Molecular Requirement of Chlorosomal Chlorophylls. Self-Organization of a Chlorophyll Derivative Possessing a Hydroxyl Group at Ring II

Aggregation of zinc 7¹-hydroxyl-13²-demethoxycarbonyl-pheophytin a (Zn-7¹-OH-Chl) was examined in relation to the structure and function of the self-aggregates of 3¹-OH-type chlorophylls (Chi) in chlorosomes of green photosynthetic bacteria. The Zn-7¹-OH-Chl aggregates yielded a Q_y absorption band at 712 nm with a 1.2-fold larger width (full width at half maximum, 500 cm⁻¹) than the monomer's (420 cm⁻¹). Infrared and NMR spectroscopies revealed that each molecule in the aggregate links together with simultaneous coordination (C7¹-OH…Zn) and hydrogen bonding (C7¹-OH … O=C13¹). A nonlinear alignment of the constituent molecules in the oligomeric structure was assumed. Despite the similar molecular linkages, linearly aligned Q_y, moments in the Zn-3¹-OH-Chl aggregate gave a chlorosome-like broader, more redshifted Q_y band (740 nm; 670 cm⁻¹, 2.1-fold larger than the monomer's). Because it is advantageous for efficient light harvesting and energy transfer to have several Q_y, spectral components, spread over a wide spectral range, that can act as the energy gradient, it is concluded that not only the intermolecular linkages but the linear locations of OH, C=0 and Mg in the molecule are crucial for photosynthetic antenna of the self-assembled chiorosomal Chl.     

PHYTOCHROME INTERMEDIATES IN VIVO–II. CHARACTERISATION OF INTERMEDIATES BY DIFFERENCE SPECTROPHOTOMETRY

Abstract— In epicotyl tissue of Pisum , irradiation of P_r at – 196°C forms a stable product P₆₉₈, whereas P_fr forms a stable product P₆₅₀. On warming P₆₉₈, dark transformation to P_r predominates. On warming P₆₅₀ to – 70°C an intermediate P₆₉₀ is formed which bleaches on further warming to –10°C. When tissue is cooled to –196°C during actinic irradiation, difference spectra for subsequent warming to –10°C indicate that P_r, P_fr and an intermediate P₇₁₀ are formed from weakly absorbing intermediates. Complete photoconversion of P_r to P_fr is not possible at temperatures below –5°C. As the temperature is reduced, the amount of P_fr produced from P_r decreases, while P₇₁₀ increases. P₇₁₀ can be photoconverted at –20°C and above, ultimately forming P_r, but in contrast to P_fr it is not photoconvertible at –196°C.     

TEMPERATURE-SENSITIVITY OF LIGHT-INDUCED PHASE SHIFTING OF THE CIRCADIAN CLOCK OF NEUROSPORA

Two new mutants of Neurospora craasa , designated hth-1 and hth-2 , have been isolated which allow clear expression of the circadian conidiation rhythm at high temperature (36°C). Both strains showed single-gene segregation and produced similar phenotypes but mapped to different genetic loci. These mutants allowed an analysis of the effect of temperature on (1) light-induced phase-shifting of the circadian rhythm, (2) period length of rhythm, and (3) growth rate. The amplitude of the phase response curve to light was drastically reduced as the temperature was increased from 25°C to 34°C. Phase advances were decreased more than phase delays. As previously reported (Sargent et al. , 1966), the period length of the rhythm is temperature-compensated below 30°C ( Q ₁₀˜ 1) but not well-compensated above 30°C ( Q ₁₀ 1.3–1.7). The decrease in amplitude of the light phase response curve occurred in both temperature ranges. Furthermore, the Q ₁₀ value was lowered by addition of yeast extract in the high temperature range but not in the low range. Q ₁₀ values for growth rate also differed in these strains both in the low temperature range (25–30°C) and the high temperature range (30–34°C).     

ABSORPTION OF METHYLBACTERIOPHEOPHORBIDE a SINGLE CRYSTALS: SPECTRAL SHIFTS DUE TO π-π INTERACTIONS

Abstract— Polarized absorption spectra were obtained for a single crystal of methylbacteriophorbide a (MeBPhide a). The Q_y band is red-shifted ∽ 1660 cm^-1 (∽ 110 nm) relative to MeBPhide a in a CH₂Cl₂/benzene solution. This is equivalent to the largest red shifts observed for in vivo bacteriochlorophyll a. The Soret band exhibits a smaller red shift and a significant reduction in intensity, and the Q_x band is not observed. The crystal spectra are qualitatively similar to spectra reported for several other aggregated (bacterio)chlorophyll and bacteriopheophytin systems. Since crystalline MeBPhide a contains no Mg, water or hydrogen bonding (Barkigia etal. , 1981), these results demonstrate that the spectral changes associated with the aggregation of photosynthetic chromophores can arise solely from IT-IT interactions between macrocycles.     

