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PubMed1989

Chlorophyll precursors in the plasma membrane of a cyanobacterium, Anacystis nidulans. Characterization of protochlorophyllide and chlorophyllide by spectrophotometry, spectrofluorimetry, solvent partition, and high performance liquid chromatography.

Plasma membranes were isolated and separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation of crude membranes prepared by French pressure cell extrusion of lysozyme-treated Anacystis nidulans. Two distinct populations of chlorophyll-free plasma membrane vesicles were obtained exhibiting buoyant densities of 1.087 and 1.100 g/cm3 as opposed to a uniform density of 1.192 g/cm3 for thylakoid membranes. Plasma and thylakoid membranes were characteristically different also with respect to fatty acid and protein composition, cytochrome oxidase activity, and pigment content as analyzed by spectrophotometry, spectrofluorimetry, and high performance liquid chromatography. Apart from carotenoids, chlorophyll a was the only major photosynthetic pigment detected in thylakoid membranes while plasma membranes contained virtually no chlorophyll a but (besides large amounts of carotenoids) protochlorophyllide a and chlorophyllide a as revealed by solvent partition (between n-hexane and acetone or methanol), room and low temperature fluorescence emission and excitation spectra, and analytical separation and identification by high performance liquid chromatography and comparison with authentic standards. The protochlorophyllide in the plasma membrane could be transformed into chlorophyllide in the dark in vitro by incubating the membrane preparation with NADPH; NADP+ effected the reverse transition.

Cell Membrane↗
PubMed2001

Chloroplast biogenesis 84: solubilization and partial purification of membrane-bound [4-vinyl]chlorophyllide a reductase from etiolated barley leaves.

[4-Vinyl] chlorophyllide a reductase (4VCR) is a key enzyme of the chlorophyll (Chl) biosynthetic pathway. It catalyzes the conversion of divinyl chlorophyllide (Chlide) a to monovinyl Chlide a by reduction of the vinyl group at position 4 of the macrocycle to ethyl. 4VCR is a membrane-bound enzyme, embedded in etioplast and etiochloroplast membranes. A study of the regulation and properties of this enzyme is mandatory for a comprehensive understanding of the biosynthetic heterogeneity of Chl biosynthesis. Solubilization and partial purification of 4VCR are described for the first time. The enzyme was solubilized with 5 mM Chaps and was partially purified by chromatography on DEAE-Sephacel and Cibacron Blue 3GA-1000 agarose. An overall 20-fold purification was achieved. The partially purified enzyme was stable for several months at -80 degrees C.

Cell Membrane↗
PubMed2001

Participation of free radicals in photoreduction of protochlorophyllide to chlorophyllide in an artificial pigment-protein complex.

The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressed photoenzyme protochlorophyllide-oxidoreductase (POR), protochlorophyllide, and NADPH were investigated by optical and ESR spectroscopy. An ESR signal (g = 2.002; H = 1 mT) appeared after illumination of the complex with intense white light at 77 K. The ESR signal appeared with simultaneous quenching of the initial protochlorophyllide fluorescence, this being due to the formation of a primary non-fluorescent intermediate. The ESR signal disappeared on raising the temperature to 253 K, and a new fluorescence maximum at 695 nm belonging to chlorophyllide simultaneously appeared. The data show that the mechanism of protochlorophyllide photoreduction in the complex is practically identical to the in vivo mechanism: this includes the formation of a short-lived non-fluorescent free radical that is transformed into chlorophyllide in a dark reaction.

Chlorophyll↗
PubMed1971

The sites of photoconversion of protochlorophyllide to chlorophyllide in barley seedlings.

The photoreduction of protochlorophyllide a to chlorophyllide a in intact 6-day-old seedlings of etiolated barley (Hordeum vulgare) exhibits a small initial phase, followed by an induction period of about 1 hour before a rapid phase of additional chlorophyll formation begins. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial phase of conversion of preformed protochlorophyllide, but it either abolishes or severely inhibits the subsequent phase of rapid chlorophyll synthesis within 45 minutes of its application to the seedlings. An analysis of the biphasic inhibition process suggests that the lifetime of the enzyme controlling protochlorophyllide synthesis (probably delta-amino-levulinic acid synthetase) is not longer than 10 minutes.The rapid phase of chlorophyll formation can be effected by a series of brief (15 second) pulses of light spaced at least 5 minutes apart. When longer dark intervals are used, no increase is observed in the yield of chlorophyll per pulse. We interpret the findings to indicate that the photoconversion takes place at distinct enzymatic sites whose concentration does not increase during a period of 4 hours following the initial illumination. The sites can be used repeatedly with a turnover time determined by the removal of the product chlorophyllide and the synthesis and placement of a new protochlorophyllide molecule.

