MECHANISM OF ORIENTATION AND LINEAR DICHROISM OF PHOTOSYNTHETIC PARTICLES IN ELECTRIC FIELDS: CHLOROPLASTS AND CHLOROPHYLL-PROTEIN COMPLEXES

Abstract— The mechanisms of orientation in pulsed and alternating electric fields of thylakoids (derived from the sonication of spinach chloroplasts) and of light-harvesting chlorophyll a/b-protein complexes (CPII) were investigated by utilizing linear dichroism techniques. Comparisons of the linear dichroism spectra of thylakoids and CPII particles suggest that the latter are oriented with their directions of largest electronic polarizabilities (and thus probably their largest dimensions) within the thylakoid membrane planes. At low electric field strengths (< 12 V cm^?1), and at low frequencies of alternating electric fields (< 0.25 Hz), thylakoid membranes tend to align with their normals parallel to the direction of the applied electric field; the mechanism of orientation involves a permanent dipole moment of the thylakoids which is oriented perpendicular to the planes of the membranes. However, at high field strengths and high frequencies of the applied alternating electric fields, the thylakoids tend to orient with their planes parallel to the applied field, thus exhibiting an inversion of the sign of the linear dichroism as the electric field strength is increased. At the higher frequencies and at higher field strengths, the orientation mechanisms of the thylakoids involve induced dipole moments related to anisotropies in the electronic polarizabilities. The polarizability is higher within the plane than along a normal to the plane, thus accounting for the inversion of the dichroism as the electric field strength is increased. The CPII particles align with their largest dimension parallel to the applied field at all field strength, indicating that the induced dipole moment dominates the orientation mechanisms in pulsed electric fields. The magnitude of the absolute linear dichroism of CPII suspensions increases with increasing dilution, indicating that aggregates of lower symmetry are formed at higher concentrations of the CPII complexes.     

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.     

ELECTRIC LINEAR DICHROISM OF P700 CHLOROPHYLL U-PROTEIN COMPLEXES

Abstract— The P₇₀₀ chlorophyll a -protein complex (CPI) isolated from green plants was oriented in aqueous solutions using pulsed electric fields of up to 6700 V cm^-1. The electric linear dichroism spectrum is reported in the range of 400–720nm. Positive peaks in the linear dichroism Δ A = A _I - A ₁ (where A_I and A₁ are the absorbance components in which the polarizer orientation is parallel and perpendicular with respect to the electric field. respectively) are observed at 443 and 686 nm. The ΔA signal at 686 nm is discussed in terms of either a specialized chlorophyll form absorbing at 686 nm. or due to an exciton component absorbing at the same wavelength.     

Influence of cation binding on the electro-optically determined electric moments of purple membranes

Analysis of the elect ro-optically determined permanent dipole moment and electric polarizability of purple membrane fragments reveals the complex nature of the membrane electric moments.The problem to distinguish between the contribution of the membrane structural charges (charged groups of the polypeptide chain and polar lipid headarouos), bound cations and the electric double layer structure deserves particular attention not only because of its importance for electro-optics but also in respect to the relation of the membrane surface electric properties to the membarans transport function.The removal of divalent cations (Ca²⁺ and Mg²⁺) bound to purple membrane in the native state induces a cat ion-free species or purple membrane (deionized - blue membrane) with drastically changed spectroscopic properties and function. The preseent paper summarizes our study on the electric moments of blue membrane and their changes during the blue to purple transition. We intended to provide an insight into the possible regulation of this reversible transition (purple-to-blue and blue-to-purple) through changes of the asymmetric charge distribution and the importance of the asymmetric interfacial charge distribution for the proton transfer in purple membranes.The changes in the electric moments (permanent and induced dipole moments) of purple membrane fragments upon di- and trivalent cations binding to cation-depleted purple membranes were studied by electric light scattering (rotational electrokinetics) in d.c. and a.c. electric fields, and by electric pulses with reversing polarity, the results show a recovery of the membrane charge asymmetry (permanent dipole moment) though not of the induced dipole moment.     

Orientation of pea chloroplast in an ac electric field

The orientation of pea chloroplast in an ac electric field was studied. The maximum linear dichroism signal at 681 nm was used as an indicator of orientation. It was found that the degrees of orientation depended on the frequency of the electric field. In the frequency range studied, there were two distinct dispersions at about 80 Hz and 750 kHz (α and β dispersion), and a maximum at about 100 kHz. The dependence of orientation on the electric field strength was used to estimate the polarizability. In agreement with the dispersion spectrum, there were stepwise decreases in polarizability at the two dispersions and an increase when moved into the region of the maximum. There was not permanent dipole detected.At a frequency below the α dispersion, an induced dipole arising from the oscillation of a counterion cloud, which is formed adjacent to the thylakoid membrane surface due to the presence of fixed surface charges, is believed to be a major cause of orientation. After the relaxation of this oscillation at higher frequency, the charge accumulation on both the inner and outer surfaces of the thylakoid membrane is believed to play a determinant role. Since the two interfacial polarizations have different dispersion frequencies, the polarizability of chloroplast increases after one dispersion (the maximum), which is then followed by a decrease at the second dispersion (the β dispersion).     

