INTERPRETATIONS OF THE SOLUTION AND ORIENTED FILM SPECTRA OF BROWN MEMBRANE OF HALOBACTERIUM HALOBIUM

Abstract— The absorption and circular dichroic spectra of the brown holo-membrane (retinal present) and apo-membrane (retinal absent) of Halobacterium halobium in solution and oriented as a film have been studied over the accessible wavelength region, 800–183nm. Since the structure of the well-studied purple membrane can be considered to be a modification of the structure of the brown membrane and much is known about the structure of the purple membrane, interpretations of the brown membrane spectra are based on our previous interpretations of similar studies of the purple membrane. The brown membrane contains two membrane proteins, bacteriorhodopsin and cytochrome b-561 in a 3:1 molar ratio in contrast to the purple membrane which contains only bacteriorhodopsin. Main findings are (a) degenerate oscillator coupling (exciton) among the retinyl chromophores of the bacteriorhodopsins, (b) a relatively strong in-plane interaction between the retinal and the bacteriorhodopsin apoprotein environment, possibly due to a dissymmetric static charge distribution, (c) the planes of the aromatic rings of some of the tryptophans must be nearly parallel to the plane of the membrane, (d) the helical axes of the bacterio-opsin polypeptide segments are significantly tilted in respect to the normal to the membrane plane in contrast to the helical axes of the bacteriorhodopsin polypeptide segments which are nearly parallel to the normal, (e) no detectable interaction between the two membrane proteins, (f) the plane of the heme of the cytochrome cannot be parallel to the membrane plane and is most likely perpendicular to it. (g) the dipole moments of the two mutually perpendicular Soret porphyrin transitions of the heme are most likely oriented at an angle to the membrane plane, (h) there seems to be a significant reduction in the symmetry of the heme group in the environment of the apoprotein, (i) the possibility of a unique geometrical arrangement and resonance interaction between the Soret porphyrin and nearby cytochrome aromatic amino acid π–π* transitions, (j) the secondary structure of the cytochrome is significantly α-helical, and (k) the helical axes of the cytochrome polypeptide segments are randomly oriented in respect to the normal to the membrane plane. A consequence of these findings is that the fine structures of the bacteriorhodopsins of the brown and purple membranes are very similar in spite of differences in the composition and the structure of the two membranes. In addition, the orientation of the helical segments of the bacteriorhodopsin polypeptides relative to the membrane plane in the brown and purple membranes can be regulated by the retinal–apoprotein interactions. Significance of this possible regulation in respect to the proton-pumping function of these membranes is discussed.     

[MESITYL]BACTERIORHODOPSIN. THE PROPERTIES OF AN ANALOGUE OF THE PURPLE MEMBRANE CONTAINING [MESITYL] RETINAL AS THE CHROMOPHORE

Abstract— A new synthesis of all-trans-[mesityl]retinal, II , (all-trans-3,7-dimethyl-9-(2',4',6'-trimethylphenyl)-2,4,6,8,-nonatetraenal) and 13-cis-[mesityl]retinal, VI , (3,7-dimethyl-9-(2'4'6'-trimethylphenyl)-2Z,4E,6E,8E-nonatetraenal) is reported. Combination of all-trans-[mesityl]retinal with bacterioopsin results in the formation of a synthetic membrane (Λ_max 460) which has photocycling properties similar to the purple membrane although its cycling rate is very much slower. An M-type intermediate can be trapped at -60°C. Photoreversal of the M-intermediate to the wavelength of initial absorption is observed. Phototransformation of the initial [mesityl]bacteriorhodopsin is accompanied by conversion of the all-frans to the 13- cis -isomer.     

