Effect of light-adaptation on the photoreaction of bacteriorhodopsin from Halobacterium halobium

Light-induced formation of the 410 nm intermediate was investigated on dark-and light-adapted bacteriorhodopsin. The amplitude of the light-induced absorption increase at 410 nm of the light-adapted bacteriorhodopsin was twice as large as that of the dark-adapted bacteriorhodopsin. The amount of protons released from bacteriorhodopsin in response to illumination was also enhanced by light-adaptation. The degree of the enhancement was independent of the temperature in the dark-adaptation. The relation between these photochemical events and the isomeric configurations of retinal is discussed.     

On the photocycle and light adaptation of dark-adapted bacteriorhodopsin.

Pulsed Nd laser (25 ns, 530 nm) photolysis experiments were carried out at room temperature in aqueous suspensions of dark- and light-adapted fragments of the purple membrane of Halobacterium halobium. It is shown that the (50%) 13-cis isomeric component (BR13-cis) of dark-adapted bacteriorhodopsin (BRDA) undergoes a photocycle involving a characteristic transient absorbing in the neighborhood of 610 nm. At relatively high excitation intensities BR13-cis is converted to the same 410 nm (M) transient that characterized the photocycle of the all-trans isomer (BRtrans) of light-adapted bacteriorhodopsin (BRLA). This process, which competes with the generation of the "610" species, is attributed to the photo-induced conversion, during the pulse, of BR13-cis (or of its primary photoproduct "X") to a species in the BRtrans photocyte. The relationship between these observations and the mechanism of BRDA hv leads to BRLA adaptation at low excitation intensities (for which a quantum yield limit, 0 less than or equal to (3.5 +/- 0.7) X 10(-2) , is established) is discussed.     

Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs.

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.     

Isolation and properties of the native chromoprotein halorhodopsin

The native chromoprotein of the light-driven chloride pump halorhodopsin (HR) was isolated from Halobacterium halobium strain L-33 which lacks bacteriorhodopsin but contains 'slow cycling rhodopsin-like pigment' (SR). A membrane fraction was prepared in low salt and dissolved in a high salt medium by the detergents Lubrol PX or octylglucoside. These conditions destroyed the chromophore of SR but not the HR pigment. Chromatography on phenyl-Sepharose and hydroxylapatite produced, in 60% yield, a 230-fold enriched monomeric chromoprotein with an apparent mol. wt. of 20,000. The chromoprotein was stable in 1 M NaCl and 1% octylglucoside and remained stable upon removal of detergent. It reacted with borohydride in the dark and with hydroxylamine in the light. The absorption maximum of the light-adapted state is at 580 + 2 nm and its molar extinction approximately 50,000/M/cm. Upon illumination in the presence of detergent it was converted into a 410 nm absorbing species with concomitant release of protons. A thermal reconversion to the 580 nm species occurred with a half time of 76 s at -6 degrees C. Blue light absorbed by the photoproduct accelerated the re-conversion as well as the re-uptake of protons. Removal of the detergent prevented the light-induced formation of the 410 nm species. Under these conditions a photochemical behaviour similar to that in intact cells and cell vesicles, i.e., a photocycle in the 10-20 ms range was observed. These findings form the basis for functional reconstitution of HR.     

Photochemistry of monomethylated and permethylated bacteriorhodopsin.

Methylation of the nonactive site lysines of bacteriorhodopsin to form permethylated bacteriorhodopsin does not interfere with the formation of the short wavelength intermediate M412 or light-induced proton release/uptake. The absorption spectrum is similar to that of the native bacteriorhodopsin. However, additional monomethylation of the active site lysine of bacteriorhodopsin causes a red shift of the absorption maximum from 568 nm in light-adapted bacteriorhodopsin [BR] to 630 nm. The photochemistry of active-site methylated BR does not proceed beyond the L-photointermediate. In particular, the photointermediate corresponding to M412 does not form, and there is no proton pumping. Moreover, there is no tyrosine deprotonation. Thus, the formation of an M-type photointermediate is required for proton pumping by BR.     

Light and dark adaptation of halorhodopsin

Dark incubation of envelope vesicles derived from a strain of Halobacterium halobium that lacks bacteriorhodopsin but contains halorhodopsin and a third rhodopsin-like pigment caused a decrease in the flash yield [the amplitude of a transient absorbance change of flash reactive component(s) by flash] of halorhodopsin but not the rhodopsin-like pigment. The flash yield decreased to reach a low steady level after incubation for about 4 days in the dark. The flash yield of halorhodopsin at any stage of dark incubation was increased by actinic illumination of the vesicles. The flash yield at 490 nm (absorbance increase) was found to be approximately proportional to that at 590 nm (absorbance decrease). These results indicate that halorhodopsin in the envelope vesicles has two forms, dark and light adapted, and that the halorhodopsin phototransient absorbing at 490 nm is originated from the light-adapted form. A difference spectrum between these two forms of halorhodopsin shows that the light-adapted halorhodopsin was red-shifted from the dark-adapted form. The light-induced membrane potential was measured by tetraphenylphosphonium uptake. The uptake by the dark-adapted vesicles was slower than that by the light-adapted vesicles, suggesting that only the light-adapted halorhodopsin has ion-transporting activity.     

