REACTIVITY OF CHLOROPHYLL a/b-PROTEINS AND MICELLAR TRITON X-100 COMPLEXES OF CHLOROPHYLLS a OR b WITH BOROHYDRIDE

The reaction of several plant chlorophyll-protein complexes with NaBH₄ has been studied by absorption spectroscopy. In all the complexes studied, chlorophyll b is more reactive than Chi a, due to preferential reaction of its formyl substituent at C-7. The complexes also show large variations in reactivity towards NaBH₄ and the order of reactivity is: LHCI > PSII complex > LHCII > PSI > P700 (investigated as a component of PSI). Differential pools of the same type of chlorophyll have been observed in several complexes.
Parallel work was undertaken on the reactivity of micellar complexes of chlorophyll a and of chlorophyll b with NaBH₄ to study the effect of aggregation state on this reactivity. In these complexes, both chlorophyll a and b show large variations in reactivity in the order monomer > oligomer > polymer with chlorophyll b generally being more reactive than chlorophyll a. It is concluded that aggregation decreases the reactivity of chlorophylls towards NaBH₄ in vitro, and may similarly decrease reactivity in naturally-occurring chlorophyll-protein complexes.     

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.     

ON THE FORMATION OF PHOTOSYNTHETIC MEMBRANES IN BEAN PLANTS*

Abstract— The formation of lamellar chlorophyll-protein complexes I and II, solubilized by sodium dodecyl sulfate, was studied by hydroxylapatite column chromatography during greening of etiolated Phaseohis vulgaris leaves.
The protein moiety of both complexes preexists in the prolamellar body of etiolated tissue. The complex II to complex I protein ratio is of the order of 0.5. During greening in intermittent illumination the 'proto'-chloroplast is agranal, and contains 'primary' thylakoids and chlorophyll a (Chl a ). At this stage the complex II to complex I protein ratio increases only slightly. Further greening of the plant tissue in continuous illumination results in grana, Chi b (chlorophyll b ) and more Chl a formation. The complex II to complex I protein ratio in unfractionated thylakoids is now of the order of 2.5, while in grana it is of the order of 4.0.
The binding of chlorophyll formed during greening to the protein moiety of the two complexes is found to be selective. The Chi a selectively formed under intermittent illumination is more strongly bound to the complex I protein. The Chi b and Chl a formed in continuous illunination are found bound to both complex I and complex II proteins.
Analysis by hydroxylapatite column chromatography of subchloroplast fractions obtained by different fractionation procedures have shown that these two chlorophyll-protein complexes are most probably derived from the PSI (photosystem I) and PSII (photosystem II) particles of the photosynthetic membrane. These findings suggest that PSI units are assembled ahead of PSII units. Moreover, they indicate that the complex I protein is the main protein component in the prolamellar body membranes, the 'primary' thylakoids. and the stroma lamellae, while in the grana membranes the major protein is the complex II protein. Finally our results show that formation of the photosynthetic membranes is a multi-step process.     

EVIDENCE FOR A HETEROGENOUS ORGANIZATION OF VIOLAXANTHIN IN THYLAKOID MEMBRANES

Abstract Two functionally different species of violaxanthin have been observed in thylakoid membranes, one that can be de-epoxidised to zeaxanthin under light and one not available for light-induced zeaxanthin formation (Siefermann, D. and H. Y. Yamamoto, 1974, Biochim. Biophys. Acta 357 , 144–150). Here the distribution of available and unavailable violaxanthin is examined between membrane subfractions obtained from Triton X-100 solubilized spinach thylakoids by isoelectric focusing: (1) Only 40% of the available violaxanthin is detected in isolated Chl-proteins, while the residual 60% occur in a fraction of'free'pigments; (2) Almost 80% of the unavailable violaxanthin is recovered from the light-harvesting Chl a/b -protein complex (36%) and from photochemically active complexes containing photosystem I (20%) or photosystem II (20%). The results suggest a heterogenous organization of available and unavailable violaxanthin in thylakoid membranes.     

