Abstract
The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-equilibrium nuclear spin polarization in frozen electron-transfer proteins upon illumination and radical-pair formation. The effect can be observed in various natural photosynthetic reaction center proteins using magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, and in a flavin-binding light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin. In the latter system, a functionally instrumental cysteine has been mutated to interrupt the natural cysteine-involving photochemistry allowing for an electron transfer from a more distant tryptophan to the excited flavin mononucleotide chromophore. We explored the solid-state photo-CIDNP effect and its mechanisms in phototropin-LOV1-C57S from the green alga Chlamydomonas reinhardtii by using field-cycling solution NMR. We observed the 13C and, to our knowledge, for the first time, 15N photo-CIDNP signals from phototropin-LOV1-C57S. Additionally, the 1H photo-CIDNP signals of residual water in the deuterated buffer of the protein were detected. The relative strengths of the photo-CIDNP effect from the three types of nuclei, 1H, 13C and 15N were measured in dependence of the magnetic field, showing their maximum polarizations at different magnetic fields. Theoretical level crossing analysis demonstrates that anisotropic mechanisms play the dominant role at high magnetic fields.
Highlights
The solid-state photo-chemically induced dynamic nuclear polarization effect generates non-equilibrium nuclear spin polarization in frozen electron-transfer proteins upon illumination and radical-pair formation
The solid-state photo-Chemically induced dynamic nuclear polarization (CIDNP) effect[7,19,29,30,31,32] can enhance nuclear magnetic resonance (NMR) signals by a factor of 100,000, allowing for a direct observation of the photo-chemical machineries of reaction centers (RCs) in membrane preparations[33], in whole cells[7,16] and even in entire plants[18]. It can be used as a sensitive analytical tool to map electronic structures of photo-active cofactors[34], given that the photo-CIDNP signal intensities are related to the local electron-spin density and the hyperfine coupling (HFC)[35]
The results reveal that 1H, 13C, and 15N photo-CIDNP enhancement curves have different magnetic field effect (MFE) maxima, Bmax, (Fig. 5) showing this trend: Bmax(1H) < Bmax(13C) < Bmax(15N), inversely proportional to their gyromagnetic ratio
Summary
The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-equilibrium nuclear spin polarization in frozen electron-transfer proteins upon illumination and radical-pair formation. The solid-state photo-CIDNP effect[7,19,29,30,31,32] can enhance NMR signals by a factor of 100,000, allowing for a direct observation of the photo-chemical machineries of RCs in membrane preparations[33], in whole cells[7,16] and even in entire plants[18] It can be used as a sensitive analytical tool to map electronic structures of photo-active cofactors[34], given that the photo-CIDNP signal intensities are related to the local electron-spin density and the hyperfine coupling (HFC)[35]. The occurrence of the solid-state photo-CIDNP effect in liquid membrane samples demonstrates that sufficient orientation allows for its induction[33]
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