Till Biskup

Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor

Among the biological phenomena that fall within the emerging field of “quantum biology” is the suggestion that magnetically sensitive chemical reactions are responsible for the magnetic compass of migratory birds. It has been proposed that transient radical pairs are formed by photo-induced electron transfer reactions in cryptochrome proteins and that their coherent spin dynamics are influenced by the geomagnetic field leading to changes in the quantum yield of the signaling state of the protein. Despite a variety of supporting evidence, it is still not clear whether cryptochromes have the properties required to respond to magnetic interactions orders of magnitude weaker than the thermal energy, kBT. Here we demonstrate that the kinetics and quantum yields of photo-induced flavin—tryptophan radical pairs in cryptochrome are indeed magnetically sensitive. The mechanistic origin of the magnetic field effect is clarified, its dependence on the strength of the magnetic field measured, and the rates of relevant spin-dependent, spin-independent, and spin-decoherence processes determined. We argue that cryptochrome is fit for purpose as a chemical magnetoreceptor.

Direct observation of a photoinduced radical pair in a cryptochrome blue-light photoreceptor.

Proteins from the photolyase/cryptochrome family share their three-dimensional fold, sequence homology, and the redox-active flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse activities.[1] In response to blue or UV-A light, they function physiologically in DNA repair, entrainment of the circadian clock, or other processes.[1-3] Members of the photolyase/cryptochrome family have been identified in various organisms ranging from bacteria to plants, animals and humans.[1] Within this family, a phylogenetic cluster of genes originally identified from Arabidopsis and Synechocystis encode cryptochrome-like proteins, which are distinct from previously characterized “classic” plant (represented by Arabidopsis HY4) or animal (represented by Drosophila and Homo sapiens) cryptochromes, yet more closely resemble the latter.[4] Remarkably, the cryptochromes from this new cluster (Cry-DASH) have now been found through all kingdoms of life.[5] While multiple biological functions have been discussed, the availability of stable, recombinantly expressed, Cry-DASH proteins from diverse species provides the means of deciphering cryptochrome protein chemistry. Results from recent experiments point to the direction that Cry-DASH could work as a transcriptional regulator,[4, 5] as well as a DNA repair enzyme for single-stranded DNA.[6] Other experimental results suggest the participation of Cry-DASH in circadian input pathways.[7, 8]

Alternative radical pairs for cryptochrome-based magnetoreception

There is growing evidence that the remarkable ability of animals, in particular birds, to sense the direction of the Earths magnetic field relies on magnetically sensitive photochemical reactions of the protein cryptochrome. It is generally assumed that the magnetic field acts on the radical pair [FAD•− TrpH•+] formed by the transfer of an electron from a group of three tryptophan residues to the photo-excited flavin adenine dinucleotide cofactor within the protein. Here, we examine the suitability of an [FAD•− Z•] radical pair as a compass magnetoreceptor, where Z• is a radical in which the electron spin has no hyperfine interactions with magnetic nuclei, such as hydrogen and nitrogen. Quantum spin dynamics simulations of the reactivity of [FAD•− Z•] show that it is two orders of magnitude more sensitive to the direction of the geomagnetic field than is [FAD•− TrpH•+] under the same conditions (50 µT magnetic field, 1 µs radical lifetime). The favourable magnetic properties of [FAD•− Z•] arise from the asymmetric distribution of hyperfine interactions among the two radicals and the near-optimal magnetic properties of the flavin radical. We close by discussing the identity of Z• and possible routes for its formation as part of a spin-correlated radical pair with an FAD radical in cryptochrome.

Variable Electron-Transfer Pathways in an Amphibian Cryptochrome: Tryptophan versus Tyrosine-Based Radical Pairs

Background: Cryptochrome/photolyase proteins maintain a tryptophan electron transfer pathway allowing for efficient light-induced reduction of the FAD cofactor. Results: When this canonical pathway is blocked, electron transfer in a frog cryptochrome occurs through a tyrosine radical, identified by EPR spectroscopy. Conclusion: Alternative electron transfer pathways provide robust photochemistry in cryptochromes. Significance: Proteins can preserve electron transfer functions through diverse compensatory pathways. Electron transfer reactions play vital roles in many biological processes. Very often the transfer of charge(s) proceeds stepwise over large distances involving several amino acid residues. By using time-resolved electron paramagnetic resonance and optical spectroscopy, we have studied the mechanism of light-induced reduction of the FAD cofactor of cryptochrome/photolyase family proteins. In this study, we demonstrate that electron abstraction from a nearby amino acid by the excited FAD triggers further electron transfer steps even if the conserved chain of three tryptophans, known to be an effective electron transfer pathway in these proteins, is blocked. Furthermore, we were able to characterize this secondary electron transfer pathway and identify the amino acid partner of the resulting flavin-amino acid radical pair as a tyrosine located at the protein surface. This alternative electron transfer pathway could explain why interrupting the conserved tryptophan triad does not necessarily alter photoreactions of cryptochromes in vivo. Taken together, our results demonstrate that light-induced electron transfer is a robust property of cryptochromes and more intricate than commonly anticipated.

Entanglement and sources of magnetic anisotropy in radical pair-based avian magnetoreceptors.

One of the principal models of magnetic sensing in migratory birds rests on the quantum spin dynamics of transient radical pairs created photochemically in ocular cryptochrome proteins. We consider here the role of electron spin entanglement and coherence in determining the sensitivity of a radical pair-based geomagnetic compass and the origins of the directional response. It emerges that the anisotropy of radical pairs formed from spin-polarized molecular triplets could form the basis of a more sensitive compass sensor than one founded on the conventional hyperfine-anisotropy model. This property offers new and more flexible opportunities for the design of biologically inspired magnetic compass sensors.

Unexpected Electron Transfer in Cryptochrome Identified by Time-Resolved EPR Spectroscopy†

Subtle differences in the local sequence and conformation of amino acids can result in diversity and specificity in electron transfer (ET) in proteins, despite structural conservation of the redox partners. For individual ET steps, distance is not necessarily the decisive parameter; orientation and solvent accessibility of the ET partners, and thus the stabilization of the charge-separated states, contribute substantially.

Origin of Light-Induced Spin-Correlated Radical Pairs in Cryptochrome

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.

Light-induced activation of class II cyclobutane pyrimidine dimer photolyases

Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD photolyase and (6-4) photolyase. By ENDOR spectroscopy, the local environment of the flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FAD(ox), have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD photolyase, light-induced generation of FADH from FAD(ox), and subsequently FADH(-) from FADH, proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I photolyases, but completely lacking from the equivalent positions of class II photolyase sequences. Nevertheless, class II photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution.

Structural basis of enzymatic benzene ring reduction

In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.

Magnetic field effects in flavoproteins and related systems.

Within the framework of the radical pair mechanism, magnetic fields may alter the rate and yields of chemical reactions involving spin-correlated radical pairs as intermediates. Such effects have been studied in detail in a variety of chemical systems both experimentally and theoretically. In recent years, there has been growing interest in whether such magnetic field effects (MFEs) also occur in biological systems, a question driven most notably by the increasing body of evidence for the involvement of such effects in the magnetic compass sense of animals. The blue-light photoreceptor cryptochrome is placed at the centre of this debate and photoexcitation of its bound flavin cofactor has indeed been shown to result in the formation of radical pairs. Here, we review studies of MFEs on free flavins in model systems as well as in blue-light photoreceptor proteins and discuss the properties that are crucial in determining the magnetosensitivity of these systems.