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

• Published in: The journal of physical chemistry. B Vol. 118; no. 40; pp. 11622 - 11632
• Main Authors: Kothe, Gerd, Lukaschek, Michail, Link, Gerhard, Kacprzak, Sylwia, Illarionov, Boris, Fischer, Markus, Eisenreich, Wolfgang, Bacher, Adelbert, Weber, Stefan
• Format: Journal Article
• Language: English
• Published: 01-01-2014
• Subjects: Dynamics; Magnetic fields; Magnetization; Photoexcitation; Polarization; Proteins; Radicals; Tryptophan
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/jp507134y

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, super(3)F, 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 super(13)C 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 super(13)C nuclei, our experiments were performed on proteins reconstituted with specifically super(13)C-labeled flavins. Two potential sources for photo-CIDNP can be identified: The photogenerated triplet state, super(3)F, and the triplet radical pair super(3)(F super(- times )W super(+ times )), formed by electron abstraction of super(3)F 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 super(3)(F super(- times )W super(+ times )) and super(3)F contribute to photo-CIDNP, whereas at high magnetic fields, the calculated enhancement factors of super(3)F 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.