PHYTOCHROME INTERMEDIATES IN VIVO– III KINETIC ANALYSIS OF INTERMEDIATE REACTIONS AT LOW TEMPERATURE

Abstract— Kinetic experiments have provided evidence for a series of light and dark reactions of phytochrome intermediates at low temperature in Pisum epicotyl tissue. A photoequilibrium exists between P_r and P₆₉₈, and between P_fr and P₆₅₀. A dark reversion of P₆₉₈P_r and P₆₅₀p_fr at –70°C has been demonstrated. When cooled to 70°C under incandescent light, most of the phytochrome in the tissue is driven into photochemically unreactive intermediates. About 2% of the phytochrome remains as weakly absorbing intermediates that form P_r and P_fr in darkness. A scheme is presented for phytochrome phototransformation in vivo.     

PHYTOCHROME INTERMEDIATES IN VIVO-I. EFFECTS OF TEMPERATURE, LIGHT INTENSITY, WAVELENGTH AND OXYGEN ON INTERMEDIATE ACCUMULATION

Abstract— Accumulation of weakly absorbing phytochrome intermediates has been demonstrated in Pisum epicotyl tissue under conditions of pigment cycling using a quasi-continuous measuring spectrophotometer. An action spectrum shows 690–700 nm to be the most efficient wavelength range in this process. Difference spectra for the decay of intermediates maintained by 690 nm light show that, if the experiment is done at 0°C, only P_fr is formed. At – 11°C, intermediates decaying to P_r can also be observed. At – 20°C, P_r is produced as well as a pigment with peak absorption at 710nm. Kinetic analysis of intermediate decay at – 11°C reveals that at least two intermediates are maintained by 690 nm light. The level of intermediate maintained by incandescent light at 0°C was 25% higher in air than in nitrogen.     

MODIFYING FACTORS OF THE CELLULAR CONCENTRATION OF PHOTOLYASE MOLECULES IN Saccharomyces cerevisiae CELLS–I. EFFECTS OF TEMPERATURE AND LIGHT

Abstract— The effects of temperature and light on the cellular concentration of photoreactivating enzyme (PRE) molecules in haploid Saccharomyces cerevisiae cells were investigated. (1) Temperature effect: The number of active PRE molecules per cell (N_PRE) in cells grown at 37°C was about 13% of that grown at 23°C, although the amount of proteins per cell remained the same. (2) Light effect: N_PRE in cells grown in light was about 2.8 times larger than that grown in the dark. The value of N_PRE in cells grown in the light decreased more rapidly during holding in buffer in the dark than in the light. The N_PRE decrease during holding in buffer in the dark was more rapid in cells grown in the light than grown in the dark. A comparable decrease was observed after holding in buffer in the presence of cycloheximide. (3) In cells harboring a plasmid containing the gene PHR1, N_PRE was larger in cells grown at 23 than at 30°C.     

FLASH INDUCED OSCILLATION OF THE LOW TEMPERATURE THERMOLUMINESCENCE BAND Zv IN CHLOROPLASTS

Using preilluminating flashes at 2°C prior to continuous excitation of isolated chloroplasts at -80°C, a period-four oscillation with maxima at the 0th, 4th and 8th flashes was observed in the amplitude of the thermoluminescence band Z_v. Inactivation of the water-splitting system by hydroxylamine- or Tris-treatments greatly diminished the Z_v band. On the other hand, its amplitude was not considerably affected by the addition of inhibitory concentration of DCMU. On the basis of the results, the possible origin of the Z_v band is discussed.     

THE CHROMOPHORES AS ENDOGENOUS SENSITIZERS INVOLVED IN THE PHOTOGENERATION OF SINGLET OXYGEN IN SPINACH THYLAKOIDS

Abstract— The photogeneration of singlet oxygen (¹O₂) from thylakoids and the chromophores involved as endogenous sensitizers were investigated using chloroplasts and thylakoids isolated from spinach. The blue light-induced inhibition kinetics of photosynthetic electron transport and that of CTvCF, ATPase were also studied. The spectral dependence of the generation of ¹O₂ from thylakoid membranes, measured by the imidazole plus RNO method, clearly demonstrated that the Fe-S centers play an important role in ¹O₂ generation, acting as sensitizers in thylakoids. The photoinhibition of the electron transport in isolated chloroplasts was strikingly depressed by a lipid-soluble '0₂ quencher and enhanced by deuterium oxide substitution, indicating that the inhibition processes are mainly mediated by ¹O₂ which is produced via photodynamic activation. The involvement of chloroplast cytochromes in the production of ¹O₂ was deduced from the action spectrum for the photodynamic inhibition of the electron carrier chain. The results obtained from the kinetic studies appear consistent with the involvement of some components such as the Fe-S centers and cytochrome chromophores of the carrier chain in the generation of ¹O₂.     

