A Study of Flavin-Protein and Flavoprotein-Ligand Interactions

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Abstract
To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein (RBP), D-amino acid oxidase (D-AO) and its complexes with ligands were observed in the spectral region of 310–600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 × 10−8 M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is suggested that the magnitude of the ellipticity in MCD spectra around 370 nm reflects the interaction of isoalloxazine with the aromatic ring instead of with the ribityl group. The CD spectra of these complexes displayed wider variation. This showed that the pattern of the CD spectra more sensitively reflected the environment around isoalloxazine than did that of the MCD spectra. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein ligand interaction. Comparison of the absorption, CD and MCD spectra (310–600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.