On the electric polarizability of purple membrane

The permanent dipole moment and electric polarizability of purple membrane have been found using electrooptic methods. Comparison of the results with those for other inorganic or biological disperse particles as well as the observed effects and dynamics for a large range of applied electric field strengths support the proposal of a surface origin for the electric polarizability of purple membrane.     

Buffer-induced changes of purple membrane surface electric properties: an electro-optical study

Buffer-induced alteration of the purple membrane electric dipole moments and electrokinetic charge was studied by electric light scattering and microelectrophoresis. The permanent dipole moment and electrophoretic mobility of purple membranes change in opposite direction in presence of 'P'- and 'N'-type buffer molecules, shown to produce 'positive' and 'negative' additional components to the bR light-induced charge displacement current. It is concluded that the two types buffer molecules distribute differently on the membrane surfaces, depending on their protonation state, as a result of different interaction with the membrane cytoplasmic and extracellular surfaces.     

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.     

LIGHT ADAPTATION OF DARK-ADAPTED BACTERIORHODOPSIN STUDIED BY NANOSECOND TIME-RESOLVED ABSORPTION SPECTROSCOPY

Abstract— The aim of the study is to clarify the mechanism of light adaptation of dark-adapted bacteriorhodopsin. Double-pulse experiment was carried out at room temperature in aqueous suspension of dark- and light-adapted fragments of the purple membranes for different excitation laser light intensities. It is demonstrated that the route of light adaptation of the dark-adapted bacteriorhodopsin depends on laser light intensity used.     

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.     

THE EFFECT OF MOLECULAR POLARIZATION ON THE ELECTROCHROMISM OF CAROTENOIDS—I. THE INFLUENCE OF A CARBOXYLIC GROUP

Abstract— Electrochromism of oriented all- trans -β-apo-8'-carotenoic acid is studied in thin capacitors. The linear electrochromism is very strong, in contrast to that of symmetrical carotenoids. It is proportional to the first derivative of the absorption spectrum. The quadratic electrochromism can be described as a superposition of fractions proportional to the first and second derivatives of the absorption spectrum. The permanent dipole moment difference between the ground state and the excited state of the carotenoic acid molecule is Δμ= 3.6 × 10^-29 C·m (±20%) (10.7 Debyes). The polarizability difference parallel to the long axis of the molecule is Δα|| = 1.17 × 10^-37 C·m²·V^-1 (±20%) (1050 ų). Furthermore, the relative permittivity of the solid carotenoic ethyl ester is _r= 3.5 ± 0.2.
Δμ is due to the polarizing force of the carboxylic group. This force is equivalent to a mean local electric field of F _t≅3 × 10⁶V/cm. Such a "local field" may also be exerted on a symmetrical carotenoid in the membrane of photosynthesis, e.g. by asymmetrical complex formation with a polarizing molecule. To obtain an effective permanent field of F _p≅ 2 × 10⁶V/cm across the membrane, as postulated in photosynthesis, a local field of F _l≅ 5.5 × 10⁵ V/cm would be sufficient. F _p is shown to be directed from inside to outside of the thylakoid. To realize this, e.g. a positive polar (i.e. electron-attracting) complex partner of the carotenoid, located more to the inside of the thylakoid, can be postulated.     

FURTHER CHARACTERIZATION OF ANESTHETIC-TREATED PURPLE MEMBRANES

Abstract— The absorption maximum of bacteriorhodopsin is shifted from 568 nm to 480 nm when halogenated volatile anesthetics (enflurane; halothane) are added to purple membranes. Analysis of the rate of formation of this new species upon addition of the anesthetic and of the back-formation of native bacteriorhodopsin upon its removal indicate that in purple membranes, the dark-adapted chromophore is much less reactive than its light-adapted counterpart. Lipid-soluble molecules thus have a lower accessibility to the dark-adapted chromophore.
In addition, activity of the 480 nm bacteriorhodopsin was investigated. Flash and steady-state photolysis experiments reveal that this blue shifted chromophore has full photochemical activity. It has a meta-intermediate absorbing maximally at 380 nm. The photocycle ofBR–480 is mainly characterized by a slow decay of the "O" intermediate, enabling the direct observation of the branching reaction between the "M" intermediate and the parentBR–480 pigment.     

