Circular dichroism studies on the structure and the photochemistry of protochlorophyllide and chlorophyllide holochrome

On the basis of absorption and circular dichroism (CD) spectral measurements, we conclude that the photoreduction of protochlorophyllide to chlorophyllide in homogenates of etiolated bean seedlings (Phaseolus vulgaris L.) involves two light steps in series. Before illumination, the active protochlorophyllide occurs in a dimeric form in the holochrome protein. The initial light reaction converts one of the protochlorophyllide molecules and forms a chlorophyllide-protochlorophyllide holochrome intermediate with a weak, characteristic CD spectrum. The second light reaction subsequently converts the second protochlorophyllide in a less efficient reaction that is temperature dependent. This produces a chlorophyllide holochrome which exhibits a strong double CD characteristic of dimers and which is stable below 1°C. At higher temperatures this dimeric chlorophyllide transforms in the dark to a monomeric form with low CD amplitude. Sucrose at high concentrations (2 M) alters the chlorophyllide holochrome CD spectrum and prevents the final dark dissociation step. Analysis of the photochemical kinetics confirms the occurrence of the two-step photoreduction and supports the stoichiometry of two (proto)chlorophyllides per holochrome protein.     

Purification of protochlorophyllide holochrome

Phototransformable protochlorophyllide holochrome was prepared from etiolated bean leaves. The detergent Triton X-100 in the presence of glycerol and tricine-KOH buffer (pH 8) enhanced the extractability, specific activity, and phototransformability of the holochrome. Purification was achieved by polyethylene glycol-6000 precipitation and hydroxyl-apatite, DEAE-cellulose, and agarose chromatography. The presence of Triton X-100 permitted removal of the carotenoid contamination from the holochrome. The 678-nm absorption maximum of newly formed chlorophyllide a holochrome shifts to 672 nm in a temperature-dependent manner. The purified holochrome contains 0.24 g of protein per μmole of protochlorophyllide. Estimation of the molecular weight of the holochrome by gel filtration on agarose revealed the presence of aggregates of approximately 550,000 and 300,000. There are at least 2 chromophores per 550,000 molecular weight.     

On the Relationship between Ribulose Diphosphate Carboxylase and Protochlorophyllide Holochrome of Phaseolus vulgaris Leaves

The relationship between ribulose diphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase [dimerizing], EC 4.1.1.39, formerly known as carboxydismutase) and protochlorophyllide holochrome of etiolated Phaseolus vulgaris leaves has been studied.A procedure for partially selective extraction of the two proteins was devised using tris-HCl buffer first without and then with Triton X-100. Ribulose diphosphate carboxylase was readily extracted from etiolated bean leaves without Triton X-100, and protochlorophyllide holochrome was extracted on the addition of Triton X-100.Optimal extraction conditions for protochlorophyllide holochrome have been found to be different for tissues of different ages.     

Rapid regeneration of protochlorophyllide(650)

The rate of regeneration of protochlorophyllide₆₅₀ was examined spectrophotometrically after a saturating light flash using 8- to 9-day-old dark-grown bean leaves. The regeneration occurred to the extent of 15% with a half rise time of about 20 seconds. Feeding δ-aminolevulinic acid to the excised leaves in the dark increased protochlorophyllides₆₃₅ but not the absorption at 650 nanometers, suggesting that the holochrome was normally saturated with protochlorophyllide and that the holochrome protein was not controlled by the level of protochlorophyllide. After a light flash, the excess protochlorophyllide, formed from exogenous δ-aminolevulinic acid, readily combined to regenerate the 650 nanometer absorbing species; the regeneration occurred to the extent of 60 to 80% with a half rise time of about 50 seconds. Regeneration was blocked at 0°, suggesting that there was some enzymic process required for regeneration, possibly the formation of a reductant component of the protochlorophyllides₆₅₀ holochrome.     

