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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↗
PubMed1999

Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity.

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.

Avena↗
PubMed1984

The proteolytic degradation in vitro of the NADPH-protochlorophyllide oxidoreductase of barley (Hordeum vulgare L.).

A cell-free membrane system has been developed from isolated barley etioplasts which displays a highly selective decrease of the NADPH-protochlorophyllide oxidoreductase in vitro which is indistinguishable from that observed previously in the intact plant. The rapid breakdown of the enzyme protein in vitro is caused by a membrane-bound proteolytic activity. The protease is essentially independent of pH in the physiological pH range of 6 to 8.5. The optimum temperature for the reaction is approximately 40 degrees C. In the presence of excessive protochlorophyllide the enzyme is no longer degraded or inactivated during illumination of dark-grown plants. In the isolated membrane fraction protochlorophyllide also enhances the stability of the enzyme, a similar effect is exerted by NADPH but not by NADH. The results suggest that the inactivation of the NADPH-protochlorophyllide oxidoreductase is influenced by the interaction of the enzyme with protochlorophyllide and NADPH. In the absence of these two components the enzyme becomes susceptible to proteolytic degradation.

Edible Grain↗
PubMed1995

Photoreduction of zinc protopheophorbide b with NADPH-protochlorophyllide oxidoreductase from etiolated wheat (Triticum aestivum L.).

A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.

Chlorophyll↗
PubMed1994

Association of protochlorophyllide with the PufQ protein of Rhodobacter capsulatus.

The PufQ protein, prepared by the recombinant expression of the pufQ gene of Rhodobacter capsulatus, has been reconstituted into liposomes in the presence of the bacteriochlorophyll precursor, protochlorophyllide. The liposomes were separated from liposomes free of the PufQ protein by sucrose density gradient ultracentrifugation and analyzed for their content of protochlorophyllide, protein, and phospholipid. The results indicated a 3.5 times higher level of association of protochlorophyllide with the PufQ protein-containing liposomes than with liposomes containing either the hydrophobic protein, apolipoprotein A-I, or the water-soluble maltose-binding protein. Only the association of the protochlorophyllide with the PufQ protein corresponded to a small but reproducible shift in its fluorescence emission maximum to a lower wavelength, consistent with a change in its environment.

Bacterial Proteins↗
PubMed2006

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro.

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.

Freezing↗
PubMed2004

Protochlorophyllide phototransformation in the bundle sheath cells of Zea mays.

The protochlorophyllide transformation process was investigated by using comparative analysis of 77 K fluorescence spectral changes occurring in isolated bundle sheath (BS) cells of etiolated Zea mays leaves after being exposed to a 200 ms saturating flash. Deconvolution analysis of the fluorescence spectra showed essential differences in the ratio of protochlorophyll(ides) and chlorophyll(ides) spectral forms indicating for BS cells to have a characteristic pathway of protochlorophyllide transformation. Bundle sheath cells showed a high ratio between non-photoactive protochlorophyll(ide)-F632 and photoactive protochlorophyllide-F655. In those cells, the 200 ms flash triggered a preferential formation of chlorophyll(ide)-F675 which remained stable in the dark for at least 90 min. Isolated BS cells showed an accumulation of chlorophyll(ide)-F675 resulting in the formation of inactive photosystem II. However for mesophyll cells of intact leaves, it was found to have a high ratio between photoactive and non-photoactive protochlorophyll(ide), showing the succession of chlorophyll(ide) forms usually known in C(3) plants. Protochlorophyllide phototransformation pathway in BS cells related to early stages of plastid differentiation triggered by light may indicate specific conditions for PSII assembly process leading to inactive PSII forms.

Cold Temperature↗
PubMed2000

The influence of glycerol and chloroplast lipids on the spectral shifts of pigments associated with NADPH: protochlorophyllide oxidoreductase from Avena sativa L.

Dark-grown angiosperm seedlings lack chlorophylls, but accumulate protochlorophyllide a complexed with the light-dependent enzyme NADPH:protochlorophyllide oxidoreductase. Previous investigators correlated spectral heterogeneity of in vivo protochlorophyllide forms and a shift of chlorophyllide forms from 680 to 672 nm (Shibata shift) occurring after irradiation, with intact membrane structures which are destroyed by solubilization. We demonstrate here that the various protochlorophyllide forms and the Shibata shift which disappear upon solubilization are restored if the reconstituted complex is treated with plastid lipids and 80% (w/v) glycerol. We hypothesize that the lipids can form a cubic phase and that this is the precondition in vitro and in vivo for the observed spectral properties before and after irradiation.

Avena↗
PubMed1998

Photodynamic action of uroporphyrin and protochlorophyllide in greening barley leaves treated with cesium chloride.