Control of particle alignment in water by an alternating electric field

We attempted to align a large number of silica particles dispersed in aqueous solution by controlling the alternating electric field between the two electrodes (400 microm apart). Relatively large particles (9.9 microm) were found to align forming strings in the direction parallel to the electric field while relatively small particles (2.0 and 4.9 microm) were observed to align making stripes in the direction perpendicular to the field. The number of stripes formed by particles between the electrodes increased with increasing frequency of the alternating field. This peculiar perpendicular particle alignment appeared when the contribution to particle alignment of electroosmotic flow exceeded that of dielectric polarization and the osmotic flow was found to be stronger around the particles than in the vicinity of the electrode surface.     

THE EFFECT OF A PERMANENT ELECTRIC FIELD ON THERMOLUMINESCENCE OF CHLOROPLASTS

Abstract— The effect of a permanent electric field on the thermoluminescence (TL) of pea chloroplasts was studied. The field was applied to suspensions at lowtemperatures(–15 to -40_oC) and to dry films of various humidity. A permanent electric field increased TL at low temperatures at the expense of the peaks above OA_oC. When the field strength was high, an increase in the quantum yield could be observed. When a field was applied at -15_OC, the band around +10_oC was reduced preferentially, but when a field was applied at – 40_AoC, TL bands emitted above OA_oC decreased to a similar extent. Analogies between the behaviour of solid suspensions and dry films of various humidity, and the isotope effect with D₂O, indicated that the efficiency and mode of action of an electric field on TL depends on the conformational mobility of the microsurroundings of the traps in the membrane.     

All-trans to 13-cis retinal isomerization in light-adapted bacteriorhodopsin at acidic pH

The flash photolysis kinetic spectra of the intermediate M(412) of bacteriorhodopsin were monitored during the process of acid titration. In the light-adapted state, the maximum peak amplitude of M(412) absorbance of bacteriorhodopsin decreased (pK(a)=3.40+/-0.05) as the pH decreased from 7.3 to 1.9. In the dark-adapted state, the maximum peak amplitude of M(412) absorbance of bacteriorhodopsin increased as the pH decreased from 6.9 to 4.1, and then decreased (pK(a)=2.85+/-0.05) as the pH dropped to 2.1. These different trends in the change in the maximum peak amplitude suggested that not only the transition of purple membrane to blue membrane had taken place in both light and dark-adapted states, but also the fraction of all-trans-bR had changed during the acid titration. The pH-dependent absorption changes at 640 nm of bacteriorhodopsin in both light- and dark-adapted states were also observed. The pK(a)-values of the purple-to-blue transition were 3.80+/-0.05 in light-adapted state and 3.40+/-0.05 in dark-adapted state, respectively. According to Balashov's method, the fraction of all-trans-bR was assayed as the pH decreased. All these results indicated that the purple-to-blue transition of light-adapted bacteriorhodopsin was accompanied by an all-trans to 13-cis retinal isomerization at acidic pH.     

Effect of phosphatidylglycerol depletion on the surface electric properties and the fluorescence emission of thylakoid membranes

To explore the possible effect of phosphatidylglycerol (PG) on the surface electric properties and chlorophyll fluorescence characteristics we used electric light scattering technique and 77 K chlorophyll fluorescence of thylakoid membranes from a cyanobacterium, Synechocystis PCC6803 (wild type) and its pgsA mutant defective in PG synthesis. We found a strong decrease in the permanent and induced electric dipole moments of the mutant thylakoids, following long-term PG depletion parallel with a decrease of the emission peak from PSI and an increase of the emission peak from PSII. Partial recovery of the electric state of thylakoid membranes was observed at re-addition of PG to the mutant cells depleted of PG for 21days. This change in the electric dipole moments is probably due to a decrease in PG content and progressive structural alterations in the macroorganization of the photosynthetic complexes induced by PG deprivation.

Our results suggest that the depletion of a lipid, which carries a negative charge, despite its small contribution to the overall lipid content, significantly perturbs the surface charge of the membranes. These changes are related with the chlorophyll fluorescence emission ratios of two photosystems and may partly explain our earlier results concerning the PG requirement for the function and assembly of photosystems I and II reaction centers.     

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.     

Orientation mechanism of palygorskite and kaolinite derived from the electric reversing pulse technique

Summary The investigation of electrooptical effects (birefringence, light scattering, dichroism) by the electric reversing pulse technique gives information on the directions of the electric moments of the particles. In case of Palygorskite and Kaolinite the experimental curves show a maximum at the instant when the field changes its polarity. The existence of this maximum demonstrates that the electric moments (permanent and induced moment) are oriented perpendicular to each other. The electric polarizability is parallel oriented to the long axis of the particles and the permanent dipole moment perpendicular to it. From the maximum the mean rotational diffusion coefficient of the particles has been calculated and the ratio of both electric moments in the case of Palygorskite after the theory ofI. Tinoco et al. andK. Yoshioka et al. has been obtained.

