F. Siebert

Evidence for the protonation of two internal carboxylic groups during the photocycle of bacteriorhodopsin: Investigation of kinetic infrared spectroscopy

The amino acid sequence of bacteriorhodopsin (BR) [ 1,2], its three-dimensional structure at low resolution [3] and projected structure at 3.7 A resolution [4] have been determined. Attempts have been made to fit the amino acid sequence into the threedimensional structure [5,6] and the retinal binding site has been established [7-91. To understand the proton pumping mechanism, molecular events in the chromophore and protein should be known as well as the structural information. Resonance Raman spectroscopy provides information on the chromophore (review [lo]), whereas kinetic infrared spectroscopy [ 11 ,121 detects molecular changes in the chromophore as well as the protein. It has been applied successfully in investigations of rhodopsin [ 131 and bacteriorhodopsin. For the latter, the BR570-M412 difference spectrum has been measured over 17001500 cm-’ and 1300-l 100 cm-’ [ 121; a preliminary study on the BR570-L550 difference spectrum in the same spectral range has been published [ 141. Having extended the spectral range beyond 1700 cm-‘, we now report evidence for the presence of two carboxylic acid residues being protonated and re-deprotonated during the photocycle. While the protonation of one group occurs simultaneously with the formation of M412, the time course for the second group is slower and does not reflect kinetics observed for chromophoric changes. In addition, the two groups can be identified by their different absorption maxima. The time constant for re-deprotonation is similar for both groups coinciding with that for the reformation of BR570 from M412. These results will be discussed with respect to a possible involvement of carboxylic groups in the proton pumping mechanism [ 15,171 and with respect to their role regarding the spectroscopic properties of BR [15,16,18-211.

Flash-induced kinetic infrared spectroscopy applied to biochemical systems.

A flash photolysis apparatus with monitoring infrared beam is described allowing measurements of relative transmission changes of 10−3 in times of a few milliseconds. The investigation of the photodissociation of CO-myoglobin confirms the results obtained by static infrared difference spectroscopy. The application of our method to the rhodopsin/Meta II transition reveals signals which can tentatively be ascribed to the disappearance of the C=C-band of the protonated N-retinylidene Schiff base in rhodopsin. The developed method will be compared with other existing methods of kinetic vibronic spectroscopy such as kinetic resonance Raman spectroscopy and kinetic Fourier infrared spectroscopy.

Two distinct rhodopsin molecules within the disc membrane of vertebrate rod outer segments.

The kinetics of the metarhodopsin I-II reaction have been measured over a wide range of temperatures (1-37C ) and pH values (4.5-8) with suspensions containing fragments of bovine rod outer segments. It was found that for all conditions the occurrence of metarhodopsin II could be described by two independent first-order processes. The fast component: slow component amplitude ratio depends upon pH and temperature. The kinetics of the lumi-metarhodopsin I reaction show the same pH dependence for the fast component: slow component amplitude ratio as the one observed for the metarhodopsin II signals. All the results observed could be described with the assumption that rhodopsin itself exists in two conformational states before bleaching which are in a pH and temperature-dependent equilibrium. This hypothesis is confirmed by its ability to explain some apparently anomalous observations in the literature.

[87] Kinetic properties of rhodopsin and bacteriorhodopsin measured by kinetic infrared spectroscopy (KIS)

Publisher Summary Kinetic infrared spectroscopy monitoring transmission changes only renders infrared spectroscopy selective. In addition, information on the dynamics of the reaction and the existence and nature of the intermediates is obtained from the kinetics of the transmission changes. The difference spectrum generated from kinetic infrared signals then contains useful information on the molecular processes in the chromophore as well as in the protein. This chapter discusses the application of flash photolysis apparatus for an infrared monitoring. This instrument is sensitive enough to detect the very small changes that occur in a small part of the total complex molecule. The preparation of samples for the investigation of the rhodopsin reaction sequence and the bacteriorhodopsin reaction cycle with kinetic infrared spectroscopy are reported, and the results obtained with the method are discussed in connection with other experimental techniques, mainly resonance Raman spectroscopy.

The influence of the 13-methyl group of the retinal on the photoreaction of rhodopsin revealed by FTIR difference spectroscopy

The photoreaction of rhodopsin regenerated with 11-cis-13-demethyl-retinal was investigated by FTIR difference spectroscopy. The measurements show that the chromophore experiences different twists in the modified bathorhodopsin as compared to normal bathorhodopsin and that the twists are relaxed in the additional intermediate batho-lumirhodopsin. Whereas the missing methyl group influences the lumimetarhodopsin-I transition, a metarhodopsin-I-metarhodopsin-II difference spectrum very similar to that of unmodified rhodopsin is observed. The significance of the steric interaction for regulating the photoreaction is discussed.

Effect of Triton X-100 and of deuteration on the amplitude of the O640-intermediate in the bacteriorhodopsin photocycle.