THE STRUCTURAL CHANGES IN BOVINE ROD OUTER SEGMENTS IN THE PRESENCE OF ATP

Abstract— We have, previously, described a light-induced near infrared (700–850 nm) light scattering transient obtained in the presence of ATP from bovine rod outer segments suspensions in which the plasma but not the disk membranes were perforated (Uhl et al ., 1979a). This transient was termed the 'A' signal. To elucidate its possible origin, we have analyzed their angular and wavelength dependencies. These data have been compared with osmotically controlled (non-light) induced light scattering changes from identical control rod outer segments suspensions. It has been found that A_D (the dark light scattering signal obtained in the presence of ATP) and A_LS (the slow component of the actinic flash induced light scattering signal, A_L) can be assigned to the swelling of the disk membranes while A_Lf (the fast component of this latter signal) can be attributed to the change in refractive index of the ROS caused by the hypsochromic spectral shift of photolyzed rhodopsin. The collective disk swelling associated with A, and A_LS is consistent with the pumping of ions into the disk lumen by the action of a disk membrane bound ATPase.     

IN VIVO FLUORESCENCE SPECTROSCOPY OF CHLOROPHYLL IN VARIOUS UNRIPE AND RIPE FRUIT

—Low temperature (77 K) fluorescence emission spectra of slices obtained from the peel and various layers of the pericarp were recorded for fruits which remain green or undergo color break during ripening.
Fluorescence emission peaks characteristic of the photosystem II antennae (λ_F 686 nm) and reaction center (λ_F 696 nm), as well as of the photosystem I antenna (λ_F 730-740 nm), were present in the peel and all parts of the green pericarp of ripe kiwi, avocado and cantaloupe, as well as in ripe tomato and tangerine after color break. The pattern of the fluorescence emission spectra of all samples except that of the kiwi fruit was similar to that obtained from green photosynthetic tissue of leaves, indicating a normal organization of the chlorophyll-containing complexes of thylakoidal membranes. This pattern is characterized by a significantly higher emission at 730-740 nm relative to that of the 696 and 686 nm peaks. In contradistinction, the fluorescence emission at 686 and 696 nm was higher than that at 730 nm in the kiwi fruit, indicating a reduction in the size of the photosystem I antenna chlorophyll. In the innermost yellowish layers of the kiwi pericarp, a further loss of this antenna occurred, as well as disorganization of the photosystem II complex. The above conclusions are suggested also by measurements of variable fluorescence kinetics.
The results presented here indicate that fluorescence spectroscopy might be used as a tool for the study of chlorophyll organization during the growth and ripening periods of fruit.     

COMPLEX FORMATION BETWEEN ALCOHOLS AND THE AROMATIC HYDROCARBONS PYRENE AND 1-METHYLPYRENE

Abstract—Pyrene and 1-methylpyrene have been shown by infrared spectrometry to form 1:I molecular complexes with alcohols at a concentration of 0.02 M in CCl₄ solutions. The association constants are of the order of 1 M^-1. The fluorescence decay profiles of 10μM pyrcnc in dilutc butanol-heptane solutions have been found to be the sum of two exponential components in agreement with ground state complex formation     

In situ SUSCEPTIBILITIES OF PHOTOSYSTEMS I AND II TO PHOTOSENSITIZED DEACTIVATION via SINGLET OXYGEN MECHANISM

Abstract— A comparative study was carried out on the in situ susceptibilities to photoinactivation of the photosystem I (PS I) and II (PS II) complexes of spinach thylakoids treated with efficient type II sensitizers. While the presence of the exogenous sensitizers caused a substantial increase in the extent of photoinactivation of whole chain electron transport, it did not affect PS I activity of thylakoids in light but exerted an enhanced photoinactivating effect only on PS II. The measurements of the action spectrum for the inhibition of PS II activity of the sensitizer-incorporated thylakoids and that for the generation of singlet oxygen (¹O₂) from them revealed that photosensitized inactivation of PS II is directly related to the photoproduction of ¹O₂ in thylakoid membranes. The results obtained in the present work clearly demonstrate an exceptional sensitivity of PS II to ¹O₂, providing circumstantial evidence that high light-induced damage to PS II may result from photosensitization reactions mediated by ¹O₂, which is not necessarily produced within the PS II complex.     