Journal Article↗
PubMed1983

Separation of monovinyl and divinyl protochlorophyllides and chlorophyllides from etiolated and phototransformed cucumber cotyledons.

A method was developed to separate the monovinyl and divinyl forms of protochlorophyllide and chlorophyllide by high pressure liquid chromatography using a silicic acid column coated with dodecyl residues and a moving phase containing the lipophilic cation, tetrabutyl ammonium. The solvent was 70% methyl alcohol containing varying amounts of methyl ethyl ketone. The separation was carried out at 0 degrees C. This method was used to test and confirm a previous report that, in cucumber cotyledons, divinyl protochlorophyllide is phototransformed to give divinyl chlorophyllide, which is biologically unstable and disappears rapidly in the dark.

Journal Article↗
PubMed1985

Biosynthesis of a chlorophyllide b-like pigment in phenanthroline-treated Chlamydomonas reinhardtii y-1.

Incubation of degreened Chlamydomonas reinhardtii y-1 cells in the dark with m-phenanthroline induced de novo synthesis of a chlorophyllide b-like pigment. The rate of synthesis of this pigment in the dark was greater than that of total chlorophyll in illuminated cells. Most of the newly synthesized pigment was excreted into the culture medium. The product was extracted from the medium as the metal-free pheophorbide, which had a fluorescence excitation maximum at 428 +/- 1 nm and an emission maximum at 657 +/- 1 nm (E428F657) in ethyl acetate (E427F657 in diethyl ether). Three pheophorbide species were extracted from the medium of green cells treated in the dark, a minor component with a spectrum (E410F670) identical to demetallated chlorophyll a, and two major species with spectral values of E428F657 and E433F657. The latter, predominant form had a spectrum identical to demetallated chlorophyll b, which was purified from the algal cells. E428F657 and E433F657 reacted with hydroxylamine and Girard's T-reagent, which caused a shift in the fluorescence emission maximum to 668 nm. Pheophytin b, which contains an aldehyde group, exhibited an identical spectral shift when treated in the same way, but pheophytin a or porphyrin biosynthetic intermediates did not. Proton NMR analysis of the E428F657 chlorin produced by yellow cells treated with m-phenanthroline confirmed the presence of an aldehydic proton. Chelating and nonchelating phenanthroline analogs equally stimulated synthesis of this product.

Chlamydomonas↗
PubMed1979

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.

Chlorophyll↗
PubMed1988

Detection of chlorophyllide in chlorophyll-free plasma membrane preparations from Anacystis nidulans.

Plasma and thylakoid membranes were isolated and purified from the cyanobacterium Anacystis nidulans. Spectrophotometric examination of acetone extracts gave major absorption bands resulting from carotenoids and chlorophyll a in plasma and thylakoid membranes, respectively. Only a very small absorption peak at 663 nm was detected in acetone extracts of plasma membranes which, in contrast to the corresponding peak from thylakoid membranes, could not be extracted into n-hexane; methanol, on the other hand, was effective with both plasma and thylakoid membranes. Aqueous membrane suspensions excited at 435 nm gave strong fluorescence emission at 662 nm for plasma membranes, but only a very small one for thylakoid membranes which had been adjusted to equal absorbance at 678 nm. Excitation spectra of the 668 nm fluorescence emission peak in acetone extracts of plasma and thylakoid membranes were strikingly different from each other. Finally, high performance liquid chromatography afforded clear-cut preparative separation of the two "chlorophyll-like" pigments in plasma and thylakoid membranes, respectively, and identification by comparison with retention characteristics known from the literature, together with a pure chlorophyll a standard. Our results indicate that the highly fluorescent and polar "chlorophyll-like" pigment in plasma membranes of Anacystis is a chlorophyll precursor, viz. chlorophyllide a.

Cell Membrane↗
PubMed2005

Mg-dechelation activity in radish cotyledons with artificial and native substrates, Mg-chlorophyllin a and chlorophyllide a.