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Zusammenfassung Die Untersuchung elektrooptischer Effekte (Doppelbrechung, Lichtstreuung, Dichroismus) im elektrischen Feld mit rechteckförmigen Impulsen wechselnder Polarität gibt die Möglichkeit, die Richtung der elektrischen Momente der Teilchen zu ermitteln. Im Fall von Palygorskit und Kaolinit zeigen die experimentellen Kurven der Doppelbrechung ein Maximum beim Wechsel der Polarität des Feldes. Dieses Maximum bedeutet, daß die elektrischen Momente (permanentes und induziertes) senkrecht zueinander orientiert sind. Die elektrische Polarisierbarkeit liegt parallel zur langen Achse der Teilchen und das permanente Dipolmoment senkrecht dazu. Aus dem Maximum der Doppelbrechung ergibt sich für Palygorskit nach der Theorie vonI. Tinoco et al. und vonK. Yoshioka et al. die mittlere Rotations-Diffusionskonstante der Teilchen und das Verhältnis beider elektrischer Momente.

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With 2 figures     

Effect of surface-active substances on the surface charge density of purple membrane fragments

The surface charge density of purple membrane fragments and its alteration upon treatment of purple membranes with several surface-active substances [sodium dodecyl sulphate (SDS), cetylpyridinium chloride (CPC) and 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulphonate (CHAPS) were examined by use of 9-amino-acridine fluorescence.The value of the surface charge density of native purple membrane fragments (0.8 electric charges/nm²) obtained by this method is comparable to previously reported values and in agreement with the structural model of the purple membrane.An increase followed by a decrease in the negative surface charge density was observed after treatment of purple membranes with the negatively charged surfactant SDS within the concentration range 0–5 mM, whereas treatment with the positively charged surfactant CPC and zwitterionic derivative of cholic acid (CHAPS) led to a decrease in the surface charge density. The large reduction of the surface charge density after treatment of purple membranes with CHAPS (i.e. partial delipidation of purple membranes) proves the significant contribution of the negative charges of the lipid polar head groups to the negative surface charge of purple membranes.     

Pulsed Electric Linear Dichroism of Triphenylmethane Dyes Adsorbed on Montmorillonite K10 in Aqueous Media

Electric linear dichroism (ELD) spectra of two cationic triphenylmethane dyes, crystal violet (CV) and malachite green (MG), bound to sodium montmorillonite K10 (MK-10) were studied at 20 degrees C in aqueous media at two mixing ratios, D/S, of 0.10 and 0.24 in the 700- to 400-nm wavelength region and in the applied electric field strength range between 0 and 3 kV/cm. The specific parallel and perpendicular dichroism (Delta A( ||)/A and Delta A( perpendicular)/A) spectra of dye-adsorbed MK-10 suspension were measured at a fixed field strength with an apparatus equipped with a 512-channel photodiode array detector. By changing the field strength over a wide range, a series of the reduced dichroism values of the bound dyes were measured at a fixed wavelength. By fitting these dichroism values to theoretical orientation functions, the intrinsic reduced dichroism (Delta A/A)(int) spectra at the limiting high fields (ELD spectrum) were determined for CV and MG bound to MK-10. No appreciable difference was observed at the two D/S values. The ELD spectra of these bound dyes are undulatory but never constant, throughout their absorption region; thus, the dye plane does not lie flatly either on the surface or between layers of MK-10 particle. The isotropic absorption spectra, A, of bound CV and MG were each deconvoluted to eight partial absorption bands, which were grouped into three differently polarized transitions, i.e., one out-of-plane and two mutually perpendicular in-plane. The optical transition dipole moment direction of each group was found to make a considerable angle with respect to the symmetry axis of the disklike MK-10 particle, whose plane (or surface) tends to orient toward the applied electric field at the limiting high fields. By simulating the observed ELD spectra of bound CV and MG with those deconvoluted bands, the roll, tilt, and inclination angles of both dyes were evaluated quantitatively with a new analytical method. The average angles (+/-θ(R), +/- θ(T), |θ(N)|) are -(34-47) degrees, 34 degrees, and 51 degrees for bound CV and -44 degrees, 32 degrees, and 53 degrees for bound MG at two D/S values; thus, the triangular plane of each dye is rolled as well as tilted with a large inclination angle. Copyright 2000 Academic Press.