Henlea earthworm bioluminescence comprises violet-blue BRET from tryptophan 2-carboxylate to deazaflavin cofactor

We recently identified the deazaflavin cofactor as a light emitter in novel bioluminescence (BL) system from Siberian earthworms Henlea sp. (Petushkov et al., 2023, Org. Biomol. Chem. 21:415-427). In the present communication we compared in vitro BL spectra in the absence and in the presence of the cofactor and found a wavelength shift from 420 to 476 nm. This violet-blue BRET to deazaflavin cofactor (acceptor of photonless transfer) masks the actual oxyluciferin as an emitter (BRET donor) in the novel BL system. The best candidate for that masked chromophore is tryptophan 2-carboxylate (T2C) found previously as a building block in some natural products isolated from Henlea sp. (Dubinnyi et al., 2020, ChemSelect 5:13155-13159). We synthesized T2C and acetyl-T2C, verified their presence in earthworms by nanoflow-HRMS, explored spectral properties of excitation and emission spectra and found a chain of excitation/emission maxima with a perfect potential for BRET: 300 nm (excitation of T2C) - 420 nm (emission of T2C) - 420 nm (excitation of deazaflavin) - 476 nm (emission of deazaflavin, BL). An array of natural products with T2C chromophore are present in BL earthworms as candidates for novel oxyluciferin. We demonstrated for the Henlea BL that the energy of the excited state of the T2C chromophore is transferred by the Förster mechanism and then emitted by deazaflavin (BRET), similarly to known examples: aequorin-GFP in Aequorea victoria and antenna proteins in bacterial BL systems (lumazine from Photobacterium and yellow fluorescent protein from Vibrio fischeri strain Y1).