Role of Aspartate-96 in Proton Translocation by Bacteriorhodopsin

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 13; pp. 4943 - 4947
• Main Authors: Gerwert, Klaus, Hess, Benno, Soppa, Jörg, Oesterhelt, Dieter
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.07.1989; National Acad Sciences
• Subjects: Absorption spectra; Analytical, structural and metabolic biochemistry; Asparagine; Aspartic Acid; bacteriorhodopsin; Bacteriorhodopsins; Bacteriorhodopsins - metabolism; Biological and medical sciences; Carboxylates; Fourier Analysis; Fundamental and applied biological sciences. Psychology; Halobacterium - metabolism; Miscellaneous; Mutant proteins; Proteins; Protons; purple membranes; Spectral bands; Spectral index; Spectral methods; Spectrophotometry, Infrared; Vibration; Vibrational spectra; Bacteriorhodopsin; Proteins; Infrared spectrometry; Fourier transformation; Membrane pump; Archaeobacteria; Point mutation; Proton transfer; Reaction mechanism; Bacteria; Halobacteriaceae; Halobacterium halobium
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.13.4943

Proton transfer reactions in bacteriorhodopsin were investigated by Fourier transform infrared spectroscopy, using a mutant protein in which Asp-96 was replaced by Asn-96. By comparison of the BR - K, BR - L, and BR - M difference spectra (BR indicating bacteriorhodopsin ground state and K, L, and M indicating photo-intermediates) of the wild-type protein with the corresponding difference spectra of the mutant protein, detailed insight into the functional role of this residue in the proton pump mechanism is obtained. Asp-96 is protonated in BR, as well as another aspartic residue, which is tentatively assigned to be Asp-115. Asp-96 is not affected in the primary photoreaction. During formation of the L intermediate it is subjected to a change in the H-bonding character of its carboxylic group, but no deprotonation occurs at this reaction step. Also, in the mutant protein a light-induced structural change of the protein interior near the Asn-96 residue is probed. The BR - M difference spectrum of the mutant protein lacks the negative carbonyl band at 1742 cm-1of Asp-96 and in addition a positive band at about 1378 cm-1, which is most likely to be caused by the carboxylate vibration of Asp-96. This argues for a deprotonation of Asp-96 in the time range of the M intermediate during its photostationary accumulation. On the basis of these results, it is suggested that the point mutation does not induce a gross change of the protein structure, but a proton-binding site in the proton pathway from the cytoplasmic side to the Schiff base is lost.