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At least 19 recordsLinked to original sources
PubMed1992

Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme.

Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues.

Chlorophyll↗
PubMed1982

Chloroplast biogenesis. Identification of chlorophyllide a (E458f674) as a divinyl chlorophyllide a.

The chemical identification of chlorophyllide (E458F674) (Belanger, F. C., and Rebeiz, C. A. (1980) Plant Sci. Lett. 18, 343-350) has been confirmed by chemical derivatization coupled to spectrofluorometric, spectrophotometric, and chromatographic analysis. Chlorophyllide (E458F674) and its demetallated analog were converted by catalytic hydrogenation into mesochlorophyllide a and mesopheophorbide a. Furthermore, methyl chlorophyllide (E458F674) was converted by partial hydrogenation into a mixture of monovinyl chlorophyllide a isomers and the latter into mesochlorophyllide a by further hydrogenation. On the other hand, chemical oxidation of methyl chlorophyllide (E458F674) converted it into methyl divinyl protochlorophyllide. Chlorophyllide (E458F674) was detected in several plant species and is proposed to be an important intermediate of the chlorophyll a biosynthetic pathway.

Chemical Phenomena↗
PubMed1995

Chloroplast biogenesis 72: a [4-vinyl]chlorophyllide a reductase assay using divinyl chlorophyllide a as an exogenous substrate.

[4-Vinyl]Chlorophyllide alpha reductase (4VCR) catalyzes the conversion of 2,4-divinyl chlorophyllide alpha (DV Chlide alpha) to 2-vinyl,4-ethyl chlorophyllide alpha (MV Chlide alpha) via an NAPDH- dependent reaction. MV Childe alpha is the immediate precursor of monovinyl chlorophyll alpha in plants. In etiolated cucumber (Cucumis sativus L.) cotyledons, 4VCR is a plastidic membrane-bound enzyme. Further research on this enzyme required the development of an assay that utilizes DV Childe alpha as an exogenous substrate. Such an assay is now described. It involves conversion of exogenous DV Chlide alpha to MV Chlide alpha at high rates by etioplast membranes of cucumber, corn (Zea mays L.), and barley (Hordum vulgare L.). 4VCR exhibits high activity between 30 and 40C and in the pH range of 6.3 to 7.0. Activity is quasilinear for the first 60 s of incubation.

Chlorophyllides↗
PubMed1984

Excitation transport and trapping in a synthetic chlorophyllide substituted hemoglobin: orientation of the chlorophyll S1 transition dipole.

Excitation transport in synthetic zinc chlorophyllide substituted hemoglobin has been observed by pico -second time-resolved fluorescence depolarization experiments. In this hybrid molecular system, two zinc chlorophyllide molecules are substituted into the beta-chains of hemoglobin, while deoxy hemes remain in the alpha-chains. The rate of excitation transfer between the two chlorophyllides is analyzed in terms of the distance and orientation dependences predicted by the F orster dipole-dipole theory. In this analysis, the beta-beta interchromophore geometry is assumed to be that of the deoxyhemoglobin crystal structure. When combined with steady-state fluorescence depolarization data of the complementary hybrid containing zinc chlorophyllide in the alpha-chains, these experiments provide the necessary information to determine the orientation of the S1 transition dipole moment in the zinc chlorophyllide molecule. We also find that the fluorescence lifetime of the zinc chlorophyllide is 1.42 ns when the heme is in the deoxy state but 3.75 ns when the heme is ligated to carbon monoxide. This is explained by irreversible excitation transfer from the S1 state of the zinc chlorophyllide to the lower energy excited states present in deoxy heme.

Chlorophyll↗
PubMed2001

Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol.

Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond. The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol. It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase. PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a. The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution.

Carboxylic Ester Hydrolases↗
PubMed1978

Reconstitution of chlorophyllide formation by isolated etioplast membranes.

1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the additon of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation. 6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35 micron respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.

Chlorophyll↗
PubMed1982

[Enzymatic system transforming chlorophyllide into chlorophyll in etiolated leaves using exogenous substrates].

The substrate specificities of the enzymatic system, which transforms chlorophyllide into chlorophyll in etioplasts of whole leaves and of the chlorophyllase enzyme, which performs the inverse reaction, i. e. chlorophyll hydrolysis, were compared. Infiltration of chlorophyllide a of b into the excised etiolated leaves of rye results in a formation of chlorophylls a and b in the dark, whereas pheophorbide a or b does not cause pheophytin to appear under the same conditions. Consequently, for the in vivo esterification reaction to occur, substitution of the methyl group by the formyl one in position 3 of the tetrapyrrole ring is not essential, but the presence of the central magnesium atom in the molecule is necessary. On the other hand, chlorophyllase from the same etiolated leaves equally hydrolyzes both chlorophylls and pheophytins. It is concluded that the enzymes present in etioplasts which catalyze chlorophyllide esterification and chlorophyll hydrolysis are not identical. The optimal temperature of the in vivo esterification of exogenous chlorophyllide to chlorophyll (37 degrees) was determined and the ability of this chlorophyll to be hydrolyzed by chlorophyllase was established.

