Photo-CIDNP of Solvent-Exposed Flavins in Flavoproteins

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is a hyperpolarization NMR technique that enhances the resonances of molecules involved in the formation of spin-correlated radical pairs. In this contribution, the method is used to selectively enhance the resonances of solvent-exposed, protein-bound flavins by adding an electron donor (tryptophan) to the sample prior to irradiation. To the best of our knowledge, this method has not been used in this way before. By applying photo-CIDNP to two different flavoproteins with solvent-exposed flavins, namely flavodoxin A from Escherichia coli and lumazine protein (LumP) from Photobacterium leiognathi in complex with riboflavin (riboflavin-LumP), we investigate the requirements for radical pair formation. The results reveal that only riboflavin-LumP shows an observable photo-CIDNP effect. Using continuous-wave photo-CIDNP on ¹H and ¹³C nuclei, flavin resonances can be selectively hyperpolarized. Signal assignment is possible by comparing hyperfine data from time-resolved photo-CIDNP and density functional theory (DFT). In addition, the anionic riboflavin radical is determined as the radical present in the geminate radical pair.