QUENCHING OF CHLOROPHYLL FLUORESCENCE BY NITROBENZENE

Abstract—Nitrobenzene quenching of chlorophyll fluorescence in ethanol has been investigated. Steady state relative quantum yields have been measured and fluorescence decay rates were determined using both nanosecond photon counting and picosecond pulses from a mode-locked Nd³⁺ glass laser.
The fluorescence decay is described by
1( t )= I ₀ exp (- t/τ−At^1/2 )
the form predicted for decay governed by the kinetics of the continuum model of diffusion controlled reactions. From the parameters of the fluorescence decay, the encounter distance is 5–7 A° the mutual diffusion coefficient is 0.62 × 10^--⁵ cm2s^-1± 12%.
Some of the fluorescence quenching is also attributed to static quenching by a nitrobenzene-chlorophyll, ground-state complex. The equilibrium constant for formation of this ground-state complex was determined to be 4.1 M ^-1. The combined dynamic and static quenching model allows calculation of quantum yields of fluorescence in good agreement with the experimentally determined quantum yields.     

APPEARANCE OF PHOTOPHOSPHORYLATION CAPACITY: THRESHOLD vs GRADED CONTROL BY PHYTOCHROME

Abstract— It is shown that in attached mustard cotyledons graded control of chlorophyll synthesis by physiologically active phytochrome (P_fr) and threshold control by P_fr of the 'potential capacity' to photophosphorylate are totally different phytochrome actions even though both controls are essential for the build-up of the same functional complex, the machinery for photophosphorylation. The essential findings are as follows: The action of P_fr (made by a 1 min red light pulse) on the capacity and efficiency of photophosphorylation is rapid—detectable after 15 min and completed after 30 min—whereas the action of P_fr on chlorophyll formation is slower—only detectable 45 min after the original red light pulse (R). Detailed escape studies (loss of full reversibility of the inductive effect of a R pulse by far-red) show that the effect of a R pulse on chlorophyll synthesis remains fully reversible for 45 min whereas the action of P_fr on the capacity for photophosphorylation is very fast (occurring within 2 min). Control of capacity for photophosphorylation is a threshold response (whereby the threshold value is approximately 1.25% P_fr based on total phytochrome at 36 h = 100%) whereas control by P_fr of chlorophyll synthesis is graded. Control of capacity for photophosphorylation by P_fr only operates if the hypocotyl hook is connected to the cotyledons for at least 2 min after the inductive R pulse, i.e. until full escape from reversibility has occurred, whereas chlorophyll formation in the cotyledons is not affected by the separation of hook and cotyledons.     

USE OF THE FLUORESCENT LANTHANIDE Tb3+ AS A PROBE FOR CATION-BINDING SITES ASSOCIATED WITH ISOLATED CHLOROPLAST THYLAKOID MEMBRANES

Abstract— The fluorescent properties of the rare-earth ion, Tb³⁺ have been utilized to probe the nature of cation-binding sites associated with thylakoid membranes. At low concentrations (< 100μ M ), Tb³⁺ was observed to inhibit the increase in chlorophyll a fluorescence normally seen on adding 5 m M MgCl₂ (or 100 m M NaCl) to isolated, broken chloroplasts. We also observed under these conditions, the appearance of a new band around 280 nm in the excitation spectrum of Tb³⁺ ion fluorescence. However, similar changes in Tb³⁺ fluorescence could be observed in the presence of a membrane-free preparation of chloroplast coupling factor protein (CF₁). From this and other results it is concluded that changes in Tb³⁺ fluorescence reflect an association of the ion with CF₁ followed by intermolecular transfer of excitation energy from protein ligands (possibly un-ionized tyrosine residues) to the lanthanide. The interaction of Tb³⁺ with sites which control chlorophyll a fluorescence does not seem to modify Tb³⁺ fluorescence, suggesting that in this case, simple membrane-bound ligands such as carboxyl or phosphate groups are involved.     