Analysis of the subunit structure of protochlorophyllide holochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The subunit structures of protochlorophyllide holochrome (PCH) and chlorophyllide holochrome (CH) were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. PCH from leaves of dark-grown (Phaseolus vulgaris var. red kidney) is a polymeric pigment-protein complex of approximately 600,000 daltons. It is composed of 12 to 14 polypeptides of 45,000 daltons, when examined prior to and immediately following photoconversion. The protochlorophyllide or chlorophyllide pigment molecules are associated with these polypeptides. Subsequent to photoconversion, the absorption maximum of newly formed chlorophyllide shifts from 678 nm to 674 nm upon standing in darkness. Following the 678 to 674 spectral shift, the chlorophyllide is associated with a polypeptide with a molecular weight of 16,000 daltons. In addition, sucrose gradient centrifugation of PCH and CH under nondenaturing conditions indicates that during the course of the dark spectroscopic shift, the 600,000 dalton CH undergoes dissociation into a small chlorophyllide protein. The dissociation of CH, the change in the molecular weight of the chlorophyllide polypeptide from 45,000 to 16,000 daltons, as well as the dark spectroscopic shift are temperature-dependent and blocked below 0 C. It was also found that each holochrome molecule of 600,000 daltons contains at least four protochlorophyllide pigment molecules.     

The Abolition of the Lag Phase in Greening Cucumber Cotyledons by Exogenous delta-Aminolevulinic Acid

Etiolated cucumber cotyledons treated with δ-aminolevulinic acid accumulated protochlorophyllide which was phototransformable to chlorophyll (ide). The phototransformation process in the δ-aminolevulinic acid-treated tissue was markedly temperature-dependent, consistent with the view that this protochlorophyllide must combine with the holochrome apoenzyme before phototransformation can occur.     

The Conversion of Protochlorophyllide636 to Protochlorophyllide630 in Leaves Treated with δ-Aminolevulinic Acid

Absorbancy changes in dark-grown, excised wheal leaves fed with δ-aminolevulinic acid are measured in vivo. The treatment with σ-aminolevulinic acid caused accumulation of protochlorophyllide, absorbing at 636 nm. After flashlight this form is found to convert in darkness to protochlorophyllide, absorbing at 650 nm. The conversion starts instantly after the leaves have been exposed to the flashlight, and the pre-existent pool of protocholorophyllidc absorbing at 650 nm will become emptied. The conversion is completed after 15–20 minutes, when a new pool of protochlorophyllide has been filled up. This new pool is transformed to chlorophyllide by a second flash and the sequence is repeated. The conversion may be composed of two reactions, a conclusion which can be drawn from the behaviour at different temperatures. One of these reactions is fairly temperature independent while the other is temperature dependent. The action of the protochlorophyllide holochrome is discussed.     

Action Spectra for Transformation and Fluorescence of Protochlorophyll Holochrome from Bean Leaves

The action spectrum from 232 to 687 nm was determined for the transformation of protochlorophyllide into chlorophyllide a in solutions of protochlorophyll holochrome Fran bean leaves. The whole ultraviolet region is effective. The peaks at 445 and 639 nm have a height ratio of 4.0. Only radiation absorbed in the protochlorophyllide itself is effective in transformation (absorption in aromatic amino acids of the protein and in carotenoids is ineffective). The activation spectra for fluorescence at 643 and 683 nm are measured for the holochrome before and after transformation, as well as the change in absorption spectrum that takes place upon transformation. By combining the various measurements the spectrum of inactive (non-transformable) protochlorophyllide (peak at 440 nm) and the holochromatic chlorophyllide a are derived. In the latter spectrum the peaks at 419 and 435 nm are of about the same height.     

The association of protein synthesis with protochlorophyllide holochrome regeneration in dark-grown barley leaves

A light-stimulated increase in incorporation of radioactive amino acids into protein associated with protochlorophyllide holochrome occurs concomitantly with the regeneration of phototransformable protochlorophyllide in dark-grown barley leaves. This increase in radioactivity and the protochlorophyllide regeneration process are both abolished by incubation of the leaves with inhibitors of cytoplasmic protein synthesis. Prelimiary data implicate protein in the molecular weight range of 45,000–60,000 daltons in this process.     