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.     

TIME RESOLUTION OF A BACK PHOTOREACTION IN BACTERIORHODOPSIN

Abstract— The back photoreaction from the M(412nm) intermediate in the photocycle of light-adapted bacteriorhodopsin, BR_LA(570 nm), is studied using pulsed laser excitation. The decay of a primarily produced species, M_P, regenerates BR_LA(570nm) in a process characterized by a half life of 200 ns at 25°C. The absorption maximum of M_P is blue shifted (Λ_max≃ 395 nm) relative to that of M(412nm). The primary photochemical step, M(412nm) → M_P, is attributed to a conformational change in the polyene residue. The energy and entropy of activation of the subsequent M_P→ BR_LA (570 nm) relaxation are reported and discussed.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

POLARIZED FLUORESCENCE OF 5-METHYLCYTOSINE SPECIES IN SOLUTION AT ROOM TEMPERATURE

Fluorescence yields (π_f,'s) and polarizations ( P ) are measured for aqueous 5-methylcytosine (˜ 0.1 m M ) at 20°C as a function of pH over the range 2–14. Both properties change abruptly and in parallel at pH's corresponding to the known pK_a values. Polarizations were also obtained for the 5-methylcytosine cation, neutral and anion species in ethylene glycol water glass at ˜180K. The weak fluorescence of the neutral and cation species at 20°C was polarized almost as highly as at low temperature. When the fluorescence lifetimes are assumed to be correctly given by the product of calculated radiative lifetimes and quantum yields, the polarizations are found to be consistent with rotational diffusion rates ˜4 times faster than predicted from Stokes-Einstein models for the neutral and anion species. The cation seemed to rotate about two times more slowly than the neutral and anion species. It was also shown that the properties of the three species are such that a plot of 1/P vs apparent π_f in the pH range 2–11 is fortuitously linear.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

THE TEMPERATURE DEPENDENCE OF PHYTOCHROME TRANSFORMATIONS

Abstract— The kinetics of phytochrome transformation were examined over a 30°C temperature range (+5° to–25°C) in 75% glycerol. Two new intermediate reaction stages are described for the transformation of the red-absorbing form, P _r to the far-red absorbing form, P _fr The free energies, enthalpies and entropies of activation were obtained for five of the six reaction stages observed in the transformation of P _r to P _fr and for the two reaction stages observed in the reverse process. All exhibited positive entropies of activation with the maximum being 25 entropy units. The results suggest that the phototransformations between P _r and P _fr consist of relaxation processes beginning with the intermediate produced immediately upon absorption of a quantum of light and ending with either P _r or P _fr.     

ACTION OF HYDROGEN PEROXIDE ON HUMAN FIBROBLAST IN CULTURE

Abstract— Human fibroblasts in culture lose the capacity of proliferating when exposed to hydrogen peroxide in the concentration range of 1 to 10 μ M . The toxicity of H₂O₂ to xeroderma pigmentosum cells (XP12RO). defective in excision repair of lesions produced by UV-irradiation, was about twice as high as to cells proficient in excision repair (VA13). This compound produces single-strand breaks in intracellular DNA but not in purified DNA. These breaks are in situ physical discontinuities rather than alkali-labile bonds, and their generation occurs at the same extent at 4°C and 37° indicating that they are not produced by an endonuclease. The results favor the hypothesis that H₂O₂ reacts in the cell producing a radical species which brings about the formation of DNA single-strand breaks. These breaks are effectively repaired by both XP12RO and VA13 fibroblasts. The possible reason for the lethality of H₂O₂ is discussed.     

PHOTOCHEMISTRY OF RETINOCHROME

Abstract— Retinochrome is a photopigment found in the visual cells of cephalopods. It has been considered to act as a supplier of the 11- cis -retinal required for synthesis of rhodopsin, because its all-trans chromophore is isomerized to 11- cis form in the light. Light and thermal reactions of squid retinochrome were investigated by low-temperature spectrophotometry.
On irradiation with green light at liquid-nitrogen temperature, retinochrome (λ_max 496 nm, – 190°C) is converted mainly to an intermediate lumiretinochrome (λ_max 475 nm, – 190°C), its chromophore being changed to 11- cis -retinal. On irradiation with blue light at - 190°C, retinochrome is changed to a photosteady–state mixture (λ_max 487 nm, – 190°C) composed mainly of retinochrome and lumiretinochrome, since lumiretinochrome is partially regenerated back to retinochrome. Similarly, irradiation of lumiretinochrome with blue light also results in the same photosteady-state mixture, which can be completely reverted to lumiretinochrome on re-irradiation with green light.
Lumiretinochrome is stable at a wide range of temperatures from – 190°C to about – 20°C. Above – 20°C, it is further converted, thermally, into metaretinochrome (λ_max 470 nm), which is the same bleached product as has been observed on irradiation of retinochrome at room temperatures. Thus, the light-bleaching process of retinochrome is rather simple compared with that of rhodopsin.     