PRIMARY PHOTOCHEMISTRY OF BACTERIORHODOPSIN: COMPARISON OF FOURIER TRANSFORM INFRARED DIFFERENCE SPECTRA WITH RESONANCE RAMAN SPECTRA

Abstract —Fourier transform infrared (FTIR) difference spectra of the BR→rK transition in bacteriorhodopsin at 77→K are compared with analogous resonance Raman difference spectra obtained using a spinning sample cell at 77→K. The vibrational frequencies observed in the FTIR spectra of native purple membrane and of purple membrane regenerated with 15-deuterioretinal are in good agreement with the frequencies observed in the Raman spectra, indicating that the lines in the FTIR difference spectra arise predominantly from retinal chromophore vibrations. This agreement confirms that the spinning cell method for obtaining resonance Raman spectra of K minimizes potential contributions from unwanted photoproducts. The unexpected similarity between the resonance Raman scattering intensities and the FTIR absorption intensities for BR and K is discussed in terms of the delocalized electronic structure of the chromophore. Finally, comparison of the Schiff base regions of the K Raman and FTIR spectra provide additional information on the assignment of its Schiff base vibration.     

BIOSYNTHESIS AND ASSEMBLY OF PURPLE MEMBRANE OF HALOBACTERIUM HALOBIUM S9: LIGHT STIMULATION OF BACTERIORHODOPSIN FORMATION

Abstract— The effect of light on purple membrane biogenesis in Halobacterium halobium S9 strain was investigated. When bacteria were grown in the dark, the 570nm absorption due to bacteriorhodopsin increased more slowly than under illumination, but eventually after longer incubation, reached the same level as that seen in the illuminated culture.
Analysis of membrane fractions by sucrose density gradient centrifugation revealed that two different membrane fractions, containing purple and brown membrane could be detected in the exponential growth phase. Another fraction whose density was higher than that of purple membrane, disappeared concomitantly with the increase in purple membrane and brown membrane, indicating that it may be related to purple membrane formation.
HPLC analysis of membrane proteins showed that there was no significant difference in de novo synthesis of bacterio–opsin between dark and illuminated cultures. This led us to conclude that light stimulated retinal binding to bacterio–opsin and/or retinal biosynthesis rather than bacterio–opsin synthesis. Bacteriorhodopsin seemed to form the brown membrane fraction first, which then spontaneously reorganized into purple membrane.
When incorporated in liposomes, bacteriorhodopsin in brown membrane was found to have rather higher proton pump activity than that in purple membrane. The H⁺ pumping activity was quite heat labile. This and the CD spectra indicated that bacteriorhodopsin in brown membrane might exist without forming normal timer unit.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.     

RESONANCE RAMAN SPECTRA OF THE "BLUE" and THE REGENERATED "PURPLE" MEMBRANES OF Halobacterium halobium

Abstract— We have obtained the resonance Raman spectra of the deionized form of the purple membrane, the so called blue membrane, as well as the purple membrane regenerated by titrating the blue membrane with either Na+, Ca²+ or La³+. All types of regenerated purple membrane have identical Raman spectra which are virtually indistinguishable from the native light-adapted bacteriorhodopsin spectrum. On the other hand, Raman data for the blue membrane suggest that it consists of essentially two pigment forms with absorption maxima around 605 and 570 nm and containing 13-cis and all-trans isomeric configurations of the chromophore. This is consistent with our chromophore extraction results which reveal that the blue membrane consists of 30% 13-cir and 70% all-trans chromophore.     

SOME OBSERVATIONS ON THE MORPHOGENESIS OF LATTICE STRUCTURE IN THE PURPLE MEMBRANE

Abstract— The formation process of purple membrane was studied morphologically by freeze-fracture and etching methods. The bacteria, H. halobium R₁, cultured in the medium containing 2m M nicotine, did not show the hexagonal structure of purple membrane, but a particle free area was frequently observed in the plasma membrane. However, within 24h after returning to the normal medium, hexagonal and triclinic lattices with spacings of 6 to 8nm occurred as small patches on the P face of the plasma membrane. Furthermore, it was revealed that the retinal was not required for the formation of the two-dimensional hexagonal lattice structure of purple membrane, since the mutant bacteria, H. halobium R₁mW, lacking the ability of retinal synthesis, displayed a similar lattice. The other mutant, H. halobium R₁mR, lacking the ability of bacterio-opsin synthesis, never showed any highly ordered arrangement. This evidence suggests that membrane proteins other than bacterio-opsin or bacteriorhodopsin do not form highly ordered lattices in the membrane.     