The Mg-dechelation activity in extracts from radish (Raphanus sativus L.) cotyledons was investigated using an artificial substrate, Mg-chlorophyllin a (Chlin) and the native substrate, chlorophyllide a (Chlide). In addition to a known a small molecular weight metal-chelating substance (MCS), Mg-releasing protein (MRP) was present when Chlin was used as the substrate. However, only MCS had Mg-dechelation activity with the native substrate. To examine the possibility of the dissociation of MRP into a protein moiety and a small molecular mass compound with an activity like MCS, extraction with low and high ionic strength buffers was carried out. No evidence was obtained that MCS is a moiety of MRP, however. Inhibitor studies showed that MCS and MRP had different susceptibilities to the inhibitors, especially to the chelators tiron and EDTA when Chlin was used as the substrate. Tiron had no effect on MRP, but it severely reduced MCS activity in both substrates. The activity of MRP increased during senescence, indicating the induction of MRP, while the activity of MCS was almost unchanged. These results suggest different reaction mechanisms by independent compounds. These findings suggest that MRP and MCS are present independently, and MCS is postulated to be a substance that catalyzes the Mg-dechelation reaction in the breakdown pathway of Chl, although MCS was not induced during senescence. The properties of MRP and MCS in relation to the small molecular mass substance obtained from strawberry fruit are also discussed.

Azetidinecarboxylic Acid↗
PubMed2000

The formation of chlorophyll from chlorophyllide in leaves containing proplastids is a four-step process.

The time course of the different esters of chlorophyllide (Chlide) during the formation of chlorophyll a (Chl) in embryonic bean leaves containing proplastids was investigated by HPLC. After the reduction of photoactive Pchlide (Pchlide) to Chlide, three intermediates, i.e. Chlide geranylgeraniol, Chlide dihydrogeranylgeraniol and Chlide tetrahydrogeranylgeraniol were detected before the formation of Chlide phytol, i.e. authentic Chl. The transformation of Chlide to Chl was found to be much faster in leaves containing proplastids than in etiolated leaves with etioplasts.

Acyclic Monoterpenes↗
PubMed2001

Carotenoid dependence of the protochlorophyllide to chlorophyllide phototransformation in dark-grown wheat seedlings.

The influence of carotenoids on partial protochlorophyllide (Pchlide) photoreduction and the successive formation of long-wavelength chlorophyllide (Chlide) forms was studied by low-temperature fluorescence spectroscopy (77 K). Wheat leaves with a decreased content of carotenoids obtained from norflurazon-treated seedlings (10 and 100 micromol l(-1)) were compared with leaves containing normal amounts of these pigments. Partial photoreduction of Pchlide was achieved by irradiation of the leaves with one light flash in combination with a number of neutral gray and/or red Perspex filters. There were significant differences between the fluorescence emission spectra (the position and height of the peaks) of dark-grown normal and carotenoid-deficient leaves irradiated with non-saturating white light of increasing intensity. The long-wavelength Chlide forms appeared first in the leaves nearly devoid of carotenoids (treated with 100 micromol l(-1) norflurazon), then in the leaves with carotenoid deficiency (treated with 10 micromol l(-1) norflurazon), and finally in normal leaves. After irradiation with non-saturating light of the same intensity, the ratio Chlide/Pchlide(657) was always the highest in the leaves nearly deficient of carotenoids, medium in the leaves with carotenoid deficiency and lowest in the normal leaves. Similarly to white light, red light of low intensity induced faster formation of long-wavelength Chlide species in the leaves with carotenoid deficiency in comparison to the normal leaves. We propose that, in leaves with reduced carotenoid content, a greater number of Pchlide molecules transform to Chlide per light flash than in normal leaves. The results are discussed in relation to the involvement of carotenoids in competitive absorption and light screening, as well as to their influence on Pchlide-Chlide interactions.

Carotenoids↗
PubMed2003

Early reactions of light-induced protochlorophyllide and chlorophyllide transformations analyzed in vivo at room temperature with a diode array spectrofluorometer.

