Resonance Raman spectroscopy of chemically modified and isotopically labelled purple membranes. I. A critical examination of the carbon-nitrogen vibrational modes.

Resonance Raman spectra of bacteriorhodopsin are compared to the spectra of this protein modified in the following ways: (1) selective deuteration at the C-15 carbon atom of retinal, (2) full deuteration of the retinal, (3) the addition of a conjugated double bond in the beta-ionone ring (3-dehydroretinal), (4) full deuteration of the protein and lipid components, (5) 15N enrichment of the entire membrane and (6) deuteration of the entire membrane (including the retinal). A detailed comparison of the 15N-enriched membrane and naturally occurring purple membrane from 800 cm-1 to 1700 cm-1 reveals that 15N enrichment affects the frequency of only two vibrational modes. These occur at 1642 cm-1 and 1620 cm-1 in naturally occurring purple membrane and at 1628 cm-1 and 1615 cm-1 in the 15N-enriched samples. Therefore, this pair of bands reflects the states of protonation of the Schiff base. However, our data also indicate that neither of these modes are simple, localized C=N-H or C=N stretching vibrations. In the case of the 1642 cm-1 band motions of the retinal chain beyond C-15 are not significantly involved. On the other hand, in the 1620 cm-1 band atomic motions in the isoprenoid chain beyond C-15 are involved.