Magnetic sensitivity of cryptochrome 4 from a migratory songbird.

Night-migratory songbirds are remarkably proficient navigators1. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass2,3. 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 birds4-7. 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.

Singlet-triplet dephasing in radical pairs in avian cryptochromes leads to time-dependent magnetic field effects.

Cryptochrome 4a (Cry4a) has been proposed as the sensor at the heart of the magnetic compass in migratory songbirds. Blue-light excitation of this protein produces magnetically sensitive flavin-tryptophan radical pairs whose properties suggest that Cry4a could indeed be suitable as a magnetoreceptor. Here, we use cavity ring-down spectroscopy to measure magnetic field effects on the kinetics of these radical pairs in modified Cry4a proteins from the migratory European robin and from nonmigratory pigeon and chicken. B1/2, a parameter that characterizes the magnetic field-dependence of the reactions, was found to be larger than expected on the basis of hyperfine interactions and to increase with the delay between pump and probe laser pulses. Semiclassical spin dynamics simulations show that this behavior is consistent with a singlet-triplet dephasing (STD) relaxation mechanism. Analysis of the experimental data gives dephasing rate constants, rSTD, in the range 3-6 × 107 s-1. A simple "toy" model due to Maeda, Miura, and Arai [Mol. Phys. 104, 1779-1788 (2006)] is used to shed light on the origin of the time-dependence and the nature of the STD mechanism. Under the conditions of the experiments, STD results in an exponential approach to spin equilibrium at a rate considerably slower than rSTD. We attribute the loss of singlet-triplet coherence to electron hopping between the second and third tryptophans of the electron transfer chain and comment on whether this process could explain differences in the magnetic sensitivity of robin, chicken, and pigeon Cry4a's.

Ultrafast flavin/tryptophan radical pair kinetics in a magnetically sensitive artificial protein.

Radical pair formation and decay are implicated in a wide range of biological processes including avian magnetoreception. However, studying such biological radical pairs is complicated by both the complexity and relative fragility of natural systems. To resolve open questions about how natural flavin-amino acid radical pair systems are engineered, and to create new systems with novel properties, we developed a stable and highly adaptable de novo artificial protein system. These protein maquettes are designed with intentional simplicity and transparency to tolerate aggressive manipulations that are impractical or impossible in natural proteins. Here we characterize the ultrafast dynamics of a series of maquettes with differing electron-transfer distance between a covalently ligated flavin and a tryptophan in an environment free of other potential radical centers. We resolve the spectral signatures of the cysteine-ligated flavin singlet and triplet states and reveal the picosecond formation and recombination of singlet-born radical pairs. Magnetic field-sensitive triplet-born radical pair formation and recombination occurs at longer timescales. These results suggest that both triplet- and singlet-born radical pairs could be exploited as biological magnetic sensors.

Magnetically Sensitive Radical Photochemistry of Non-natural Flavoproteins.

It is a remarkable fact that ∼50 µT magnetic fields can alter the rates and yields of certain free-radical reactions and that such effects might be the basis of the light-dependent ability of migratory birds to sense the direction of the Earth's magnetic field. The most likely sensory molecule at the heart of this chemical compass is cryptochrome, a flavin-containing protein that undergoes intramolecular, blue-light-induced electron transfer to produce magnetically sensitive radical pairs. To learn more about the factors that control the magnetic sensitivity of cryptochromes, we have used a set of de novo designed protein maquettes that self-assemble as four-α-helical proteins incorporating a single tryptophan residue as an electron donor placed approximately 0.6, 1.1, or 1.7 nm away from a covalently attached riboflavin as chromophore and electron acceptor. Using a specifically developed form of cavity ring-down spectroscopy, we have characterized the photochemistry of these designed flavoprotein maquettes to determine the identities and kinetics of the transient radicals responsible for the magnetic field effects. Given the gross structural and dynamic differences from the natural proteins, it is remarkable that the maquettes show magnetic field effects that are so similar to those observed for cryptochromes.

Engineering an Artificial Flavoprotein Magnetosensor.

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.

TREPR spectra of micelle-confined spin correlated radical pairs: II. Spectral decomposition and asymmetric line shapes.

In the second paper, spectral decomposition is used to explain the origin of the asymmetry of the anti-phase structure (APS) and its temperature dependence in dynamic spin correlated radical pairs (SCRPs) created via the photoreduction of benzophenone (BP) in sodium dodecyl sulfate (SDS) micelles. It is shown that the main parameters defining the spectral shape of the TREPR spectra are the effectiveness of the electron spin exchange in contact pairs, and the ratio of the frequency of enforced encounters (Z) to the frequency of singlet-triplet mixing (q) in the separated radical pairs. The Z/q ratio is particularly important for the creation of the APS asymmetry. The existence of different q values in the same TREPR spectrum in this system affords the observation of SCRPs in both regimes: exchange broadening (large |q|/Z) and exchange narrowing (small |q|/Z). An important observation, supported by the successful simulation of the TREPR spectra, is that the S-component of the APS can be shifted in a direction opposite to that predicted by the earlier Closs-Forbes-Norris (CFN) model. This result is naturally explained in terms of a spectral exchange approach. Dispersion-like components in the spectra further amplify the asymmetry of the APS.

TREPR spectra of micelle-confined spin correlated radical pairs: I. Molecular motion and simulations.