Incubation of greening barley leaves with cesium chloride (CsCl) results in photodynamic leaf lesions within 24 h due to an inactivation of uroporphyrinogen III decarboxylase, an enzyme of tetrapyrrole biosynthesis, and transient accumulation of uroporphyrin (ogen). To examine the mechanism of porphyrinogenesis, time kinetics of the accumulating tetrapyrrole intermediates uroporphyrin (ogen) and protochlorophyllide were performed with leaves which were cut from 7-day-old dark-grown barley seedlings and incubated in 15 mM CsCl or water under different light regimes. In the presence of CsCl chlorophyll and carotenoids accumulation was inhibited in the first 24 h of continuous light and the pigment content decreased dramatically during extended illumination. When CsCl=treated leaves were transferred to darkness, accumulated uroporphyrinogen was completely converted to protochlorophyllide. Low temperature fluorescence spectroscopy confirmed that uroporphyrinogen almost completely accumulated in the reduced form. The oxidised form, uroporphyrin, was detectable after 24 h of illumination. The photodynamic leaf lesions became visible at the same time. Protochlorophyllide synthesised from accumulated uroporphyrinogen III in dark incubated leaves had a fluorescence maximum at 635 nm which is indicative for its non-photoconvertible form. Re-illumination of the barley leaves resulted in a rapid degradation of proteins and pigments and an intense lipid peroxidation within less than two hours due to the photodestructive potential of non-metabolised protochlorophyllide.

Carotenoids↗
PubMed1980

Substrate-specificity studies on protochlorophyllide reductase in barley (Hordeum vulgare) etioplast membranes.

1. The substrate specificity of the enzyme protochlorophyllide reductase in barley (Hordeum vulgare) etioplasts was investigated. 2. It was shown that naturally occurring esterified protochlorophyllide and chemically prepared protochlorophyllide methyl ester are not substrates for the enzyme, suggesting an important role for the C-7 carboxylic acid group in binding of the porphyrin to the enzyme. 3. Removal of magnesium from the protochlorophyllide leads to inactivity of the compound as a substrate for the enzyme. However, activity can be restored by replacing the magnesium with zinc, whereas nickel, copper or cobalt failed to restore substrate activity. 4. Binding of the second substrate, NADPH, to the enzyme probably occurs through the 2'-phosphate group in the coenzyme.

Chloroplasts↗
PubMed1998

Chlorophyll alpha synthesis upon interruption and deletion of por coding for the light-dependent NADPH: protochlorophyllide oxidoreductase in a photosystem-I-less/chlL- strain of Synechocystis sp. PCC 6803.

The gene coding for the light-dependent NADPH:protochlorophyllide oxidoreductase (POR) was interrupted or deleted in a Synechocystis sp. PCC 6803 strain lacking photosystem I (PS I) as well as ChlL, which takes part in light-independent catalysis of protochlorophyllide reduction. Interruption of por by a kanamycin-resistance cartridge between the codons for M263 and V264 (about 83% into the coding region) did not abolish POR activity, but resulted in a decrease in the protochlorophyllide-(PChlide)-binding capacity of POR. Deletion of por in the PS I-less/chlL- strain generated a mutant [PS I-less/chlL-/por (del)] which accumulated both monovinyl-PChlide and divinyl-PChlide and excreted PChlides into the medium. This mutant also synthesized small amounts of protochlorophyllide dihydrogeranylgeraniol ester (protochlorophyll) when it was grown under light-activated heterotrophic growth conditions. However, the mutant was still able to synthesize small amounts of normal chlorophyll a under weak continuous illumination, even though the quantum yield of chlorophyll a formation was reduced. Either protochlorophyll or PChlide reduction by an unspecific reductase or by a ChlB/ChlN complex could account for chlorophyll a synthesis in the PS I-less/chlL-/por (del) strain. Functional photosystem II (PS II) was assembled in this mutant, but the PS II/chlorophyll ratio was fourfold lower than in the PS I-less strain with normal chlorophyll synthesis. The PS I-less/chlL-/por (del) mutant had a 77-K fluorescence emission maximum at 685 nm but no peak or shoulder at 695 nm when the cells were excited at 435 nm. Much of the chlorophyll in the PS I-less/chlL-/por (del) mutant therefore seems to be associated with components other than PS II.

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↗
PubMed2000

NADPH:Protochlorophyllide oxidoreductase uses the general import route into chloroplasts.

Chloroplast differentiation in angiosperm plants depends on the light-dependent conversion of protochlorophyllide to chlorophyllide by NADPH:protochlorophyllide oxidoreductase (PORA; EC 1.6.99.1), a nuclearly encoded protein. The protein import of the precursor form of PORA into plastids was shown previously to strictly depend on the presence of its substrate protochlorophyllide. PORA seemed to follow a novel, posttranslationally regulated import route. Here we demonstrate that the precursor of PORA from barley is imported into isolated barley plastids independently of protochlorophyllide. PORA as well as PORB import is competed for by the precursor of the small subunit of Rubisco. The data demonstrate that the PORA precursor uses the general import pathway into plastids. Furthermore, en route into chloroplasts the pea POR precursor can be cross-linked to the protein import channel in the outer envelope Toc75 from pea.

