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.

Absorbance changes in the infrared and visible spectral range were measured in parallel during the photocycle oflight-adapted bacteriorhodopsin, which is accompanied by a vectorial proton transfer. A global fit analysis yielded the same rate constants for the chromophore reactions, for protonation changes of protein side groups, and for the backbone motion. From this result we conclude that all reactions in various parts of the protein are synchronized to each other and that no independent cycles exist for different parts. The carbonyl vibration of Asp-85, indicating its protonation, appears with the same rate constant as the Schiff base deprotonation. The carbonyl vibration of Asp-96 disappears, indicating most likely its deprotonation, with the same rate constant as for the Schiff base reprotonation. This result supports the proposed mechanism in which the protonated Schiff base, a deprotonated aspartic acid (Asp-85) on the proton-release pathway, and a protonated aspartic acid (Asp-96) on the proton-uptake pathway act as internal catalytic proton-binding sites.Recently, the structure of two membrane proteins, the photosynthetic reaction center and bacteriorhodopsin (bR), have been determined at near-atomic and molecular resolution, respectively (1, 2). In order to understand the structurefunction relationship on an atomic level, time-resolving methods have to be applied to yield insight into the intramolecular reactions. Here, the light-driven proton pump bR (3) is investigated by time-resolved Fourier-transform infrared (FTIR) difference spectroscopy (4, 5).The proton-transfer reactions of bR are initiated by a light-induced isomerization reaction of the chromophore retinal. This reaction is followed by protonation changes of the protonated Schiff base binding site between chromophore and protein and internal aspartic acids of the protein (6, 7). Based on these results, it has been proposed that the interplay between the protonated Schiff base and a deprotonated and a protonated internal aspartic acid describes the principal features of the pump mechanism (5, 6). By using mutant proteins in which internal aspartic acids were altered, these two residues were identified as Asp-85 and Asp-96 by static FTIR difference spectroscopy (8, 9). Substitution of these two residues results in defective proton pumping by the mutated proteins (10, 11).To determine simultaneously the light-induced kinetics in various parts of the protein, including the protonation changes of Asp-85 and Asp-96 relative to the Schiff base, we performed time-resolved FTIR experiments and, in parallel, measured absorbance changes in the visible spectral range. MATERIALS AND METHODSPurple membrane was isolated as described (12). Wet, highly concentrated purple membrane pellets in distilled water were squeezed between two CaF2 windows, separated by a 2.5-,um spacer in a homemade sample chamber. The final OD was -0.5 and the pH -6. No salt was added. IR spectra were recorded on a Bruker IFS 88 instrument with the modifications and addition...

Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 mus, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k(7) = 0.3 ms, k(4) = 2.3 ms, k(3) = 6.9 ms, k(6) = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k(4)), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M(1) to M(2) transition. However, experimentally two different M intermediates are not resolved, and M(2) and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M(2)) transition. This reaction is proposed to be involved in the "reset switch" of the pump, (b) In the M to N (conceptionally M(2)) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm(-1) and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O.

No abstract

No abstract