Indication for a radical intermediate preceding the signaling state in the LOV domain photocycle

The blue light photoreceptor phototropin mediates crucial processes in plants leading to optimization of photosynthesis. Phototropin comprises two flavin mononucleotide-binding LOV (light-, oxygen-, or voltage-sensitive) domains. The LOV domains undergo a photocycle upon illumination, in which two intermediates have been detected by UV/Vis spectroscopy. The triplet excited state of flavin is formed and decays within a few microseconds into a photoadduct with an adjacent cysteine, which represents the signaling state of the LOV domain. For bond formation of the photoadduct, several reaction pathways have been proposed, but evidence for an intermediate at ambient conditions has not been found. Here, we performed nanosecond time-resolved UV/Vis spectroscopy on the phototropin-LOV1 domain from Chlamydomonas reinhardtii. We designed a flow cell which was used to efficiently replace the sample after each photoexcitation because the cycling time is in the order of hundreds of seconds. The comparison of difference spectra of the wild type with those of the C57S mutant that produces only the triplet excited state revealed the existence of an additional intermediate between the triplet and the adduct state. This intermediate exhibits spectral properties similar to a neutral flavin radical. This finding supports a reaction mechanism involving a neutral radical pair.