INTERACTION OF DIBUCAINEHCl LOCAL ANESTHETICS WITH BACTERIORHODOPSIN IN PURPLE MEMBRANE: A SPECTROSCOPIC STUDY

Several spectroscopic techniques (absorption, emission, transient absorption and differential scanning calorimetry--DSC) were used to investigate the deprotonation of dibucaine.HCl in a hydrophobic environment, and the interaction sites and mechanisms of the local anesthetic dibucaine.HCl on bacteriorhodopsin (bR) in purple membrane. The important results are summarized as follows: (1) the visible absorption features of native (lambda max = 568 nm) and deionized (lambda max = 608 nm) bR are sensitive to the amount of dibucaine.HCl added; (2) the emission spectrum of dibucaine.HCl embedded in the retinal-free mutant bR is similar to that of dibucaine free base in Triton X-100 micellar solutions; (3) the phosphorescence emission of dibucaine at 77 K is completely quenched by bR and the fluorescence quenching rate for the incorporated dibucaine.HCl in bR was determined as kq = 4.09 x 10(13) M-1 s-1; (4) the incorporation of dibucaine.HCl in bR inhibits the slow component rate of formation of M412 and decreases the amount of M412 formation in the photochemical cycle of bR; and (5) the thermal stability of native bR was measured by DSC in the presence and absence of dibucaine and yielded an endothermic transition at 95.9 +/- 1.0 degrees C with 13.6 J/g (3.25 +/- 0.12 cal/g) of enthalpy changes. All observations suggest that the action site of the local anesthetic, dibucaine.HCl, is near or at the chromophore, i.e. the retinal Schiff base of bR. The anesthetic action on bR purple membrane is probably via a specific site binding, but not a conformational mechanism.     

THE pH DEPENDENCE OF TRANSIENT CHANGES IN THE CURVATURE OF THE PURPLE MEMBRANE*

Abstract– We have observed transient changes in the curvature of purple membrane fragments upon photoexcitation as a function of the pH of the suspending medium. The room temperature suspensions have low ionic strengths, and the bending is observed by changes in the scattered light at 320 nm. The photoexcitation is from a 20 ns laser pulse at 532 nm. We use a simple first-order approximation to subtract any changes in the scattered light associated with transient absorption changes during the photocycle. The resultant scattering curves are then fitted to the sum of three fundamental bending processes. Each process has an exponential rise, an exponential decay and an amplitude. We model two of the processes as transient forces correlated with the charge motion during the photocycle. The third process is probably cuased by local changes in the pH as the protein pumps protons. However, this is not proved rigorously. Additional experiments are proposed.     

THE USE OF NEUTRAL RED TO MONITOR THE SURFACE POTENTIAL OF THE PURPLE MEMBRANE

Abstract— The binding of neutral red to purple membrane has been studied. The intrinsic p K _a and the apparent p K _a, of bound neutral red were determined by titration and by measuring the binding ratio of neutral red to purple membrane as a function of pH. The surface potential of purple membrane was inferred from the difference between these two p K _as. The H⁺/M412 ratio at different ionic strengths was also measured and compared with the surface potential. The results show that the H⁺/M412 decreased as the surface potential increased due to decreased salt concentrations. However, this correlation holds only for KCl concentrations higher than 30 m M . At lower salt concentrations, the change in surface potential is always less than the variation in the H⁺/M412 ratio.     

LIGHT INDUCED ISOMERISATION, AT ACIDIC pH, INITIATES HYDROLYSIS OF BACTERIORHODOPSIN TO BACTERIO-OPSIN AND 9-CIS-RETINAL

Abstract— Bacteriorhodopsin (BR) from the purple membrane of Haiobacterium halobium contains covalently bound retinal in the 13- cis and all- trans configurations. Several forms of bacteriorhodopsin are known, with different absorption maxima which are designated as BRλ_max (nm). At acidic pH, BR605 is formed from BR560. The following sequence of reactions was found, which is initiated by irradiation of BR605 with red light:

An all- trans /13- cis to 9- cis isomerisation occurs in the light induced reaction BR605 ∼ BR500. BR500 seems to contain covalently bound retinal, whereas BR390 contains free retinal. By irradiation with light, BR500, BR450 and BR390 can be reconverted to BR560.     

