The interaction of flavins with egg white riboflavin-binding protein

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The properties of the riboflavin-binding site in the riboflavin-binding protein from egg white have been elucidated by determining constants for binding of flavin analogs to the protein and by changes in absorption spectra of free and bound flavins. The spectral changes and unfavorable interaction of the protein with charged species indicate that the overall flavin environment in the holoprotein is hydrophobic. Modification of either ring or side-chain portions of flavin usually results in a decrease of binding energy. Although no one portion of the structure is absolutely essential, both 7- and 8-methyl groups and 2′-hydroxyl group contribute most significantly to binding. The binding site at the region of C-2 and N-3 of the isoalloxazine is rather insensitive to the relative site of a substituent and thus relatively open, whereas considerable steric limitation is imposed at C-8, N-10, especially C-1′, and 4carbonyl positions. The hydroxyl groups of the N-10 side chain contribute in a stereoselective manner by formation of hydrogen bonds. Studies with model compounds that represent only a part of flavin suggest that the dimethylbenzenoid portion of the ring is involved in primary interactions of binding, and relatively buried in the protein. The quenching of protein fluorescence upon binding is mainly due to ground-state stacking interaction between a trytophanyl residue at the binding site and the quinoxaline portion, and not to Förster energy transfer.