Aminolevulinic Acid↗
PubMed1980

Chloroplast biogenesis. Detection of divinyl protochlorophyllide in higher plants.

It is shown that the protochlorophyllide pool of etiolated higher plants is made up of both monovinyl and divinyl protochlorophyllide. Although the two pigments exhibited similar emission maxima, they were distinguishable by their Soret excitation maxima, which were found at 436 to 437 and 443 to 444 nm, respectively, in ether at 77 K. The two pigments were partially separated on thin layers of polyethylene. They were shown to be accompanied by two unknown fluorescent compounds. The latter were designated compound (E451 F626) and compound (E453 F640) where E refers to the Soret excitation maxima and F to the fluorescence emission maxima of the two unknown compounds. Furthermore, it was shown under several different growth conditions that divinyl protochlorophyllide constituted the major component of the protochlorophyllide pool.

Chlorophyll↗
PubMed1977

Chloroplast biogenesis. Net synthesis of protochlorophyllide from magnesium-protoporphyrin monoester by developing chloroplasts.

Developing chloroplasts were isolated from greening Cucumis cotyledons in a cofactor-enriched medium and were incubated in the dark with 14C-labeled and unlabeled magnesium-protoporphyrin monoester. The metabolic pools between protoporphyrin and protochlorophyllide were monitored spectrofluorometrically. The incorporation of the 14C label into protochlorophyllide was also determined. It was shown that magnesium-protoporphyrin monoester, a postulated intermediate of the chlorphyll biosynthetic pathway, was convertible into protochlorophyllide with relatively high yields. Since protochlorophyllide is the immediate precursor of chlorophyll a it was concluded that magnesium-protoporphyrin monoester was indeed an intermediate of the chlorophyll biosynthetic pathway.

Chlorophyll↗
PubMed1977

Chloroplast biogenesis. Net synthesis of protochlorophyllide from protoporphyrin IX by developing chloroplasts.

Developing chloroplasts were isolated from greening Cucumis cotyledons in a co-factor-enriched medium and were incubated in the dark with 14C-labeled and unlabeled protoporphyrin IX. The metabolic pools between protoporphyrin and protochlorophyllide were monitored spectrofluorometrically. The incorporation of the 14C label into protochlorophyllide was also determined. It was shown that protoporphyrin IX, a postulated intermediate of the chlorophyll biosynthetic pathway, was convertible into protochlorophyllide. Since protochlorophyllide is the immediate precursor of chlorophyll a it was concluded that protoporphyrin IX was indeed an intermediate of the chlorophyll biosynthetic pathway.

Chlorophyll↗
PubMed1998

The two spectroscopically different short wavelength protochlorophyllide forms in pea epicotyls are both monomeric.

The spectral properties of the protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634-636, 644 and 655 nm. At room temperature (293 K) the 628 and 634-636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634-636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634-636 nm (0,0) emission bands originate from two monomeric forms of protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound protochlorophyllide and the 628 nm form as a protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.

Journal Article↗
PubMed1997

Purification and kinetic analysis of pea (Pisum sativum L.) NADPH:protochlorophyllide oxidoreductase expressed as a fusion with maltose-binding protein in Escherichia coli.

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key reaction in the chlorophyll biosynthetic pathway. To facilitate structure-function studies, POR from pea (Pisum sativum L.) has been overexpressed in Escherichia coli as a fusion with maltose-binding protein (MBP) at 5-10% of the total soluble cell protein. The fusion protein (MBP-POR) has been purified to greater than 90% homogeneity by a two-step affinity-purification procedure. This represents the first successful overexpression and purification of a plant POR. MBP-POR was found to be active, and the kinetic properties were determined using a continuous assay in which the rate of chlorophyllide formation was measured. The Vmax was 20.6+/-0.9 nmol.min-1.mg-1 and the Km values for NADPH and protochlorophyllide were 8.7+/-1.9 microM and 0.27+/-0.04 microM respectively. These results represent the first determination of the kinetic properties of a pure POR and the first report on the kinetics of POR from a dicotyledenous plant. The experiments described here demonstrate that the enzyme is not a 'suicide' enzyme, and the only components required for catalysis are NADPH, protochlorophyllide and light. Size-exclusion chromatography on a Superose 6 HR column indicated that MBP-POR has a molecular mass of 155 kDa (compared with the molecular mass of 80 kDa estimated by SDS/PAGE), indicating that it behaves as a dimer in solution. This is the first direct determination of the oligomerization state of POR.

ATP-Binding Cassette Transporters↗