A photo-induced radical pair of FADH· and Y8· and in BLUF protein SyPixD was studied by pulsed electron paramagnetic resonance (EPR) spectroscopy. Blue light illumination at 150 K for 30 min followed by cooling to 50 K during illumination induced the stable radical pair. The EPR signal has been characterized by a Pake doublet signal with complete S = 1 spin state. The radical pair was utilized as a probe to analyze the oligomer of SyPixD. The relative arrangement of PixD proteins in the complex was investigated by pulsed electron-electron double resonance (PELDOR) with the orientation selection. Based on the decameric structure in the crystal, the possible structure for the PELDOR results was discussed.
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