The steps of protochlorophyllide (Pchlide) photoreduction and subsequent chlorophyllide (Chlide) transformations which occur in the seconds to minutes time-scale were studied using a diode array spectrofluorometer in dark-grown barley leaves. The intensity of the excitation light was varied between 3 and 2,500 micromol m(-2) s(-1) and a series of fluorescence spectra were recorded at room temperature in the seconds and minutes time scales. In certain experiments, 77-K emission spectra were measured with the same equipment. The high quality of the spectra allowed us to run spectral resolution studies which proved the occurrence, at room temperature, of multiple Pchlide and Chlide forms found previously in 77-K spectra. The comparison of the 77-K and room-temperature spectra showed that the fluorescence yields of the nonphotoactive 633-nm Pchlide form and of the Chlide product emitting at 678 nm were temperature independent. The fluorescence intensity of aggregated NADPH-pigment-POR complexes (photoactive 656-nm Pchlide and 693-nm Chlide forms) were strongly increased at 77 K, while that of the NADP(+)-Chlide-POR (684-686-nm Chlide form) was much less affected by temperature. Information was obtained also about the dynamics of the transformation of pigment forms in the light at different photon densities. At low light intensities, the phototransformation of the 642-644-nm Pchlide form was faster than that of the 654-656-nm form. The relative amplitudes of Gaussian components related to different Chlide forms found after exposure to a constant amount of photons strongly depended on the light intensity used. Strong quenching of all Chlide components occurred upon prolonged exposure to high intensity light. These effects are discussed by considering the interconversion processes between different forms of the pigment-protein complexes, their relative fluorescence yields and energy migration processes.

Chlorophyllides↗
PubMed1983

Chlorophyllide-substituted hemoglobin tetramers and hybrids: preparation, characterization, and energy transfer.

Three chlorophyllide-substituted human hemoglobin (Hb) complexes have been prepared: the tetrameric complex in which zinc pyrochlorophyllide alpha (ZnPChl alpha) is substituted for all four hemes and the two complementary hybrids in which ZnPChl alpha is substituted for heme in either the alpha- or beta-chains, while heme remains in the other chains. In each of these complexes, intramolecular Chl-Chl singlet energy transfer occurs. A variety of probes demonstrate that ZnPChl alpha-deoxyheme hybrids and the ZnPChl alpha-Hb complexes consistently exhibit properties associated with the well-known T-state tertiary and quaternary structure of deoxyHb itself. Using the known crystal structure of human deoxyHb, we have analyzed the steady-state fluorescence anisotropy of these complexes within the framework of the Förster energy-transfer theory. The result is the determination of the orientation of the Qy transition dipole moment of ZnPChl alpha. Nuclear magnetic resonance data for the hybrids offer insight into specific tertiary structural changes in the heme pocket surrounding the diamagnetic ZnPChl alpha, which accompany changes in the ligation state of the heme on the opposite chain.

Chlorophyll↗
PubMed2005

Photoinduced electroreduction of chlorophyllide on alkanethiol-coated mercury.

Monolayers of n-alkanethiols of chain length from C12 to C18 were self-assembled on a hanging mercury drop electrode, and a film of chlorophyllide (Chlide) was adsorbed on top of them. The reduction photocurrents following illumination of the Chlide film were measured over the potential range in which the Chlide is electroinactive in the dark, and their action spectra were determined. Plotting the derivative of the photocurrents with respect to the applied potential against potential yields bell-shaped curves that can be fitted to a Gaussian. The potential of the Gaussian maximum was used to determine the reorganization energy lambda for the Chlide electroreduction process. An increase in the thiol chain length causes lambda to decrease regularly and the photocurrent to decay exponentially with the monolayer thickness, with a decay constant beta of about 0.17 A(-1).

Biomimetic Materials↗
PubMed2004

Chlorophyllide a Oxygenase mRNA and Protein Levels Correlate with the Chlorophyll a/b Ratio in Arabidopsis thaliana.

