Assignment of NMR resonances of protons covalently bound to photochemically active cofactors in photosynthetic reaction centers by C-H photo-CIDNP MAS-J-HMQC experiment.

Modified versions of through-bond heteronuclear correlation (HETCOR) experiments are presented to take advantage of the light-induced hyperpolarization that occurs on C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. Such C-H photo-CIDNP MAS-J-HMQC and photo-CIDNP MAS-J-HSQC experiments are applied to acquire the 2D C-H correlation spectra of selectively C-labeled photochemically active cofactors in the frozen quinone-blocked photosynthetic reaction center (RC) of the purple bacterium Rhodobacter (R.) sphaeroides wild-type (WT). Resulting spectra contain no correlation peaks arising from the protein backbone, which greatly simplifies the assignment of aliphatic region. Based on the photo-CIDNP MAS-J-HMQC NMR experiment, we obtained assignment of selective H NMR resonances of the cofactors involved in the electron transfer process in the RC and compared them with values theoretically predicted by density functional theory (DFT) calculation as well as with the chemical shifts obtained from monomeric cofactors in the solution. We also compared proton chemical shifts obtained by photo-CIDNP MAS-J-HMQC experiment under continuous illumination with the ones obtained in dark by classical cross-polarization (CP) HETCOR. We expect that the proposed approach will become a method of choice for obtaining H chemical shift maps of the active cofactors in photosynthetic RCs and will aid the interpretation of heteronuclear spin-torch experiments.