SPECTRAL CHARACTERIZATION OF LIGHT-REDUCIBLE CYTOCHROME IN A PLASMA MEMBRANE-ENRICHED FRACTION AND IN OTHER MEMBRANES FROM CAULIFLOWER INFLORESCENCES

Abstract— Low temperature spectroscopy has been used to characterize microsomal fractions obtained from cauliflower inflorescences ( Brasska oleracea L.) by differential centrifugation and partition in an aqueous polymer two-phase system. The plasma membrane-enriched fraction (U₃) was found to contain one dominant b -cytochrome, which could be reduced both by blue light and by dithionite. An action spectrum of the blue light-induced absorbance change [LIAC, Δ(A₄₃₀—A₄₁₀)] associated with the reversible reduction of this b -type cytochrome indicated that the primary light-receptor was a flavin-like compound. Another microsomal fraction (L₃) containing membranes from mitochondria, endoplasmic reticulum and other organelles also contained light-reducible cytochrome. One of these could be identified as cytochrome c oxidase, and another may be identical to cytochrome b ₅ of the endoplasmic reticulum.     

CHLOROPHYLL b: AN INTEGRAL COMPONENT OF PHOTOSYSTEM I OF HIGHER PLANT CHLOROPLASTS

Abstract— An undissociated photosystem I complex may be isolated from spinach thylakoids by mild gel electrophoresis (CP1a) or Triton X-100. CP1a has a Chl a / b ratio of 11 and a Chl/P700 ratio of 120. while the Triton X-100 PS I complex (Chl a / b ratio of 5.9) has a larger antenna unit size (Chl/P700 ratio of 180). None of the Chl a / b -proteins of the main light-harvesting complex (apoproteins of 30–27 kD) are present in CP1a, and they account for less than 10% of the total chlorophyll in the Triton X-100 PS I complex. Instead, these PS I complexes have specific, but as yet little characterized, Chi a / b -proteins (apoproteins in the 26–21 kD range). With both PS I complexes, Chi b transfers light excitation to the 735 nm low temperature fluorescence band characteristic of photosystem I. We suggest that Chi b is an integral but minor component of photosystem I.     

PHOTORESPONSES OF SEMICONDUCTOR POLYCRYSTALLITES SEPARATING TWO AQUEOUS SOLUTIONS

Solar cells using polycarbonate membranes, with CdS deposited on them, were made by a very simple way; the CdS-containing membrane separates a Lucite cell into two compartments. On illumination, about 150 mV photovoltage ( V _op) and 0.5 μA cm^-2 photocurrent ( I _sc) could be produced; one side of the membrane acted as photoanode, and the other side as photocathode. By means of coating Victoria Blue B (VBB) onto the membrane before CdS deposition, the maximum V _op and I _sc of the CdS-deposited membrane could reach 500 mV and 3.0 (μA cm^-2, respectively. A mixture of CdS and CdSe deposited membrane has also been tested and found to have both the advantages of high photovoltage (over 400 mV) and good stability after modification. Even more interesting results were also obtained with CdSe pellets in place of the CdS-deposited membrane, in which V _op and I _sc of the cell were 1.2 V and 6 mA cm^-2, respectively. The essential aspect of the system, modelled after the photosynthetic thylakoid membrane, contains an asymmetrical, ultrathin semiconductor crystallite layer separating two aqueous solutions.     

LASER PHOTOLYSIS OF 3-METHYLLUMIFLAVIN

Abstract— Using high-intensity actinic light, the chlorophyll a fluorescence transient from HCO^-₃-depleted chloroplasts shows a rapid initial rise (O → I) followed by a slow phase (I → P). In the presence of HCO^-₃, the O → I rise is delayed but the I → P phase is much more rapid. Using low-intensity actinic light, the chlorophyll a fluorescence transient from 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU)-treated chloroplasts is delayed in the presence of HCO^-₃. Bicarbonate increases the amount of delayed light emission from chloroplasts given 10 s illumination with weak blue light (0·4 W/m²). DCMU greatly increases the amount of delayed light seen in the presence of HCO^-₃ under these conditions but decreases the amount seen in the absence of HCO^-₃. It is suggested that HCO^-₃ may somehow form or stabilize, in the dark, a number of reaction centers corresponding to the S₁ state in the model of B. Forbush, B. Kok and M. McGloin ( Photochem. Photobiol. 14, 307–321, 1971).     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN IN SOLUTION