Energy transfer from protochlorophyllide to chlorophyllide during photoconversion of etiolated bean holochrome

The photoconversion of protochlorophyllide to chlorophyllide in etiolated bean leaves or leaf extracts exhibits complicated kinetics that are neither simple first-order nor second-order with respect to the reactant. By comparing the chlorophyllide absorbance with the intensity of chlorophyllide fluorescence excited at wavelengths where both pigments absorb, we demonstrate that the kinetic complexity results from the transfer of electronic excitation from protochlorophyllide to chlorophyllide. Measurements of the polarization of chlorophyllide fluorescence indicate that efficient excitation transfer occurs at room temperature over pigment aggregates containing at least four molecules. The relative quantum efficiency of chlorophyllide-excited chlorophyllide fluorescence remains constant during photoconversion of holochrome or etioplast preparations. This result does not support the proposal of increasing exciton interaction between chlorophyllides during the course of photoconversion.     

Transformation of Protochlorophyllide, Formed from Exogenous δ-Aminolevulinic Acid, in Continuous Light and in Flashlight

δ-Aminolevulinic acid supplied to dark grown isolated leaves or wheat causes an accumulation of protochlorophyllide which is only partly transformed to chlorophyllide α in continuous light At the same time a considerable photodestruction of both pigments takes place. By a suitable combinations of short lights flashes and dark periods it is possible, however, to obtain at least double the amount of the protochlorophyllide transformed without photodestruction. The transformation isshown to be dependent on the dark interval between the light flashes. Possible connections with the formation of the protein part of the protochlorophyllide holochrome are discussed.     

Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

Studies on the regeneration of protochlorophyllide after brief illumination of etiolated bean leaves

The effects of various inhibitors of nucleic acid and protein synthesis on protochlorophyllide synthesis in dark-grown Phaseolus vulgaris var. Red Kidney have been studied. Actinomycin D, chloramphenicol, and puromycin inhibit the regeneration of protochlorophyllide holochrome (detected as a 650 mμ absorption peak) in vivo in darkness after photoconversion of endogenous protochlorophyllide a to chlorophyllide a; this inhibition does not occur in similarly treated leaves supplied with δ-aminolevulinic acid.

These data suggest that the regeneration of protochlorophyllide results from the synthesis of RNA and enzymes required for the production of δ-aminolevulinate.

     

Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of ?15 – ?80°C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.     

On the aggregational states of protochlorophyllide and its protein complexes in wheat etioplasts

The inner membranes from wheat ( Triticum aestivum L. cv. Walde) etioplasts were separated into membrane fractions representative of prolamellar bodies and prothylakoids by differential and gradient centrifugations. The isolated fractions were characterized by absorption-, low-temperature fluorescence-, and circular dichroism (CD) spectroscopy, by high performancy liquid chromatography and by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
The prolamellar body fraction was enriched in NADPH-protochlorophyllide oxidoreductase (E.C. 1.6.99.1), and in protochlorophyllide showing an absorption maximum at 650 nm and a fluorescence emission maximum at 657 nm. Esterified protochlorophyllide was mainly found in the prothylakoid fraction. The carotenoid content was qualitatively the same in the two fractions. On a protein basis the carotenoid content was about three times higher in the prolamellar body fraction than in the prothylakoid fraction. The CD spectra of the membrane fractions showed a CD couplet with a positive band at 655 nm, a zero crossing at 643–644 nm and a negative band at 623–636 nm. These results differ from earlier CD measurements on protochlorophyllide holochrome preparations. The results support the interpretation that protochlorophyllide is present as large aggregates in combination with NADPH and NADPH-protochlorophyllide oxidoreductase in the prolamellar bodies.     