Radical pairs created by the photoreduction of benzophenone (BP) in sodium dodecyl sulfate (SDS) micelles exhibit strong asymmetry in the line shapes of their time-resolved electron paramagnetic resonance (TREPR) signals. The asymmetry is strongly dependent on the temperature from 16 °C to 66 °C. Simulations of the anti-phase structure (APS) line shape of these spin correlated radical pairs (SCRPs), based on a numerical solution of the Stochastic Liouville Equation with the spin exchange interaction depending exponentially on the distance between radicals, are presented and discussed. The proposed model takes into account the diffusive motion of the radicals along with the motion of the transverse magnetization and accounts satisfactorily and self-consistently for the asymmetry of the observed TREPR signals.

Kinetic analysis of nitroxide radical formation under oxygenated photolysis: toward quantitative singlet oxygen topology.

Reaction kinetics for two sterically hindered secondary amines with singlet oxygen have been studied in detail. A water soluble porphyrin sensitizer, 5,10,15,20-tetrakis-(4-sulfunatophenyl)-21,23H-porphyrin (TPPS), was irradiated in oxygenated aqueous solutions containing either 2,2,6,6-tetramethylpiperidin-4-one (TMPD) or 4-[N,N,N-trimethyl-ammonium]-2,2,6,6-tetramethylpiperidinyl chloride (N-TMPCl). The resulting sensitization reaction produced singlet oxygen in high yield, ultimately leading to the formation of the corresponding nitroxide free radicals (R2NO) which were detected using steady-state electron paramagnetic resonance (EPR) spectroscopy. Careful actinometry and EPR calibration curves, coupled with a detailed kinetic analysis, led to a simple and compact expression relating the nitroxide quantum yield ΦR2NO (from the doubly-integrated EPR signal intensity) to the initial amine concentration [R2NH]i. With all other parameters held constant, a plot of ΦR2NOvs. [R2NH]i gave a straight line with a slope proportional to the rate constant for nitroxide formation, kR2NO. This establishment of a rigorous quantitative relationship between the EPR signal and the rate constant provides a mechanism for quantifying singlet oxygen production as a function of its topology in heterogeneous media. Implications for in vivo assessment of singlet oxygen topology are briefly discussed.

Investigating Cu-Site Doped Cu-Sb-S Nanoparticles Using Photoelectron and Electron Paramagnetic Resonance Spectroscopy.

Tetrahedrite (Cu12Sb4S13) and famatinite (Cu3SbS4) are good candidates for green energy applications because they possess promising thermoelectric and photovoltaic properties as well as contain earth-abundant and nontoxic constituents. Herein, X-ray photoelectron spectroscopy (XPS), ultraviolet photoelectron spectroscopy (UPS), and electron paramagnetic resonance spectroscopy (EPR) methods examined inherent electronic properties and interatomic magnetic interactions of Cu-site doped tetrahedrite and famatinite nanomaterials. An energy-efficient modified polyol method was utilized for the synthesis of tetrahedrite and famatinite nanoparticles doped on the Cu-site with Zn, Fe, Ni, Mn, and Co. This is the first parallel study of tetrahedrite and famatinite nanomaterials with XPS, UPS, and EPR methods alongside a systematic analysis of dopant-dependent effects on the electronic structure and magnetic interactions for each material. XPS showed that the Cu and Sb species in tetrahedrite and famatinite possess different oxidation states, while UPS characterization reveals larger dopant-dependent shifts in the work function for tetrahedrite nanoparticles (4.21 to 4.79 eV) than for famatinite nanoparticles (4.57 to 4.77 eV). Finally, all famatinite nanoparticles display an EPR signal, indicating trace amounts of paramagnetic Cu(II) present below the detection limit of XPS. For tetrahedrite, EPR signatures were observed only for the Zn-doped and Mn-doped nanoparticles, suggesting signal broadening from Cu-Cu spin exchange or spin-lattice relaxation. This study demonstrates the complementary nature of XPS and EPR techniques for studying the oxidation states of metals in solid-state nanomaterials. Comparing the electronic and magnetic properties of tetrahedrite and famatinite while studying the impact of dopant incorporation will guide future endeavors in designing sustainable, high-performance materials for renewable energy applications.

Electrostatic control of spin exchange between mobile spin-correlated radical pairs created in micellar solutions.

A series of photoinduced H-atom abstraction reactions between anthraquinone-2,6,-disulfonate, disodium salt (AQDS) and differently charged micellar substrates is presented. After a 248 nm excimer laser flash, the first excited triplet state of AQDS is rapidly formed and then quenched by abstraction of a hydrogen atom from the alkyl chain of the micelle surfactant, leading to a spin-correlated radical pair (SCRP). The SCRP is detected 500 ns after the laser flash using time-resolved (direct detection) electron paramagnetic resonance (TREPR) spectroscopy at X-band (9.5 GHz). By changing the charge on the surfactant headgroup from negative (sodium dodecyl sulfate, SDS) to positive (dodecyltrimethylammonium chloride, DTAC), TREPR spectra with different degrees of antiphase structure (APS) in their line shape were observed. The first derivative-like APS line shape is the signature of an SCRP experiencing an electron spin exchange interaction between the radical centers, which was clearly observable in DTAC micelles and absent in SDS micellar solutions. Solutions with surfactant concentrations well below the critical micelle concentration (cmc) or solutions where micellar formation had been disrupted (1:1 v/v CH(3)CN/H(2)O) also showed no APS line shapes in their TREPR spectra. These results support the conclusion that electrostatic forces between the sensitizer (AQDS) charge and the substrate (surfactant) headgroup charge are responsible for the observed effects. The results represent a new example of electrostatic control of a spin exchange interaction in mobile radical pairs.