MODIFYING FACTORS OF THE CELLULAR CONCENTRATION OF PHOTOLYASE MOLECULES IN Saccharomyces cerevisiae—II. EFFECTS OF PREILLUMINATION WITH LIGHT FLASHES

Abstract The effects of preillumination with photoreactivating light flashes before UV-irradiation on the number of photoreactivable complexes consisting of UV-induced DNA-damages and active photolyase molecules (N_PREact), on the fluence decrements, ΔD_PRE, that are obtained from two UV-survival curves without and with 1 flash photoreactivation and proportional to N_PREact were determined in haploid Saccharomyces cerevisiae cells. ΔD_PRE increased by preillumination from 0.115 Jm^–2 to 0.460 Jm^–2 and from 0.376 Jm^–2 to 0.494 Jm^–2 in cells in logarithmic growth phase and in stationary growth phase, respectively. Δ_PRF in log-cells that were preilluminated before and after resuspension in buffer at 40°C for 60 min was larger than ΔD_PRE in log-cells preilluminated only after resuspension in buffer.     

CHLOROPHYLL a FLUORESCENCE YIELD IN LIVE CELLS AND SOLUTIONS

Abstract— Fluorescence yields of chlorophyll a in ether and methanol solutions ( F_s ), on the one hand, and in Chlorella pyrenoidosa and Anacystis nidulans cells (F_c), on the other hand, were determined by excitation at 600 mμ (instead of at 436 mμ, as was done in earlier research in our, and other, laboratories). The ratio of the yields Fs/F_c , was found to be 5.9 for Chlorella (compared to an ether solution of chlorophyll a ), and of 4.5 (as compared to a methanol solution—not too different from the corresponding ratios of fluorescence lifetimes, τ_s /τ_c which were determined earlier as 3.1 and 4, respectively. The much higher values of the yield ratio, previously reported for Chlorella (about 13, compared to chlorophyll a in ether solution), may have been due to disregard of light absorption in carotenoids in live cells; and perhaps also to quenching of chlorophyll excitation by carotenoids. The latter can occur (as suggested in an earlier publication) when chlorophyll is excited to its second singlet excited state.
For Anacystis , the yield ratios were now found to be 5.1 and 3.8, when compared to ether and methanol solutions respectively; while the previously determined lifetime ratios were 3.7 and 4.9, respectively. It remains to be seen whether the remaining differences between the lifetime ratios and the yield ratios are real and significant.     

Structure-Based Calculations of the Optical Spectra of the LH2 Bacteriochlorophyll-Protein Complex from Rhodopseudomonas acidophila

Abstract— The molecular structure of the light-harvesting complex 2 (LH2) bacteriochlorophyll-protein antenna complex from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas acidophila , strain 10050 provides the positions and orientations of the 27 bacteriochlorophyll (BChl) molecules in the complex. Our structure-based model calculations of the distinctive optical properties (absorption, CD, polarization) of LH2 in the near-infrared region use a point-monopole approximation to represent the BChl Q_y transition moment. The results of the calculations support the assignment of the ring of 18 closely coupled BChl to B850 (BChl absorbing at 850 nm) and the larger diameter, parallel ring of 9 weakly coupled BChl to B800. All of the significantly allowed transitions in the near infrared are calculated to be perpendicular to the C9 symmetry axis, in agreement with polarization studies of this membrane-associated complex. To match the absorption maxima of the B800 and B850 components using a relative permittivity (dielectric constant) of 2.1, we assign different site energies (12 500 and 12260 cm⁻¹, respectively) for the Q_y transitions of the respective BChl in their protein binding sites. Excitonic coupling is particularly strong among the set of B850 chromophores, with pairwise interaction energies nearly 300 cm between nearest neighbors, comparable with the experimental absorption bandwidths at room temperature. These strong interactions, for the full set of 18 B850 chromophores, result in an excitonic manifold that is 1200 cm⁻¹ wide. Some of the upper excitonic states should result in weak absorption and perhaps stronger CD features. These predictions from the calculations await experimental verification.