A Short-lived Intermediate Form in the in Vivo Conversion of Protochlorophyllide 650 to Chlorophyllide 684

When dark-grown leaves of Phaseolus vulgaris, Hordeum vulgare, Zea mays and Pisum sativum were irradiated for 3 sec at 2° the first product of protochlorophyllide 650 conversion had an absorption maximum at 678 nm. This form was then converted in a dark reaction to chlorophyllide 684, the form generally observed and regarded as the in vivo product of the photoreaction. The dark conversion at 2° was complete in 6 to 10 min in the various plants. The time course of the dark reaction was followed at 690 nm near the maximum of the difference spectrum for the conversion. There was a constant relationship between the initial amount of chlorophyllide 678 and the final amount of chlorophyllide 684. The rates of the dark reaction at 2° varied 3-fold among the plants treated. The reaction was not first order. At 25° the reaction followed at 690 nm was complete in 20 to 60 sec. Q₁₀'s varied from 2.8 to 3.7 between 2° and 25°. Phytochrome absorbancy changes were shown to be too low to interfere with these measurements except in pea leaves. In a subsequent stage of greening newly regenerated protochlorophyllide went through the same sequence upon photoconversion. Chlorophyllide 678 probably corresponds to the product formed in vitro from the protochlorophyllide holochrome. The dark reaction appears to represent the first interaction between the photoconverted holochrome and other elements of the proplastid. The lack of this dark reaction could also account for the spectral properties of certain albino mutants.     

Spectrophotometric quantitation of protochlorophyll(ide): Specific absorption and molar extinction coefficients reconsidered

To allay doubt about how to calculate molar extinction eoeffieients from the specific absorption coefficients of protoehlorophyll(ide), dark-grown leaf segments and saponin protochlorophyll(ide) holochrome subunits frotn barley ( Hordeum vulgare L.) and cotyledons of cucumber ( Cucumis sativus L.) were extracted with acetone without or following prior, brief illumination. Absorbance data and eoeffieients of chlorophyll a were used to derive extinetion eoeffieients of protoehlorophyll(ide); 626 nm (range: 30 to 35 1 mmol^-1 cm^-1) without recourse to published coefficients c protochlorophyll(ide). These results combined with evaluation of how the origin; coefficients were obtained, argue for using the molecular weight of protochlorophy rather than protochlorophyllide to calculate molar extinction eoeffieients from th specific absorption coefficients.     

Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil.

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of -15 -- -80 degrees C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.     

The Conversion of Photoinactive Protochlorophyllide(633) to Phototransformable Protochlorophyllide(650) in Etiolated Bean Leaves Treated with delta-Aminolevulinic Acid

The relationship of phototransformable protochlorophyllide to photoinactive protochlorophyllide has been studied in primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Various levels of photoinactive protochlorophyllide, absorbing at 633 nm in vivo, were induced by administering δ-aminolevulinic acid to the leaves in darkness. Phototransformable protochlorophyllide, absorbing at 650 nm in vivo, was subsequently transformed to chlorophyllide by a light flash, and the regeneration of the photoactive pigment was followed by monitoring the absorbance increase at 650 nm in vivo. A small increase in the level of protochlorophyllide₆₃₃ causes a marked increase in the extent of regeneration of protochlorphyllide₆₅₀ following a flash. High levels of the inactive pigment species, however, retard the capacity to reform photoactive protochlorophyllide. A nonstoichiometric and kinetically complex decrease in absorbance at 633 nm in vivo accompanied the absorbance increase at 650 nm. The half-time for protochlorophyllide₆₅₀ regeneration in control leaves was found to be three times longer than the half-time for conversion of chlorophyllide₆₇₈ to chlorophyllide₆₈₃ at 22 C. The results are consistent with the hypothesis that protochlorophyllide₆₃₃ is a direct precursor of protochlorophyllide₆₅₀ and that the protein moiety of the protochlorophyllide holochrome acts as a “photoenzyme” in the conversion of protochlorophylide to chlorophyllide.