Chlorophyll↗
PubMed1995

A light-induced protease from barley plastids degrades NADPH:protochlorophyllide oxidoreductase complexed with chlorophyllide.

The NADPH:protochlorophyllide oxidoreductase precursor protein (pPorA) of barley (Hordeum vulgare L. cv. Carina), synthesized from a full-length cDNA clone by coupling in vitro transcription and translation, is a catalytically active protein. It converts protochlorophyllide to chlorophyllide in a light- and NADPH-dependent manner. At least the pigment product of catalysis remains tightly bound to the precursor protein. The chlorophyllide-pPorA complex differs markedly from the protochlorophyllide-pPorA complex with respect to sensitivity to attack by a light-induced, nucleus-encoded, and energy-dependent protease activity of barley plastids. The pPorA-chlorophyllide complex is rapidly degraded, in contrast to pPorA-protochlorophyllide complexes containing or lacking NADPH, which are both resistant to protease treatment. Unexpectedly, pPorA devoid of its substrates or products was less sensitive to proteolysis than the pPorA-chlorophyllide complex, suggesting that both substrate binding and product formation during catalysis had caused differential changes in protein conformation.

Adenosine Triphosphate↗
PubMed1981

Solution properties of synthetic chlorophyllide--and bacteriochlorophyllide--apomyoglobin complexes.

Well-defined 1:1 complexes have been formed between apomyoglobin (apoMb) and a number of chlorophyllide derivatives. The chlorophyllides substitute for heme in the pocket of myoglobin. These include magnesium chlorophyllide a, magnesium and zinc pyrochlorophyllide a, zinc pyrochlorophyllide b, zinc pyrochlorophyllide d, zinc pyromesochlorophyllide a, zinc 2-acetyl-2-devinylpyrochlorophyllide a, zinc protopyrochlorophyllide a, and zinc bacteriopyrochlorophyllide a. The effects of the protein on the electronic absorption, circular dichroism (CD), magnetic circular dichroism, and triplet state electron spin resonance spectra and fluorescence lifetimes in solution are compared with appropriate models in organic solvents. With the exception of the CD spectra, the protein causes shifts and intensity changes which are within the range observed for solvent effects. The CD spectra change substantially: the signs of several transitions are entirely reversed in the chlorins, and 3-6-fold intensity increases are observed with zinc bacteriochlorophyllide a. High-field 1H NMR spectra of ring current shifted Val-E11 methyl protons for the series porphyrin-, chlorin-, and bacteriochlorin-apoMb are used to establish the probable absolute orientation of the chromophore in the heme pocket. Doubled peaks in the NMR spectra of certain complexes are shown to arise from interconvertible species. The temperature dependence of the peak intensities and saturation transfer studies show that the species giving rise to the doubled peaks exchange on the time scale of about 1-60 s. Arguments are presented against inversion of the macrocycle in the heme pocket by either an inter- or an intramolecular mechanism as the origin of doubled peaks, and simple two-site exchange is ruled out by the NMR data. We suggest that the data are consistent with the idea that at least two slowly interconverting conformational substrates of the protein are populated, depending sensitively on small changes in rings I and II of the macrocycle and temperature.

Apoproteins↗
PubMed1995

Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.).

NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.

Amino Acid Sequence↗
PubMed1976

[Chlorophyll a and b biosynthesis in the dark in etiolated leaves infiltrated by exogenous chlorophyllide a].

Exogenous chlorophyllide a was introduced into etiolated rye leaves by the vacuum-infiltration technique. Appearance and accumulation of chlorophylls a and b within the leaves are observed during continued darkening, protochlorophyllide photoreduction being avoided. The pigments are identified by the solubility in petroleum ether, paper chromatograms, the fluorescence maxima, the peculiarities of exciting light 430 and 460 nm effects on fluorescence intensity, the specific interaction with hydrochloric hydroxylamine. The conclusion is made that before illumination etioplasts already contain enzyme systems and substrates which provide esterification of chlorophyllide a to chlorophyll a and conversion of chlorophyll a into chlorophyll b.

Chlorophyll↗
PubMed1991

[The effect of the acidity of the medium on the magnitude of the light-dependent antibacterial activity of chlorophyllides].

The influence of medium acidity on the intensity of light-dependent antibacterial activity of chlorophyllides isolated from biomass of microscopic green alga Westella botryoides has been studied. This light-dependent antibacterial activity has been shown to be maximum at low pH values. The ability to suppress the bacterial growth under the action of light decreases in neutral and alkaline media. It has been concluded that the reason of increase of light-dependent antibacterial activity of chlorophyllides at low pH values is formation of pheophorbides out of them

Anti-Bacterial Agents↗
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↗
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↗