Determination of retinal Schiff base configuration in bacteriorhodopsin.
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 ( approximately 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 (<5-cm(-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.
Smith
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SO
Department of Chemistry, University of California, Berkeley, CA 94720.
Myers
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Pardoen
J A
JA
Winkel
C
C
Mulder
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PP
Lugtenburg
J
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Mathies
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