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 FADOX 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 FADOX 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.
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