Conformational motion in bacteriorhodopsin: the K to L transition

By comparison of the time dependence of linear dichroism and transient absorption in light-adapted bacteriorhodopsin over the first 10 microseconds following excitation, conformational motion in the protein has been detected. Time-resolved linear dichroism and transient absorption scans are reported for several wavelengths that probe the K610 and L550 intermediates in the bacteriorhodopsin photocycle. The transient absorption scans are insensitive to conformational motion and yield the lifetimes of the K610 and L550 intermediates. In contrast, the time-resolved linear dichroism scans demonstrate orientational motion of the chromophore with a 1.7-microsecond rotational time. The wavelength dependence of the least-squares fitting parameters establishes that this motion is associated with L550. This motion is discussed in relation to a protein conformational change in the course of the bacteriorhodopsin photocycle. No evidence is observed for orientational motion on the time scale of the L550----M410 transition.     

Kinetics of the 580-nm ultrafast bacteriorhodopsin transient.

We have observed, by low-temperature picosecond spectroscopy, a photo-induced transient at 580 nm in light-adapted bacteriorhodopsin. The transient was characterized by bleaching in the 550-585-nm regions within 6 ps and recovery in approximately 20 ps. The spectral intensity of the transient is found to be enhanced at lower temperatures, and the lifetime slightly elongated.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.     

Photoconversion from the light-adapted to the dark-adapted state of bacteriorhodopsin.

Dark and light adaptation of bacteriorhodopsin in purple membrane multilayers at less than 100% relative humidity differs from that seen in suspensions. Equilibrium between the two bacteriorhodopsin isomers (bR cis 550 and bR trans 570) in the light-adapted state becomes dependent on the wavelength of actinic light. Excitation at the red edge of the visible absorption band causes dark adaptation in a light-adapted sample. Using polarized actinic and measuring light, we show that acceleration of the dark adaptation through heating by actinic light cannot explain this observation. A light-driven bR trans 570 to bR cis 550 reaction that competes with the well-known 13 cis-to-all-trans light adaptation reaction must exist under our experimental conditions. Trans-to-cis conversion is a one-photon process distinct from the two photon process observed by others in purple membrane suspensions (Sperling, W., C. N. Rafferty, K. D. Kohl, and N. A. Dencher, 1978, FEBS (Fed. Eur. Biochem. Soc.) Lett. 97:129-132). Its quantum efficiency increases monotonously on reducing the hydration level, and is paralleled by an increase in the lifetime of the M410 intermediate of the trans photocycle. We suggest that at this point a branch leads from the all-trans into the 13-cis photocycle. It is probably the same reaction that causes the reduced light adaptation in monomeric bacteriorhodopsin (Casadio, R., H. Gutowitz, P. Mowery, M. Taylor, and W. Stoeckenius, 1980, Biochim. Biophys. Acta. 590:13-23; Casadio, R., and W. Stoeckenius, 1980, Biochemistry. 19:3374-3381).     

Photocycles of bacteriorhodopsin in light- and dark-adapted purple membrane studied by time-resolved absorption spectroscopy.

Nanosecond time-resolved absorption spectra have been measured throughout the photocycle of bacteriorhodopsin in both light-adapted and dark-adapted purple membrane (PM). The data from dark-adapted samples are interpretable as the superposition of two photocycles arising independently from the all-trans and 13-cis retinal isomers that coexist in the dark-adapted state. The presence of a photocycle in dark-adapted PM which is indistinguishable from that observed for light-adapted PM under the same experimental conditions is demonstrated by the observation of the same five relaxation rates associated with essentially identical changes in the photoproduct spectra. This cycle is attributed to the all-trans component. The cycle of the 13-cis component is revealed by scaling the data measured for the light-adapted sample and subtracting it from the data on the dark-adapted mixture. At times less than 1 ms, the resulting difference spectra are nearly time-independent. The peak of the difference spectrum is near 600 nm, although there appears to be a slight (approximately 2 nm) blue-shift in the first few microseconds. Subsequently the amplitude of this spectrum decays and the peak of the difference spectrum shifts in two relaxations. Most of the amplitude of the photoproduct difference spectrum (approximately 80%) decays in a single relaxation having a time constant of approximately 35 ms. The difference spectrum remaining after this relaxation peaks at approximately 590 nm and is indistinguishable from the classical light-dark difference spectrum, which we find, in experiments performed on a much longer time scale, to peak at 588 nm. The decay of this remaining photo-product is not resolvable in the nanosecond kinetic experiments, but dark adaptation of a completely light-adapted sample is found to occur exponentially with a relaxation time of approximately 2,000 s under the conditions of our experiments.     

