Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500 -630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.

From biological complexes to devices based on organic semiconductors, spin interactions play a key role in the function of molecular systems. For instance, triplet-pair reactions impact operation of organic light-emitting diodes as well as photovoltaic devices. Conventional models for triplet pairs assume they interact only weakly. Here, using electron spin resonance, we observe long-lived, strongly-interacting triplet pairs in an organic semiconductor, generated via singlet fission. Using coherent spin-manipulation of these two-triplet states, we identify exchange-coupled (spin-2) quintet complexes co-existing with weakly coupled (spin-1) triplets. We measure strongly coupled pairs with a lifetime approaching 3 µs and a spin coherence time approaching 1 µs, at 10 K. Our results pave the way for the utilization of high-spin systems in organic semiconductors. The dynamics of spin-dependent reactions impact organic systems across scales of complexity. In vivo radicalpair recombination has been implicated in the biological mechanism for avian navigation and in photosynthesis, while in organic semiconducting materials triplet spin-reactions can determine efficiencies in light-emitting diodes and photovoltaics 1-5. One such process, singlet fission, enables efficient production of two triplet excitons from an initially excited singlet state 6-8. This carrier multiplication process has enabled photovoltaic devices with over 100% external quantum efficiencies and holds promise as a means of harnessing the solar spectrum more efficiently 9,10. Fission proceeds from a photogenerated singlet exciton to an overall spin-zero triplet-pair state, conserving spin and enabling efficient triplet-pair formation. This initial pure singlet state can further decohere into the triplet-pair eigenstates, which we study here. Understanding how these triplet-pair states interact, annihilate, and move is critical for harnessing them in optoelectronic or spintronic applications. The fate of triplet pairs depends not only on their electronic degrees of freedom, but also on their spin properties, such as the pair spin coherence time. To date, spin dynamics of triplet pairs have predominantly been explored passively via photoluminescence experiments 11-13 , which do not allow for direct triplet-pair manipulation. Spin resonance techniques allow for active spin control but have previously been limited to continuous-wave (cw) studies of triplet pair-states 14,15 , although transient spin resonance has provided insight into triplet-transfer and triplet-charge interactions 16,17. Here we focus on the early-time behaviour of the non-equilibrium population of tripletpair states formed following singlet fission and before thermalization. We report the observation of exchange-coupled triplet pairs forming pure spin-quintet (total spin S = 2) states. Quintet states have been observed previously, for example in synthetic compounds that utilize directly bonded radical species 18 or in materials with degenerate ground state orbitals 19. Here we observe, i...

Cryptochrome (Cry) photoreceptors share high sequence and structural similarity with DNA repair enzyme DNA-photolyase and carry the same flavin cofactor. Accordingly, DNA-photolyase was considered a model system for the light activation process of cryptochromes. In line with this view were recent spectroscopic studies on cryptochromes of the CryDASH subfamily that showed photoreduction of the flavin adenine dinucleotide (FAD) cofactor to its fully reduced form. However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA, which is absent for other members of the cryptochrome/photolyase family. Thus, CryDASH may have functions different from cryptochromes. The photocycle of other members of the cryptochrome family, such as Arabidopsis Cry1 and Cry2, which lack DNA repair activity but control photomorphogenesis and flowering time, remained elusive. Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH ⅐ ) accumulates upon blue light irradiation. Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering. These results demonstrate that the active form of Cry2 contains FADH ⅐ (whereas catalytically active photolyase requires fully reduced flavin (FADH ؊ )) and suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair.Blue light signaling has been historically of great interest in plants, because distinct blue light receptors are involved in such key processes as photomorphogenesis, phototropism, and initiation of flowering (1). Cryptochromes (Crys) 2 are UV-A/blue light photoreceptors (also found in animals and bacteria (2)) and are implicated in several of these responses. Because of their high sequence and structural similarity (3-6) and identical flavin cofactor content as DNA-photolyase (7, 8), it was hypothesized that cryptochromes might use the same mechanism for signaling as does photolyase for catalysis, which is light-driven electron transfer from the fully reduced flavin FADH Ϫ (2, 4, 7-9). Indeed, spectroscopic studies on Vibrio cholerae Cry1 (10) and Arabidopsis Cry3 (11) have shown that blue light illumination results in the formation of fully reduced flavin similar to the photoactivation process of DNA-photolyase (2). However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA (12), which is absent for other members of the cryptochrome/photolyase family.The photoreduction of flavin in DNA-photolyase involves several aromatic amino acid residues (mostly tryptophans) that transfer electrons from the protein surface to the flavin adenine dinucleotide (FAD), which is buried in a U-shaped conformation in the ␣-helical domain of the protein (13-16). The cryptochrome structures solved so far, namely Synechocystis Cry-DASH (3), the photolyase-related d...

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.

The following dinuclear exchange-coupled manganese complexes are investigated:  [dtneMnIIIMnIV(μ-O)2μ-OAc](BPh4)2 (dtne = 1,2-bis(1,4,7-triazacyclonon-1-yl)ethane), [(CH3)4dtneMnIIIMnIV(μ-O)2μ-OAc](BPh4)2 ((CH3)4dtne = 1,2-bis(4,7-dimethyl-1,4,7-triazacyclonon-1-yl)ethane), [(CH3)4dtneMnIVMnIV(μ-O)2μ-OAc](ClO4)3, [(tacn)2MnIIIMnIV(μ-O)2μ-OAc](BPh4)2 (tacn = 1,4,7-triazacyclononane), [bpy4MnIIIMnIV(μ-O)2](ClO4)3 (bpy = 2,2‘-bipyridyl), and [phen4MnIIIMnIV(μ-O)2](ClO4)3 (phen = 1,10-phenanthroline). For three of these complexes, X-ray structural data obtained on single crystals are reported here. All complexes are strongly antiferromagnetically coupled, with exchange coupling constants ranging from J = −110 cm-1 (bis-μ-oxo-μ-acetato-bridged) to −150 cm-1 (bis-μ-oxo-bridged). EPR investigations at X- and Q-band frequencies are reported for all five mixed-valence MnIIIMnIV complexes. G tensors and 55Mn hyperfine coupling constants (hfc's) were obtained by simultaneous simulation of the EPR spectra at both frequency bands. By using the vector model of exchange-coupled systems, tensor axes could be related to the molecular structure of the complexes. Hyperfine coupling constants from 55Mn cw-electron−nuclear double-resonance (ENDOR) spectra were in agreement with those obtained from the simulation of the EPR spectra. Ligand hyperfine couplings (1H and 14N) were also measured using cw-ENDOR spectroscopy. Electron spin−echo envelope modulation spectroscopy (ESEEM) spectra yielded information about small 14N hyperfine and quadrupole coupling constants that could not be resolved in the ENDOR spectra. On the basis of specifically deuterated complexes and results from orientation-selection ENDOR spectra, some proton hfc's could be assigned to positions within the complexes. Using an extended point-dipole model and the coordinates provided by the X-ray structure analysis, all dipolar hfc's of the complexes were calculated. Comparison of these hfc's with experimentally obtained values led to a consistent assignment of most hf tensors to molecular positions. The electronic structures of the investigated complexes are compared with each other, and the relevance of the results for metalloenzymes containing at least a dinuclear manganese core is discussed.