Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

• Published in: Biochemistry (Easton) Vol. 27; no. 18; pp. 7097 - 7101
• Main Authors: Fodor, Stephen P. A, Ames, James B, Gebhard, Ronald, Van den Berg, Ellen M. M, Stoeckenius, Walther, Lugtenburg, Johan, Mathies, Richard A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 06.09.1988
• Subjects: Bacteriology; Bacteriorhodopsins; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Growth, nutrition, cell differenciation; Microbiology; Models, Chemical; Photochemistry; Protons; Schiff Bases; Space life sciences; Spectrum Analysis, Raman; Molecular structure; Archaeobacteria; Biological transport; Biomembrane; Photoprotein; Halobacterium halobium; In vitro; Bacteriorhodopsin; Proton; Bacteria; Raman spectrometry; Halobacteriaceae; Mechanism of action; Chromophore
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00418a064

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.