Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle

Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has been identified as a candidate magnetoreceptive molecule due to its expression in the retina and its ability to form stable flavin neutral radicals (FADH^●) upon blue light absorption. Herein, we used millisecond flash photolysis to investigate the cCRY4 photocycle, in both the presence and absence of dithiothreitol (DTT); detecting the anion radical form of FAD (FAD^●-) under both conditions. Using spectral data obtained during flash photolysis and UV-visible photospectroscopy, we estimated the absolute absorbance spectra of the photointermediates, thus allowing us to decompose each spectrum into its individual components. Notably, in the absence of DTT, approximately 37% and 63% of FAD^●- was oxidized to FAD_OX and protonated to form FADH^●, respectively. Singular value decomposition analysis suggested the presence of two FAD^●- molecular species, each of which was destined to be oxidized to FAD_OX or protonated to FADH^●. A tyrosine neutral radical was also detected; however, it likely decayed concomitantly with the oxidation of FAD^●-. On the basis of these results, we considered the occurrence of bifurcation prior to FAD^●- generation, or during FAD^●- oxidization, and discussed the potential role played by the tyrosine radical in the radical pair mechanism.