Plants can change the size of their light harvesting complexes in response to growth at different light intensities. Although these changes are small compared to those observed in algae, their conservation in many plant species suggest they play an important role in photoacclimation. A polyclonal antibody to the C-terminus of the Arabidopsis thaliana chlorophyllide a oxygenase (CAO) protein was used to determine if CAO protein levels change under three conditions which perturb chlorophyll levels. These conditions were: (1) transfer to shaded light intensity; (2) limited chlorophyll synthesis, and (3) during photoinhibition. Transfer of wild-type plants from moderate to shaded light intensity resulted in a slight reduction in the Chl a/b ratio, and increases in both CAO and Lhcb1 mRNA levels as well as CAO protein levels. CAO protein levels were also measured in the cch1 mutant, a P642L missense mutation in the H subunit of Mg-chelatase. This mutant has reduced total Chl levels and an increased Chl a/b ratio when transferred to moderate light intensity. After transfer to moderate light intensity, CAO mRNA levels decreased in the cch1 mutant, and a concomitant decrease in CAO protein levels was also observed. Measurements of tetrapyrrole intermediates suggested that decreased Chl synthesis in the cch1 mutant was not a result of increased feedback inhibition at higher light intensity. When wild-type plants were exposed to photoinhibitory light intensity for 3 h, total Chl levels decreased and both CAO mRNA and CAO protein levels were also reduced. These results indicate that CAO protein levels correlate with CAO mRNA levels, and suggest that changes in Chl b levels in vascular plants, are regulated, in part, at the CAO mRNA level.

CAO↗
PubMed2002

Altering the expression of the chlorophyllase gene ATHCOR1 in transgenic Arabidopsis caused changes in the chlorophyll-to-chlorophyllide ratio.

The Arabidopsis gene ATHCOR1, which encodes the CORI1 (coronatine-induced) protein, was expressed in bacterial cells. Soluble recombinant CORI1 was purified and shown to possess chlorophyllase (Chlase) activity in vitro. To determine its activity in vivo, wild-type Arabidopsis and coi1 mutant, which lacks ATHCOR1 transcripts, were transformed with sense and antisense forms of the gene. Wild-type and coi1 plants overexpressing ATHCOR1 showed increased contents of chlorophyllide (Chlide) without a substantial change in the total amount of the extractable chlorophyll (Chl). These plants presented high Chlide to Chl ratios in leaves, whereas antisense plants and nontransformed coi1 mutant showed undetectable ATHCOR1 mRNA and significantly lower Chlide to Chl ratios, relative to wild-type control. Overexpression of ATHCOR1 caused an increased breakdown of Chl a, as revealed by the Chlide a to b ratio, which was significantly higher in sense than wild-type, coi1 mutant, and antisense plants. This preferential activity of CORI1 toward Chl a was further supported by in vitro analyses using the purified protein. Increased Chlase activity was detected in developing flowers, which correlated to the constitutive expression of ATHCOR1 in this organ. Flowers of the antisense plant showed reduced Chlide to Chl ratio, suggesting a role of CORI1 in Chl breakdown during flower senescence. The results show that ATHCOR1 has Chlase activity in vivo, however, because coi1 flowers have no detectable ATHCOR1 mRNA and present Chlide to Chl ratios comparable with the wild type, an additional Chlase is likely to be active in Arabidopsis. In accordance, transcripts of a second Arabidopsis Chlase gene, AtCLH2, were detected in both normal and mutant flowers.

Arabidopsis↗
PubMed2001

Spectroscopic analysis of desiccation-induced alterations of the chlorophyllide transformation pathway in etiolated barley leaves.

Effects of water deficit on the chlorophyllide (Chlide) transformation pathway were studied in etiolated barley (Hordeum vulgare) leaves by analyzing absorption spectra and 77-K fluorescence spectra deconvoluted in components. Chlide transformations were examined in dehydrated leaves exposed to a 35-ms saturating flash triggering protochlorophyllide (Pchlide) and Chlide transformation processes. During the 90 min following the flash, we found that dehydration induced modifications of Chlide transformations, but no effect on Pchlide phototransformation into Chlide was observed. During this time, content of NADPH-Pchlide oxydoreductase in leaves did not change. Chlide transformation process in dehydrated leaves was characterized by the alteration of the Shibata shift process, by the appearance of a new Chlide species emitting at 692 nm, and by the favored formation of Chl(ide) A(668)F(676). The formation of Chl(ide) A(668)F(676), so-called "free Chlide," was probably induced by disaggregation of highly aggregated Chlide complexes. Here, we offer evidence for the alteration of photoactive Pchlide regeneration process, which may be caused by the desiccation-induced inhibition of Pchlide synthesis.

Adaptation, Physiological↗
PubMed2004

Domain structures of chlorophyllide a oxygenase of green plants and Prochlorothrix hollandica in relation to catalytic functions.

Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b-protein complexes in land plants.

Amino Acid Sequence↗