Detecting a New Source for Photochemically Induced Dynamic Nuclear Polarization in the LOV2 Domain of Phototropin by Magnetic-Field Dependent 13C NMR Spectroscopy

Phototropin is a flavin mononucleotide (FMN) containing blue-light receptor, which regulates, governed by its two LOV domains, the phototropic response of higher plants. Upon photoexcitation, the FMN cofactor triplet state, 3F, reacts with a nearby cysteine to form a covalent adduct. Cysteine-to-alanine mutants of LOV domains instead generate a flavin radical upon illumination. Here, we explore the formation of photochemically induced dynamic nuclear polarization (CIDNP) in LOV2-C450A of Avena sativa phototropin and demonstrate that photo-CIDNP observed in solution 13C NMR spectra can reliably be interpreted in terms of solid-state mechanisms including a novel triplet mechanism. To minimize cross-polarization, which transfers light-induced magnetization to adjacent 13C nuclei, our experiments were performed on proteins reconstituted with specifically 13C-labeled flavins. Two potential sources for photo-CIDNP can be identified: The photogenerated triplet state, 3F, and the triplet radical pair 3(F–•W+•), f...