Absorption spectra of intermediates of bacteriorhodopsin measured by laser photolysis at room temperatures

Picosecond laser spectroscopic analysis was applied to determine how many intermediates existed in the primary photochemical process of trans-bacteriorhodopsin (light-adapted bacteriorhodopsin) at room temperature (18°C) and to calculate their absorption spectra. Irradiation of bacteriorhodopsin with a laser pulse (wavelength, 532 nm; pulse width, 25 ps) yielded the K intermediate (K) which was produced through a precursor, having an absorption maximum (λ_max) longer than that of K. K was stable during a picosecond time range (50–900 ps). The λ_max was located at 610 nm and the extinction coefficient (?_max) was 0.92-times that of bacteriorhodopsin. The same K intermediate was produced from bacteriorhodopsin even when it was excited with a high-energy pulse by which a saturation effect was induced. A transient difference spectrum measured at 150 ns after the excitation of bacteriorhodopsin was different in shape from that of the K intermediate, suggesting that an intermediate was formed by thermal decay of K. This intermediate, tentatively called the KL intermediate (KL), had a λ_max at 596 nm and an ?_max 0.80-times that of bacteriorhodopsin. KL decayed to the L intermediate (L) with a time constant of 2.2 μs. L has a λ_max at 543 nm and an ?_max 0.66-times that of bacteriorhodopsin.     

Light-induced reorientation in the purple membrane.

Reorientation of bacteriorhodopsin in the native purple membrane was studied by time-resolved linear dichroism spectroscopy (TRLD) over the millisecond time regime. The time responses observed in TRLD are distinctly different from the isotropic transient absorption (TA) at wavelengths in the range 550-590 nm, where the bacteriorhodopsin ground state absorbs. In contrast, the TA and TRLD responses have nearly identical time dependence at 410 and 690 nm, where the intermediates M and O, respectively, principally contribute. These results demonstrate ground-state bacteriorhodopsin reorientation triggered by the photocycle. The TRLD and TA data are analyzed to test models for reorientational motion. Rotational diffusion of ground-state bacteriorhodopsin cannot account for the details of the data. Rather, the results are shown to be consistent with a reversible reorientation of "spectator" (nonexcited) members of the bacteriorhodopsin trimer in the purple membrane in response to the photocycling member of the trimer. This response may be associated with cooperativity in the trimer.     

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.     

Resonance Raman study of the dark-adapted form of the purple membrane protein.

The resonance Raman spectrum of the dark-adapted form of the purple membrane protein (bacteriorhodopsin) has been obtained and is compared to the light-adapted pigment and model chromophore spectra. As in the light-adapted form, the chromophore-protein linkage is found to be a protonated Schiff base. Electron delocalization appears to play the dominant role in color regulation. The dark-adapted spectrum indicates a conformation closer to 13-cis than the light-adapted spectrum.     

The quantum efficiency of the bacteriorhodopsin photocycle.

The quantum yield of the primary photoprocess in light-adapted bacteriorhodopsin (phi 1) was determined at room temperature with low-intensity 530 nm neodymium laser excitation, with bovine rhodopsin as a relative actinometer. The observed value of phi 1 - 0.25 +/- 0.05, and the previously determined parameter phi 1/phi 2 - 0.4 [where phi 2 denotes the quantum efficiency of the back photoprecess from the primary species K (590)] imply that phi 1 + phi 2 approximately equal 1. This feature, also characterizing the photochemistry of rhodopsin, bears on the nature and mechanism of the primary event in both systems.     

Modulation-excitation spectrophtometry of bacteriorhodopsin

The spectra and kinetics of transient species in the photocyte of bacteriorhodopsin have been studied using modulation excitation spectroscopy. Kinetic analysis of the 410 nm species decay and 570 nm species grow-in showed that both processes are composed of two simultaneous first-order events in agreement with previous flash spectroscopy measurements. On the basis of the kinetic data a model for the biphasic kinetics is suggested.     

Effect of high pressure on the absorption spectrum and isomeric composition of bacteriorhodopsin.

The effects of high pressure upon the absorption spectra and isomeric composition of the dark (bRD) and light adapted (bRL) forms of bacteriorhodopsin were examined. Pressure favors the 13-cis form of bacteriorhodopsin (bR13-cis). The equilibrium isomeric composition and absorption spectra of bacteriorhodopsin samples at a given pressure were the same starting from either light or dark adapted bacteriorhodopsin. From the effect of pressure on the equilibrium constant between bRall-trans in equilibrium bR13-cis in the dark, the molar volume change between bRall-trans and bR13-cis was found to be -7.8 +/- 3.2 ml/mol. This volume change suggests a difference in conformation between dark- and light-adapted bacteriorhodopsin, but the magnitude of the change is small, involving only a small number of the protein residues.     

竹红菌乙素对菌紫质荧光的影响

用荧光光谱方法研究了竹红菌乙素与菌紫质的相互作用。通过竹红菌乙素对菌紫质吸收光谱,对菌紫质荧光猝死的浓度和温度的依赖性以及竹红菌乙素对明暗适应菌紫质的荧光猝灭等研究表明,竹红菌乙素是一种新型的荧光猝灭剂,它对菌紫质的荧光猝灭属于动态猝死过程。竹红菌乙素对明暗适应菌紫质荧光猝灭的差别,说明这种猝灭剂可用于生物膜体系的蛋白质构象变化的研究。