Photochemically induced dynamic nuclear polarization study on FAD and flavoproteins

Flavin adenine dinucleotide and some flavoproteins ranging in relative molecular mass from 15000 to 100000 were investigated by the photochemically induced dynamic nuclear polarization (photo-CIDNP) technique. At neutral pH values,where FAD forms a strong intramolecular complex, no photo-CIDNPsignal is observed. Lowering the pH values of an aqueous solution of FAD leads to the gradual appearance of photo-CIDNP signals. The pH dependence of the intensity of the signal follows that of the fluorescence quantum yield of FAD, indicating that in both types of experiments the same mechanism is operating. This is an example of where a photochemically active constituent of a complex is used to induce polarization in its partner. The flavoproteins investigateddid not exhibit a photo-CIDNP signal in the absence of external free flavin so that information on aromatic amino acid residues located in the neighborhood of the bound prosthetic group could not be obtained. If intermediate bi radical complexes are formed in these cases, they are not able to dissociate which is mandatory for the observation of CIDNP signals. Addition of free aromatic amino acids to solutions of flavoproteins yields the corresponding CIDNP signals ofthe amino acids upon excitation of protein-bound flavins. Addition of free flavins to solutions of flavoproteins leads to the observation of CIDNP signals of aromatic amino acid residues. Various flavodoxins and other flavoproteins wereinvestigated by using as an external dye free flavins carrying different charges. In general positively and negatively charged free flavins give rise to CIDNP signals originating from different aromatic amino acid residues. It is shown that the technique allows assignments of proton resonances of aromatic amino acid residues where the sequence and preferably the three-dimensional structure of flavoproteins are known. This is demonstrated with a few flavodoxins from various sources.