in the discussion of potential health hazards of electromagnetic radiation. Here, we demonstrate the powerful capabilities of optical cavity-enhanced techniques, such as cavity ring-down spectroscopy (CRDS) in monitoring radical recombination reactions and associated magnetic field effects (MFEs). These include submicrosecond time-resolution, high sensitivity (baseline noise on the order of 10-6 absorbance units) and small (μL) sample volumes. Combined, we show that these represent significant advantages over the single-pass flash-photolysis techniques conventionally applied. The studies described here focus on photoinduced radical pair reactions involving the protein lysozyme and one of two possible photosensitizers: anthraquinone-2,6-disulphonate and flavin mononucleotide. CRDS-measured MFEs are observed in pump-probe experiments and discussed in terms of the sensitivity gains and sample-volume minimization afforded by CRDS when compared with flash photolysis methods. Finally, CRDS is applied to an in vitro MFE study of intramolecular electron transfer in the DNA-repair enzyme, Escherichia coli photolyase, a protein closely related to cryptochrome which has been proposed to mediate animal magnetoreception. [ABSTRACT FROM AUTHOR]