Abstract— Bimolecular rate constants for the quenching of singlet oxygen O*₂(¹Δ_g), have been obtained for several transition-metal complexes and for β-carotene. Laser photolysis experiments of aerated solutions, in which triplet anthracene is produced and quenched by oxygen, yielding singlet oxygen which then sensitizes absorption due to triplet carotene, firmly establishes diffusion-controlled energy transfer from singlet oxygen as the quenching mechanism in the case of β-carotene. The efficient quenching of singlet oxygen by two trans-planar Schiff-base Ni(II) complexes, which have low-lying triplet ligand-field states, most probably also occurs as a result of electronic energy transfer, since an analogous Pd(II) complex and ferrocene, which both have lowest-lying triplet states at higher energies than the O*₂(¹Δ_g), state, quench much less effectively.     

OXYGEN QUENCHING OF CHLOROPHYLL FLUORESCENCE IN CHLOROPLASTS

Abstract— Three phases of chlorophyll a fluorescence quenching by O₂ are observed in green plants. The effects of various inhibitors on photosynthetic partial processes in chloroplasts were investigated in attempts to (1) localize the O₂-quenching sites and (2) assess possible physiological significance of O₂-quenching. Our results localize the most sensitive (and presumably functionally important) phase to a site between plastoquinone and the photosystem I acceptor, chlorophyll (P₇₀₀), possibly plastocyanin. It is suggested that PC may transfer electrons to oxygen in addition to P₇₀₀.     

ENHANCEMENT OF INTRAMOLECULAR EXCIMER FORMATION OF 1,3-BICHROMOPHORIC PROPANES VIA APPLICATION OF HIGH PRESSURE and VIA COMPLEXATION WITH CYCLODEXTRINS. PROTECTION FROM OXYGEN QUENCHING

Abstract— The emission of several 1,3-bichromophoric(BC) systems has been investigated in aqueous solutions containing various cyclodextrins (CD's). In all cases where molecular models reveal that the correspondence of the size and shape of the eclipsed conformation of the BC system and the cavity CD is high, a stable CD (host)-BC (guest) complex is formed and in these cases, the ratio of excimer to monomer emission intensity UE/IM) is much larger than that found in homogeneous solution. The effect of application of high pressure on the I_E/I_M ratio was investigated, as was the influence of 0₂ quenching of excimer and monomer in homogeneous solutions, in aqueous micellar solutions, and in aqueous solutions of CD complexes. A strong protection from 0₂ quenching is observed in the latter case.     

A PULSED LASER AND PULSE RADIOLYSIS STUDY OF AMPHIPHILIC CHLOROPHYLL DERIVATIVES WITH PDT ACTIVITY TOWARD MALIGNANT MELANOMA

Two amphiphilic derivatives of chlorophyll, which have high potential as photodynamic therapy sensitizers for malignant melanoma have been investigated by a combination of laser flash photolysis and pulse radiolysis. It is shown that direct excitation of monomeric forms of these molecules in both hydrophilic and hydrophobic environments produces significant yields of the corresponding triplet states, which have been characterized in terms of spectral and kinetic parameters. In both environments, scavenging of the triplets by oxygen produces singlet oxygen, O₂(^lΔ₈), with essentially unit efficiency as evidenced by time-resolved IR luminescence measurements.     

FLUORESCENCE LIFETIMES OF CHLOROPHYLL a: SOLVENT, CONCENTRATION AND OXYGEN DEPENDENCE

Fluorescence lifetimes (τ_f) of chlorophyll a (Chi a ) have been measured by the single-photon-counting technique over a wide range of concentrations (˜10^-7˜10^-4 M ) in deoxygenated pyridine, diethyl ether, toluene and methanol. At pigment concentrations ˜1 μ M , reabsorption of fluorescence induces significant artifacts on measured values of τ_f which are dependent on detection wavelength and the specific geometry of the experiment. There is a clear dependence of τ_f on the nature and degree of solvation, including both coordination of the central magnesium and hydrogen-bonding of the solvent (viz. alcohols) to the macrocycle. Quenching of the excited singlet state by molecular oxygen was measured quantitatively in ether, and a bimolecular rate constant markedly slower than the diffusion-controlled limit was obtained.