Determination of retinal Schiff base configuration in bacteriorhodopsin

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Abstract
Resonance Raman spectra of the BR 568 , BR 548 , K 625 , and L 550 intermediates of the bacteriorhodopsin photocycle have been obtained in 1 H 2 O and 2 H 2 O by using native purple membrane as well as purple membrane regenerated with 14,15- 13 C 2 and 12,14- 2 H 2 isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C 14 —C 15 stretch to the normal modes in the 1100- to 1400-cm -1 fingerprint region and to characterize the coupling of the C 14 —C 15 stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C[unk]N cis configuration the C 14 —C 15 stretch and the NH rock are strongly coupled, resulting in a large (≈50-cm -1 ) upshift of the C 14 —C 15 stretch upon deuteration of the Schiff base nitrogen. In the C[unk]N trans geometry these vibrations are weakly coupled and only a slight (-1 ) upshift of the C 14 —C 15 stretch is predicted upon N -deuteration. In BR 568 , the insensitivity of the 1201-cm -1 C 14 —C 15 stretch to N -deuteration demonstrates that its retinal C[unk]N configuration is trans . The C 14 —C 15 stretch in BR 548 , however, shifts up from 1167 cm -1 in 1 H 2 O to 1208 cm -1 in 2 H 2 O, indicating that BR 548 contains a C[unk]N cis chromophore. Thus, the conversion of BR 568 to BR 548 (dark adaptation) involves isomerization about the C[unk]N bond in addition to isomerization about the C 13 [unk]C 14 bond. The insensitivity of the native, [14,15- 13 C 2 ]-, and [12,14- 2 H 2 ]K 625 and L 550 spectra to N -deuteration argues that these intermediates have a C[unk]N trans configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR 568 → K 625 ) involves isomerization about the C 13 [unk]C 14 bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.