QUANTUM YIELD RATIO OF THE FORWARD and BACK LIGHT REACTIONS OF BACTERIORHODOPSIN T LOW TEMPERATURE and PHOTOSTEADY-STATE CONCENTRATION OF THE BATHOPRODUCT K*

The maximum photosteady state fraction of K, x_K^max, and the ratio of the quantum yields of the forward and back light reactions, trans-bacteriorhodopsin (bR) hArr; K, φ_bR/φ_K, were obtained by measuring the absorption changes produced by illumination of frozen water-glycerol (1:2) suspensions of light-adapted purple membrane at different wavelengths at -165°C. An independent method based on the second derivative of the absorption spectrum in the region of the β-bands was also used. It was found that The quantum yield ratio (0.66 ± 0.06) was found to be independent of excitation wavelength within experimental error in the range510–610 nm. The calculated absorption spectrum of K has its maximum at603–606 nm and an extinction 0.85 ± 0.03 that of bR. At shorter wavelengths there are P-bands at 410, 354 and 336 rim. Using the data of Hurley et al. (Nature 270,540–542, 1977) on relative rates of rhodopsin bleaching and K formation, the quantum yield of K formation was determined to be 0.66 ± 0.04 at low temperature. The quantum efficiency of the back reaction was estimated to be 0.93 ± 0.07. These values of quantum efficiencies of the forward and back light reactions of bR at - 165°C coincide with those recently obtained at room temperature. This indicates that the quantum efficiencies of both forward and back light reactions of bacteriorhodopsin are temperature independent down to -165°C.     

PURPLE MEMBRANE ANALOGS SYNTHESIZED FROM C17 ALDEHYDE

Abstract— All- trans , 11- cis and 9- cis isomers of the C₁₇ aldehyde analogs of retinal bound with purple membrane apoprotein, probably through a Schiff base linkage at the normal retinal binding site. The complex formed from C₁₇ aldehyde and purple membrane apoprotein was slowly decomposed by 10m M hydroxylamine. The C₁₇ aldehyde competitively inhibited the regeneration of purple membrane from all- trans -retinal and purple membrane apoprotein. The differential ability of the different isomers to inhibit the regeneration suggests that purple membrane has a binding site for the side chain of retinal in addition to the Schiff base binding site.     

POLARIZED SPECTRA OF ORIENTED PIGMENT-LIPOPROTEIN COMPLEXES FROM THE PURPLE BACTERIUM: Chromatium minutissimum

Abstract— Polarized absorption, fluorescence and photoacoustic spectra of bacteriochlorophyll (BChl)-lipoprotein complexes from the purple bacterium Chromatium minutissimum oriented in stretched polyvinylalcohol films were measured at room temperature and 85 K. The preparations contain large amounts of the B800-820 antenna complexes. From polarized absorption spectra taken under various light beam incidence angles with respect to the film plane, conclusions concerning arrangement of pigment molecules in B800-820 complex are obtained. The transition moments of the BChl Q_y band are not exactly parallel to the membrane plane. It seems that there are pools of differently oriented BChl chromophores absorbing in both 800 nm and 820 nm regions. Change in temperature strongly influences linear dichroism of carotenoids and BChl Q_y bands. The reversible changes in absorption, linear dichroism and photoacoustic spectra caused by the variation in sample temperature suggest strongly the reversible twisting of carotenoid molecules, related probably to modification of the interactions between carotenoids and proteins. Various carotenoids exhibit different yield of thermal deactivation and this yield is also temperature dependent.     

Photocycle of 13-cis-retinal in bacteriorhodopsin caused by destruction to the cooperative effect of purple membrane

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TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

THE QUANTUM EFFICIENCY FOR THE INTERPHOTOCONVERSION OF THE BLUE and PINK FORMS OF PURPLE MEMBRANE

When the cations bound to purple membrane are removed it turns blue, and when this blue membrane is irradiated its color changes to pink. Irradiation of pink membrane leads to the reformation of blue membrane. We have determined that the quantum efficiency for the formation of pink membrane from deionized blue membrane is 1.6 ± 0.6 ± 10 ⁴ at 0^oC, pH 5.0. We also found that the quantum efficiency for the back photoconversion, i.e. the formation of blue membrane from pink membrane, is 8.8 ± 1.6 ± 10^-3 at 0^oC, 55 times greater than that of the forward photoconversion reaction. The extinction coefficients of the pink membrane and blue membrane were determined to be 44 500 ± 670 cm^-1 M^-1 at 491 nm and 54 760 ± 830 cm^-1 M ^-1 at 603 nm, respectively, assuming light-adapted purple membrane is 63 000 cm^-1 M ^-1 at 568 nm. The quantum efficiency for forming pink membrane from blue membrane is much lower than that for forming the photointermediate of the blue membrane's photocycle. Their relationship is similar to that of light-adaptation and photocycle of the dark-adapted purple membrane.     

