Characterization of a flavin radical product in a C57M mutant of a LOV1 domain by electron paramagnetic resonance

In the flavin mononucletide-binding LOV1 domain of the Phot1-receptor from Chlamydomonas reinhardtii the photoreactive cysteine C57 has been replaced by methionine. Photoexcitation of this C57M mutant yields a metastable photoproduct (C57M-415) that thermally decomposes into a stable paramagnetic species (C57M-675) with extremely red-shifted absorption in the visible range. In this contribution, we describe the characterization of this radical by multi-frequency electron paramagnetic resonance and electron-nuclear double resonance. The main features of the spectra identify the paramagnetic species as a flavin neutral radical. However, detailed analysis shows that the isoalloxazine moiety of the flavin is alkyl substituted at N(5), rather than protonated as is usually the case. The implication of these observations on the likely mechanism of photoproduct generation in wild-type LOV domains is discussed.