Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism.
By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 +/- 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 +/- 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13 = C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode to 1618 cm-1 in D2O indicates that the Schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C = N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)
Fodor
S P A
SPA
Department of Chemistry, University of California, Berkeley 94720.
Ames
J B
JB
Gebhard
R
R
van den Berg
E M M
EMM
Stoeckenius
W
W
Lugtenburg
J
J
Mathies
R A
RA
D001436:Bacteriorhodopsins; D008956:Models, Chemical; D010777:Photochemistry; D011522:Protons; D012545:Schiff Bases; D013059:Spectrum Analysis, Raman
D011522:Protons; D012545:Schiff Bases; D001436:Bacteriorhodopsins