Proton pumping only occurs in the BRLA cyclic reaction. The coupling of the ejection of protons on one side and proton uptake on the other side of the membrane to the intermediates of the photocycle as well as the stoichiometry of the pumping, i.e., the ratio of protons pumped to BR-molecules cycling, have been investigated repeatedly [4-81. Experiments which have shown deprotonation of the protonated Schiff base linkage of all-trans retinal to bacteriorhodopsin at the Mdrz-intermediate [9] have led to the assumption that the Schiff base proton might be involved in the pumping, thus leading to a maximum stoichiometry of 1. The stoichiometry of the pumping depends strongly on external parameters such as salt concentration and pH. At physiological conditions, i.e., at high [salt], stoichiometries of -2 have been reported [6,8]. At lower [salt] the H’/M 412 ratio observed is generally smaller and was found to be O-l [6]. The formation of M4r2, however, does not depend on [salt]. This indicates that another pathway may be involved in the proton pumping. The pH-dependence of the stoichiometry of proton extrusion from Halobacterium cells and cell envelopes suggests that this might be a pathway of the O-intermediate, since it shows the same increasing yield for lower pH [5,7,10]. Again, the formation of M4r2 is not influenced by variation

Proteins of bovine rod outer segments characterized by SDS-polyacrylamide gel electrophoresis.

Abstract Proteins of bovine rod outer segments (ROS) were subjected to SDS-polyacrylamide gel electrophoresis after repeated washing. Three major proteins are found in the molecular weight region of 30,000 to 42,000. The rhodopsin content of ROS disc membranes is estimated to be only 50 % of total protein.

Time-resolved infrared studies of light-induced processes in plant thylakoids and bacterial chromatophore membranes: evidence for the function of water molecules and the polypeptides in energy dissipation

Abstract Spinach thylakoids and chromatophores from the photosynthetic bacterium Rhodopseudomonas capsulata were investigated by means of time-resolved infrared spectroscopy, using thin water-containing membrane films which fully maintained their photochemical activity. Upon flash excitation, reversible infrared absorbance changes were obtained and their difference spectra were recorded. In spinach thylakoids, these transient signals could be described by a sum of two exponential decay functions with half-times of about 2 and 30 ms, respectively. They were insensitive to the addition of benzyl viologen, ferricyanide or ferricyanide + DCMU. They are ascribed by their dependence on intensity and wavelength range of the actinic flash to processes in the antenna pigment-protein complexes. In chromatophores from photosynthetic bacteria, similar infrared signals in the millisecond time range were obtained. Their spectral distribution was investigated for three mutants of the photosynthetic bacterium and is different for membranes lacking carotenoids. Both signals, in thylakoids and chromatophores, reflect the proportion of absorbed flash energy which is neither channelled to the reaction center nor emitted as light, but is dissipated through radiationless decay. A common feature of the difference spectra from spinach thylakoids and bacterial chromatophores are bands identified by deuteration as being due to H 2 O. Some bands are interpreted in terms of water going transiently from the hydrogen-bonded to the free state. Other bands are assigned to the polypeptides of the light-harvesting complexes, and thus indicate their participation in energy dissipation. Membranes from photosynthetic bacteria containing a photochemical reaction center show a distinct slow signal component decaying in about 1 s. It saturates at low flash intensity and is abolished upon chemical oxidation of the primary electron donor. Two bands in the difference spectrum of this component are tentatively assigned to the ester C = O and keto C = O vibrations of photooxidized bacteriochlorophylls in the reaction center. The data suggest that chromophoric and non-chromophoric infrared absorbance changes contribute to the difference spectra, and thus may represent a clue to the processes at the active sites of polypeptides in photosynthesis.

Non-chromophoric spectral changes during the bacteriorhodopsin photocycle probed by kinetic infrared spectroscopy

The photocycle of light-adapted bacteriorhodopsin, BR57 O, has been investigated by our recently developed method of kinetic infrared spectroscopy (1,2). From time-resolved absorbance changes IR-difference spectra were obtained at a time scale from microseconds to seconds. On the basis of IR-spectra of model compounds and the kinetics of these absorbance changes, some of them can be attributed to chromophore molecular changes due to the BR570-L550 and the BR570-M412transition.

Kinetic Infrared Investigation on the Photodissociation of Sperm Whale CO-Myoglobin

With our method of kinetic infrared spectroscopy we investigated the photodissociation of sperm whale CO-myoglobin in the region of the CO-vibration band. With the infrared beam at a wavelength of the CO-vibration of bound CO we observed flash induced signals whose time courses correspond to the photodissociation of CO-myoglobin and its rebinding as measured at the soret band. The amplitudes of the flash induced signals reproduced the static measured difference spectrum of CO-myoglobin vs. myoglobin, with a maximum at 1944 cm−1 and a shoulder at 1932 cm−1. This shoulder has generally been taken as an evidence for a second binding site for CO in myoglobin. It could also be that there exists an equilibrium between two types of binding at the same binding site. We hoped to get information about this problem by measuring the spectral dependence of the rebinding kinetics. Within the experimental errors no dependence could be detected. The most plausible explanation for this would be a fast equilibration between two types of binding at the same binding site. Photolysed CO dissolved in water cannot be detected due to the large intermolecular interaction with the water molecules. At the time scale of our measurements we have no evidence for an intermediate binding site of CO, since no band developed (no absorbance increase was measured), which could be attributed to the CO-vibration.