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

Abstract

Blue-light excitation of cryptochromes and homologs 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, e.g., 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.

Cryptochrome in Sponges

“…Parallel to this process, the membrane-bound cryptochrome is associated with NOSIP, suggesting that the cryptochrome pathway is coupled to the NO synthase signaling cascade. As initially described by us ) and subsequently confirmed (Rivera et al 2012), light response of the sponge cryptochrome results in a photoreduction, the photoresponsiveness of the flavoprotein, under formation of FADH 2 (reduced flavin adenine dinucleotide) from flavin adenine dinucleotide (FAD (Weber et al 2010;Müller and Ahmad 2011). Essentially, this reaction also takes place in the modular eukaryotic NOS isoforms that are composed of (1) the C-terminal reductase domain that binds reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), and FAD and (2) the N-terminal oxygenase domain (see Förstermann and Münzel 2006).…”

Determination of Radical–Radical Distances in Light‐Active Proteins and Their Implication for Biological Magnetoreception

“…[12d, 15] Herein, we present data from pulsed out-of-phase electron-spin-echo envelope modulation (OOP-ESEEM) EPR [16] spectroscopic studies that were performed to directly measure the strength of the exchange and dipolar interactions in the short-lived RP states of the Drosophila melanogaster cryptochrome (DmCry) and Escherichia coli DNAp hotolyase (EcPL), for which ET along the conserved Tr pt riad has been extensively studied by transient optical absorption spectroscopy. [11b,17] OOP-ESEEM has previously been applied to examine spin-correlated RPs in photosynthetic reaction centers, [18] donor-acceptor systems for artificial photosynthesis, [19] and, more recently,c omposite materials designed for organic photovoltaic devices.…”

Variable Electron Transfer Pathways in an Amphibian Cryptochrome

“…To test this potential second ET pathway, we performed TREPR spectral simulations based on the correlated coupled RP mechanism (63)(64)(65), assuming that the RP in XCry is generated in a spin-correlated fashion from a singlet state precursor (10,66). In a first approach, all parameters affecting the TREPR spectra (g-tensors, dipolar and exchange spin-spin couplings, hyperfine-governed and residual line width parameters) were taken from simulations of the WT TREPR data, except for the g-tensor components of the amino acid radical, where principal values for Tyr rather than Trp were used (supplemental Fig.…”

“…Therefore, the detected TREPR signal is assigned to the final RP state, which is initially also singlet configured. Recently, TREPR measurements of the RP formed in WT XCry performed at two microwave frequencies (X-band, 9.7 GHz, and Q-band, 34 GHz) together with spectral simulations have been reported, presenting clear evidence for the FAD ⅐ ⅐⅐⅐Trp-324 ⅐ RP originating from a pure singlet state precursor (66). In light of the obvious similarities among the TREPR signals of WT XCry and W400F and W377F mutants in terms of their overall polarization pattern, this assumption seems to be justified as well for simulating the TREPR spectra of these two mutant proteins.…”