Resonance Raman spectra of carbon-13- and nitrogen-15-labeled riboflavin bound to egg-white flavoprotein

Abstract

The resonance Raman spectra of [2-13C]-, [4a-13C]-, [4-13C]-, [10a-13C]-, [2,4,4a,10a-13C]-, [5-15N]-, [1,3-15N]-, and [1,3,5-15N]riboflavin bound to egg-white proteins were observed for N(3)-H and N(3)-D forms with spontaneous Raman technique by using the 488.0-nm excitation line of an argon ion laser. The fluorescence of riboflavin was quenched by forming a complex with egg-white riboflavin binding protein. The in-plane displacements of the C(2), C(4a), N(1), N(3), and N(5) atoms during each Raman active vibration were calculated from the observed isotopic frequency shifts. The 1252-cm-1 mode of the N(3)-H form involved large vibrational displacements of the C(2) and N(3) atoms and was strongly coupled with the N(3)-H bending mode. This line can be used as an indicator for state of N(3)-H.cntdot..cntdot..cntdot.protein interaction. The 1584-cm-1 mode, which is known to be resonance-enhanced upon excitation near the 370-nm absorption band, was accompanied by the displacement of the N(5) atom in particular. The 1355-cm-1 mode was most strongly resonance-enhanced by the 450-nm absorption band and involved the displacements of all C atoms of ring III. Both lines can be used as structure probes for elucidating the structure of electronically excited states of isoalloxazine.