Riboflavin Bound to Egg-White Flavoproteint

The resonance Raman spectra of [2-13C]-, [4a-I3C] -, [4-I3C]-, [ 1 Oa-I3C]-, [2,4,4a, 10a-I3C]-, [ 5-I5N] -, [ 1,3-I5N]-, and [ 1,3,5-'5N]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-' mode of the N(3) -H form was found to involve large vibrational displacements of the C(2) and N(3) % elucidate the mechanism of flavoprotein catalysis, physicochemical properties of flavins and their changes upon complex formation with the apoenzyme or upon complex formation of the holoenzyme with substrate should be investigated. Details of the physicochemical nature of flavins have been studied by absorption, fluorescence, and N M R spectroscopies. However, the resonance Raman spectra of flavins have hardly been studied because of strong fluorescence. Only recently, the resonance Raman spectra of flavoproteins have become observable with coherent anti-Stokes Raman scattering (CARS) ' (Dutta et al., 1977, 1978) or the fluorescence-quenched spontaneous Raman scattering technique (Nishina et al., 1978). In resonance Raman scattering from flavoproteins, the vibrational spectra of the isoalloxazine can be selectively observed without the interference of those of the surrounding amino acid residues of apoprotein. Due to sensitive dependence of the vibrational frequencies upon molecular structure, Raman spectroscopy is potentially a powerful tool for elucidation of the interactions mentioned above. For successful application, however, correct assignments of the resonance Raman lines have to be established on sound experimental basis. Qualitative assignments of the resonance Raman lines were previously carried out by us on the basis of the observed frequency shifts caused by the replacement of the peripheral groups of the isoalloxazine; some Raman lines were assigned to each of three rings (Nishina et al., 1978). To obtain more detailed assignments, observation of the isotopic frequency shifts for ring atoms and subsequent normal coordinate calculations are indispensable. Therefore, we synthesized I3Cor I5N-labeled riboflavin. Although free riboflavin (RF) is too fluorescent to be subjected to conventional Raman spectroscopy, the fluorescence is completely quenched when R F is bound to egg-white riboflavin binding protein (apo-RBP) atoms and to be strongly coupled with the N(3) -H bending mode. This line can be used as an indicator for state of N(3)-H--protein interaction. The 1584-cm-' 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-' mode was most strongly resonance-enhanced by the 450-nm absorption band and involved the displacements of all carbon atoms of ring 111. Both lines can be used as structure probes for elucidating the structure of electronically excited states of isoalloxazine. (Nishikimi & Kyogoku, 1973). Accordingly, in the present study, we observed the resonance Raman spectra of the isotope-labeled R F in the bound state to apo-RBP, and the vibrational displacement of the C(2), C(4a), N ( l ) , N(3), and N(5) atoms for each of the resonance Raman active modes are reported. Materials and Methods Synthesis of [2-I3C]-, [4a-13C]-, [4-I3C]-, [ 10a-I3C]-, [2,4,4a,10a-'3C]-, [5-I5N]-, [ 1,3-I5N], and [1,3,5-I5N]RF was carried out as reported previously (Yagi et al., 1976a,b), and the more than 98% isotopic substitutions were confirmed by 13C and I5N nuclear magnetic resonance. [4-I3C]RF and [ 10a-I3C]RF could not be chemically separated, and their 1:l mixture was used. Egg-white apo-RBP was prepared according to Rhodes et al. (1959) and purified as described previously (Nishina et al., 1977). R F and apo-RBP were dissolved in 0.1 M sodium phosphate buffer a t p H 7.0 (or pD 7.0 for the D 2 0 solution). Concentrations of R F and apo-RBP were 6.0 X M for the H 2 0 solution and 8.0 X and 2.2 X M for the D 2 0 solution; their concentrations were determined spectrophotometrically with the molar extinction coefficients of 1.25 X lo4 M-' cm-' a t 445 nm for RF (Whitby, 1953) and 4.9 X lo4 M-' cm-' a t 282 nm for apo-RBP (Nishikimi & Kyogoku, 1973). Raman scattering was excited at 488.0 nm by an argon ion laser (Spectra Physics, Model 164) and recorded on a JEOL-400D Raman spectrometer equipped with an HTVR649 photomultiplier. The frequency calibration of the Raman spectrometer was performed with indene (Hendra & Loader, 1968). Through the measurements of the Raman spectra, 300 WL of sample solution was placed in a thermostated longitudinal cell kept a t 15 f 2 OC, and excitation light with sufficiently low power (60 mW) was irradiated from the bottom of the cell. and 2.3 X From the Institute for Protein Research, Osaka University, Suita, Osaka, 565, Japan (T.K. and Y.K.), the Department of Biochemistry, Wakayama Medical College, Wakayama, 640, Japan (Y.N.), the Department of Biochemistry, Medical School, Osaka University, Osaka, 530, Japan (T.Y.), and the Institute of Biochemistry, Faculty of Medicine, University of Nagoya, 466, Japan (N.O., A.T.-S., and K.Y.). Receioed December I . 1978. 0006-2960/79/0418-1804$01 .OO/O Results The resonance Raman spectra of nonlabeled, [2-I3C]-, [4a-I3C]-, [5-15N]-, and [1,3-'5N]RF bound to RBP are shown in Figures 1 and 2, where the 1 : l mixture of [4-13C]and [ 1Oa-l3C]RF is also included. Atomic and ring numberings