QUANTUM EFFICIENCY FOR THE PHOTOCONVERSION OF BACTERIORHODOPSIN AT VERY LOW TEMPERATURES

Abstract— Quantum efficiencies for photoconversion of bacteriorhodopsin (trans-bR) to a bathochromic product (batho-bR^t) and its photoreversion in purple membrane at 77 and 9 K were investigated with low temperature spectrophotometry. The kinetics of the photoconversion and its photoreversion cannot be expressed by a single exponential curve. The photoconversions at 77 and 9 K showed the same slope in the early stage. The kinetics of the photoreversions were identical at the two temperatures. These results indicate that the quantum efficiencies for the conversion of trans-bR to batho-bR^t or for its photoreversion are identical at the two temperatures.
The fact that the photoreversion cannot be expressed by a single exponential curve suggests the existence of several conformational states of batho-bR^t due to the trimer structure of the purple membrane.     

PHOTODESTRUCTION OF BACTERIORHODOPSIN

Abstract— Suspensions of purple membrane fragments showed obvious signs of degradation after illumination with intense pulses of light from 10 ns frequency doubled Nd: YAG laser at 532 nm with intensity densities in excess of 1 MW/cm². Using controlled illumination, a small fraction of the bacteriorhodopsin protein molecules were randomly destroyed in samples with a low salt concentration (12.5 m M ) and pH = 7.9. Calculations using information from the changes in the optical absorbance spectrum and transient changes in the optical absorbance spectrum during the photocycle support a model where one protein molecule of the bacteriorhodopsin trimer is photodestroyed, the other two protein molecules switch to a blue state . In the blue state , the protein molecules have a red shifted absorption, with a peak near 600 nm. The blue state molecules show transient absorption changes at 656 nm that are similar to the native bacteriorhodopsin, except the O state is missing or altered. Additionally, the changes in curvature of the purple membrane fragments that occur during the photocycle of intact protein molecules are severely depressed. The addition of salts to the photodestroyed suspension can change the blue state molecules back to a state with an absorption maximum at 568 nm. The salt ions probably shield the other members of the trimer from the photodestroyed protein. In these reconstituted samples, the O state is observed at 656 nm; however, the membrane bending is not observed.     

PICOSECOND KINETICS and A MODEL FOR THE PRIMARY EVENTS OF BACTERIORHODOPSIN

The picosecond absorption kinetics of light adapted bacteriorhodops in (BR) are reported for water and glycerol-water suspensions of purple membrane from Halobacieriwn hulobium at different temperatures (77-300 K) and pH (5-10). It is shown that a photoproduct of BR, called P-BR (pseudo-bacteriorhodopsin). is responsible for the 16 ± 2 ps relaxation lifetime observed under steady state irradiation (i.e. with a train of ps pulses) at room temperature. At 77 K the absorption recovery lifetime is 62 ± 5 ps in good agreement with previous fluorescence lifetime studies. The observed signal is very sensitive to the sample flow rate and at the highest flow rates a fast absorbance increase (≤ 2 ps) is observed at λ > 620 nm. while at 576 nm a similarly fast absorption recovery alter bleaching is observed. These results imply that the reaction BR → K-BR occurs within 2 ps. In order to explain the simultaneous formation of P-BR and K-BR at 77 K. a model for the primary events including a traus-cis isomerization and a protein relaxation about the chromophore is suggested. The same model can also be used in explaining the simultaneous formation of batho- and hypsorhodopsin in some rhodopsins. Finally, a scheme for the last steps in the photocycle of bacteriorhodopsin including protonation, protein relaxation and cis-trans isomerization is proposed