ASSIGNMENT AND THE EFFECT OF HYDROGEN-BONDING ON THE VIBRATIONAL NORMAL-MODES OF FLAVINS AND FLAVOPROTEINS

For a number of years we have studied the resonance Raman spectra of flavins and flavoproteins; these studies included investigation of the solvent effect on the 13 resonance Raman (RR) bands between 1700 and 1100 cm-1. These investigations led us to the conclusion that two bands, II and X, in the Raman spectrum of flavins are strongly influenced by the degree of hydrogen bonding. Other investigators have made similar suggestions based on shifts in these two bands and in others. We report the QCFF-π calculations of 87 normal modes of lumiflavin (7,8,10-trimethylisoalloxazine). We have also calculated resonance Raman intensities and isotope shifts of each resonance Raman active mode. The generally good agreement between calculated and observed values supports our assignment of the experimental bands. The QCFF-π assignments are in general agreement with earlier calculations leading to the conclusion that the investigators in the field are reaching a consensus on the assignment of flavin RR bands in the 1700-1100-cm-1 region. We have calculated the effect of hydrogen bonding at N1, C2=O, N3-H, C4=O, and N5 upon the resonance Raman spectrum, and these calculations indicate that the upward shift in frequency of band X is one of the two largest and that band II has one of the two largest downward shifts in frequency. Both of these predictions are in keeping with our previous observations on model systems; however, predictions of the frequency shift in band VI do not agree with our observations. This does not surprise us since the QCFF-π assignments of this band and band III appear to us to be incorrect. Our calculations show that shifts in band II reflect hydrogen bonding at N1, C4=O, and N3-H and that shifts in band X reflect hydrogen bonding at C2=O and N3-H. These specifics of spectral interpretation have allowed us to compare structural conclusions concerning hydrogen bonding reached on the basis of X-ray crystallography, NMR spectroscopy, and RR results. The structural conclusions are similar for flavodoxin, glutathione reductase, and fatty acyl-CoA dehydrogenase. However, the RR results lead to much different conclusions for the hydrogen-bonding structure of riboflavin binding protein. © 1990 American Chemical Society.