Investigation of the Primary Photochemistry of Bacteriorhodopsin by Low‐Temperature Fourier‐Transform Infrared Spectroscopy

Abstract

The method of Fourier-transform infrared differencc spectroscopy was applied t o investigate the transition at 77 K of bacteriorhodopsin in its light-adapted form to K, the first intermediate which is stable at low tcmperature.In addition to unmodified bacteriorhodopsin, bacteriorhodopsin in ' H 2 0 and bacteriorhodopsin containing [ 15-2H]retinal was used. The results show that major rearrangements occur in the Schiff base in this transition. It is not possible to identify a C = N stretching vibration of the Schiff base in K,,,. The identification of an N-H bending vibration in K,,, shows that the nitrogen of the previous Schiff base still has a proton attached. The fingerprint region exhibits very unusual features for Kbl, and bears no similarity t o protonated retinylidene Schiff base model compounds of any isomeric composition. Therefore, no conclusions on the isomeric state of the retinal in KbI, can be drawn. The spectra show that the terminal part ofthe retinal is predominantly reflected in the difference spectra. This indicates that the most polar part ofthe retinal is located near the Schiff base. We have evidence for protein molecular changes occurring in this transition a t 77 K.Bacteriorhodopsin (BR), the only protein component of thc purple membrane of Hulohucteriunz hulohiurn, represents a light-driven proton pump, translocating protons across the cell's plasma membrane and thus transforming light into electrochemical energy [I J (and references cited therein). Alltruns retinal is the chromophore of the light-adapted functionally active form. It is linked to the protein via a Schiff base t o the e-amino group of a lysine. Resonance Raman experiments iiidicate that this Schiff base is protonated [2-41. Upon absorption of light the pigment undergoes a cyclic reaction involving at least three intermediates:where BRSh8 represents the light-adapted form; the subscript numbers refer t o the approximate absorption maxima (in nm) of the intermediates. K,,, is the first iiitcrmediate which can be stabilized at low temperature. A still earlier species has been detected by picosecond techniques a t liquid helium temperature [5]. Whether this represents the excited state of BR,,, or even a photoproduct is not yet clear. Nevertheless, K,,, deserves special interest since it is the first intermediate which can be reliably measured by spectroscopic techniques and still has stored enough light energy t o drive the subsequent thermal reactions and consequently has sufficient energy for the translocation of protons. K,,, appears within 11 ps a t room temperature but this is slowed down to 56 ps at 18 K, where the time of appearance becomes almost temperature-independent, suggesting that a tunnel process is involved [5]. Together with the kinetic isotope effect measured for the appearance of K, , , A short note on these measuremcnts was presented at a n informal discussion meeting on visual pigments and rclated topics, held in Monti-kal. October 5-9, 1981.Ahhrcvicrtion.s. BR, bacteriorhodopsin; FTIK speciroscopy, Fou...

Fourier‐transform infrared spectroscopy applied to rhodopsin

“…Without additional labelling techniques an assignment of these bands to specific vibrations is hardly possible. In the BR568 -K610 difference spectrum of bacteriorhodopsin we have identified a band at 1350 cm-I as the N-H in-plane bending vibration of the protonated Schiff base of BRS6, [16]. In the rhodopsin -bathorhodopsin difference spectrum a sharp band at 1391 cm-' and in the isorhodopsinbathorhodopsin difference spectrum a broader band at the same position is removed by 'H,O.…”

“…It is not easily conceivable how 'H,O influences the C=C stretching vibration. However, we recently reported an effect of 'H,O on the C=C stretching band of &lo, the first photoproduct of bacteriorhodopsin [16]. There, a splitting was induced.…”

“…It is, therefore, tempting to assign these bands also to the N-H bending vibration of rhodopsin and isorhodopsin. However, a definitive assignment requires a closer investigation of the HC=NH group with the method of isotopic labelling, as it was done in the case of bacteriorhosopsin [16,30] experiments in this line are in progress.…”

“…The rhodopsin -bathorhodopsin and rhodopsin -isorhodopsin difference spectra were measured at 77 K. For this a cryostat developed by us for low temperature infrared spectroscopy, allowing the illumination of the sample with visible light in situ, was inserted into the spectrophotometer. A more detailed description of the arrangement is given in [16].…”

“…For each single-beam spectrum 1024 scans were accumulated. They were divided into four blocks of 256 scans each for baseline control, as described in [16]. The spectral resolution is 2 cm-', for plotting of the spectra a zero-filling factor of two was used.…”