The cryptochrome/photolyase protein family possesses a conserved triad of tryptophans that may act as a molecular wire to transport electrons from the protein surface to the FAD cofactor for activation and/or signaling-state formation. Members from the animal (and animal-like) cryptochrome subclade use this process in a light-induced fashion in a number of exciting responses, such as the (re-)setting of circadian rhythms or magnetoreception; however, electron-transfer pathways have not been explored in detail yet. Therefore, we present an in-depth time-resolved optical and electron-paramagnetic resonance spectroscopic study of two cryptochromes from Chlamydomonas reinhardtii and Drosophila melanogaster. The results do not only reveal the existence of a fourth, more distant aromatic amino acid that serves as a terminal electron donor in both proteins, but also show that a tyrosine is able to fulfill this very role in Chlamydomonas reinhardtii cryptochrome. Additionally, exchange of the respective fourth aromatic amino acid to redox-inactive phenylalanines still leads to light-induced radical pair formation; however, the lifetimes of these species are drastically reduced from the ms- to the μs-range. The results presented in this study open up a new chapter, to our knowledge, in the diversity of electron-transfer pathways in cryptochromes. Moreover, they could explain unique functions of animal cryptochromes, in particular their potential roles in magnetoreception because magnetic-field effects of light-induced radical pairs strongly depend on distance and orientation parameters.

Light-generated short-lived radial pairs have been suggested to play pivotal roles in cryptochromes and photolyases.Cryptochromes are very probably involved in magnetic compass sensing in migratory birds and the magnetic-fielddependent behavior of insects.W ee xamined photo-generated transient states in the cryptochrome of Drosophila melanogaster and in the structurally related DNA-repair enzyme Escherichia coli DNAp hotolyase.U sing pulsed EPR spectroscopy, the exchange and dipolar contributions to the electron spin-spin interaction were determined in astraightforward and direct way.W itht hese parameters,r adical-pair partners maybeidentified and the magnetoreceptor efficiency of cryptochromes can be evaluated. We present compelling evidence for an extended electron-transfer cascade in the Drosophila cryptochrome,a nd identified W394 as ak ey residue for flavin photoreduction and formation of as pincorrelated radical pair with as ufficient lifetime for highsensitivity magnetic-field sensing.Biological magnetoreception remains enigmatic with very different concepts under discussion. Among them is aradical pair (RP) based mechanism, [1] which gained considerable attention [2] after the discovery of cryptochromes. [3] Cryptochromes are versatile proteins present in all kingdoms of life with intriguing functions, [2b, 4] such as the resetting of the circadian clock [5] or the sensing of magnetic fields to perceive direction.[6] Their photoreceptor function is most likely driven by ar eduction of the flavin adenine dinucleotide (FAD) cofactor. [7] Even though the exact role of cryptochromes in magnetoreception is still unclear,t heir ability to readily conduct electron transfer (ET) upon blue-light exposure makes them prime candidates for ar ealization of the RP mechanism. Thei ntraprotein ET forms ap air of correlated spins,o ne situated on the FAD, the electron acceptor in the protein core,and the other one on an amino acid residue,the ultimate electron donor, which, in most cases,isatryptophan (Trp) residue at the protein surface.[8] Thee volution of the spin multiplicity of the RP,n amely singlet (S) versus triplet (T), determines the fate of this excited state,w hich either undergoes charge recombination from the singlet configuration or generates longer-lived paramagnetic products from the triplet manifold because direct charge recombination is spin-forbidden from the triplet state.[1] In amagnetoreceptor, the interconversion between the two spin multiplicities,S,T, is governed by the strength of the external magnetic field and furthermore sensitively depends on the couplings of the magnetic dipole moments associated with the electron spins to those of close-by nuclear spins,a nd on their mutual spinspin interaction, which has contributions from magnetic dipolar coupling and electronic exchange.Whereas the magnetic parameters of the individual trapped radicals of FADa nd Trph ave been characterized rather extensively in terms of their hyperfine interactions and g-matrices [9] by electron paramagnet...

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We investigated the lumazine protein from Photobacterium leiognathi in complex with its biologically active cofactor, 6,7-dimethyl-8-ribityllumazine, at different redox states and compared the results with samples containing a riboflavin cofactor. Using anaerobic photoreduction, we were able to record optical absorption kinetics from both cofactors in similar protein environments. It could be demonstrated that the protein is able to stabilize a neutral ribolumazine radical with ∼35% yield. The ribolumazine radical state was further investigated by W-band continuous-wave EPR and X-band pulsed ENDOR spectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings (hfcs) could be obtained. Remarkably, the g-tensor's principal components are similar to those of the respective riboflavin-containing protein; however, the proton hfcs show noticeable differences. Comparing time-resolved optical absorption and fluorescence data from ribolumazine-containing samples, solely fluorescence but no signs of any intermediate radical or a triplet state could be identified. This is in contrast to lumazine protein samples containing the riboflavin cofactor, for which a high yield of the photogenerated triplet state and some excited flavin radical could be detected using time-resolved spectroscopy. These results clearly demonstrate that ribolumazine is a redox-active molecule and could, in principle, be employed as a cofactor in other enzymatic reactions.

The chlorophyll precursor protochlorophyllide (Pchlide), which is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase, has unique excited-state properties that facilitate photocatalysis. Previous time-resolved spectroscopy measurements have implied that a long-lived triplet state is formed during the excited-state relaxation of Pchlide, although direct evidence of its existence is still lacking. Here we use time-resolved electron paramagnetic resonance (EPR) in combination with time-resolved absorption measurements at a range of temperatures (10-290 K), solvents, and oxygen concentrations to provide a detailed characterization of the triplet state of Pchlide. The triplet decays in a biphasic, oxygen-dependent manner, while the first reported EPR signature of a Pchlide triplet displays both emissive and absorptive features and an antisymmetric spectrum similar to other porphyrin triplet states. This work demonstrates that the Pchlide triplet is accessible to various cryogenic spectroscopic probes over a range of time scales and paves the way for understanding its potential role in catalysis.