A model-independent approach to assigning bacteriorhodopsin's intramolecular reactions to photocycle intermediates

• Published in: Biophysical journal Vol. 65; no. 5; pp. 1929 - 1941
• Main Authors: Hessling, B., Souvignier, G., Gerwert, K.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 01.11.1993; Biophysical Society
• Subjects: Analytical, structural and metabolic biochemistry; Bacteriorhodopsins - chemistry; Bacteriorhodopsins - radiation effects; Binding Sites; Biological and medical sciences; Biophysical Phenomena; Biophysics; Fundamental and applied biological sciences. Psychology; Models, Chemical; Non metallic chromoproteins, photoproteins; Photochemistry; Protein Conformation; Proteins; Proton Pumps - chemistry; Spectrophotometry; Spectrophotometry, Infrared; Bacteriorhodopsin; Cyclic photoreaction; Intramolecular reaction; Infrared spectrometry; Models; Binding site; Mutation
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(93)81264-5

By using factor analysis and decomposition, bacteriorhodopsin's intramolecular reactions have been assigned to photocycle intermediates. Independent of specific kinetic models, the pure BR-L, BR-M, BR-N, and BR-O difference spectra were calculated by analyzing simultaneously two different measurements in the visible and infrared spectral region performed at pH 6.5, 298 K, 1 M KCl, and pH 7.5, 288 K, 1 M KCl. Even though after M formation L, M, N, and O intermediates kinetically overlap under physiological conditions, their pure spectra have been separated by this analysis in contrast to other approaches at which unphysiological conditions or mutants have been used or specific photocycle models have been assumed. The results now provide a set reference spectra for further studies. The following conclusions for physiologically relevant reactions are drawn: (a) the catalytic proton release binding site, asp 85, is protonated in the L to M transition and remains protonated in the intermediates N and O; (b) the catalytic proton uptake binding site asp 96 is deprotonated in the M to N transition and already reprotonated in the N to O transition; (c) proton transfer between asp 96 and the Schiff base is facilitated by backbone movements of a few peptide carbonyl groups in the M to N transition.