Kothe, G., et al., Detecting a New Source for Photochemically Induced Dynamic Nuclear Polarization in the LOV2 Domain of Phototropin by Magnetic-Field Dependent 13C NMR Spectroscopy. The Journal of Physical Chemistry B, 2014. 118(40): p. 11622-11632.
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+?), formed by electron abstraction of 3F from tryptophan W491. To separate the two contributions, photo-CIDNP studies were performed at four different magnetic fields ranging from 4.7 to 11.8 T. Analysis revealed that, at fields <9 T, both 3(F??W+?) and 3F contribute to photo-CIDNP, whereas at high magnetic fields, the calculated enhancement factors of 3F agree favorably with their experimental counterparts. Thus, we have for the first time detected that a triplet state is the major source for photo-CIDNP in a photoactive protein. Since triplet states are frequently encountered upon photoexcitation of flavoproteins, the novel triplet mechanism opens up new means of studying electronic structures of the active cofactors in these proteins at atomic resolution.