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

Abstract

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

Novel Pulsed Electron Paramagnetic Resonance Techniques for the Studies of Structure and Dynamics of Photo‐excited Triplet State of Organic Molecules: A Professional Journey

“…The results of these spin dynamic studies allow the assessment of the nature of crossing of spin states under the influence of a fast switching magnetic field. We have further applied the results observed at LAC to other studies, such as nuclear quantum coherence and quantum computing, to be discussed in later sections 16a,49 . Recently, a similar FFS technique was applied to perform NMR experiments in the earth's magnetic field and in rapid switching magnetic field to examine the magnetic field effects on radical pair reactions …”

“…Detailed analysis reveals that pulsed light excitation also initiates oscillatory nuclear spin polarization. This opens new perspectives for the analysis of chemically induced dynamic nuclear polarization (CIDNP) in mechanistic studies of photoactive proteins …”

Sensitivity Enhancement in Solution NMR via Photochemically Induced Dynamic Nuclear Polarization

“…The enhancement factors are quite large for these systems where the RC showed an 10,000-fold NMR sensitivity enhancement 38 ( Fig. 4A), whereas the LOV2-C450A showed up to a 6,000-fold enhancement 39 (Fig. 4B).…”

Controlling Cells with Light and LOV

“…Mutation of the PAS core cysteine that forms a covalent bond with the flavin chromophore upon photoexcitation (C450 in the As LOV2) has been shown to strongly enhance the fluorescent property of various LOV domains, while impairing their natural photocycle . In context of As LOV2‐based optogenetic tools, constructs bearing a C450 to alanine or methionine mutation are therefore often used as negative control (pseudodark mutant), as moderate light intensities and short illumination times are usually unable to activate them.…”

“…In context of As LOV2‐based optogenetic tools, constructs bearing a C450 to alanine or methionine mutation are therefore often used as negative control (pseudodark mutant), as moderate light intensities and short illumination times are usually unable to activate them. It is important to note, however, that even in an As LOV2 C450 mutant, the FMN chromophore can still be excited toward the characteristic triplet state, an intermediate in the natural LOV2 photocycle . Strikingly, the As LOV2 C450M pseudodark mutant even turns into an irreversible lit state‐like conformation upon long‐term irradiation (several hours) due to formation of a stable covalent bond between the methionine thiol group and the chromophore.…”