Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

DETECTION OF CHLOROPHYLL C1 AND MAGNESIUM-2,4-DIVINYLPHEOPORPHYRIN A5 MONOMETHYLESTER IN CRYPTOPHYTES1

Three different chlorophyll (chl) c-type pigments were isolated from two cryptophyte species by silica thin-layer chromatography or polyethylene high-performance liquid chromatography. Chroomonas sp. Hansgirg contained chl c₁ and magnesium-2,4-divinylpheoporphyrin a, mono-methylester; chl c₂ and magnesium-2,4-divinylpheoporphyrin a₅ monomethylester were found in Cryptomonas maculata (syn. Rhodomonas maculata Butcher). These identifications were based on spectral characteristics and on comparison with reference pigments isolated from the synurophycean Synura petersenii Korshikov and the prasinophyte Mantoniella squamata Manton & Park. Neither of the cryptophyte species contained chl c₁ and chl c₂. The significance of chl c₁ as a major pigment and the occurrence of magnesium-2,4-divinylpheoporphyrin a₅ monomethylester in cryptophytes are discussed.     

A new spectrally distinct component in preparations of chlorophyll c from the micro-alga Emiliania huxleyi (Prymnesiophycease)

A new chlorophyll c pigment designated chlorophyll c₃ has been isolated from the coccolithophorid Emiliania huxleyi (Prymnesiophyceae) using reverse-phase high-performance thin-layer chromatography (HPTLC). Its spectral properties were compared with chlorophylls c₁ and c₂ from standard sources. Visible absorption maxima of the new pigment in diethyl ether were at 451, 585 and 625 nm with band ratios of 30.77, 3.79 and 1.00, respectively. Chlorophyll c₂ was present in approximately equal proportions to chlorophyll c₃, with maxima in diethyl ether at 447, 579 and 628 nm and band ratios of 12.26, 1.17 and 1.00, respectively. No chlorophyll c₁ was detected. The visible absorption spectra of the magnesium-free derivatives of both chlorophylls c₂ and c₃ from E. huxleyi in acetone were also recorded. The new chlorophyll c₃ pigment was chromatographically and spectrally distinct from a similar pigment, magnesium 2,4-divinylpheoporphyrin a₅ monomethyl ester, present in prasinophyte algae, with which it could have been confused.     

In Vitro Synthesis of the Chlorophyll Isocyclic Ring : Transformation of Magnesium-Protoporphyrin IX and Magnesium-Protoporphyrin IX Monomethyl Ester into Magnesium-2,4-Divinyl Pheoporphyrin A(5)

Developing chloroplasts of Cucumis sativus L., cv Beit Alpha which were incubated with either Mg-protoporphyrin IX or Mg-protoporphyrin IX monomethyl ester in darkness produced a partially phototransformable protochlorophyllide species that was tentatively identified as Mg-2,4-divinyl pheoporphyrin a₅. S-Adenosylmethionine stimulated Mg-2,4-divinyl pheoporphyrin a₅ formation irrespective of the starting material used. In the case of Mg-protoporphyrin IX monomethyl ester, this stimulation was attributed to the need to remethylate substrate that had been hydrolyzed by an endogenous methylesterase which converts part of the added Mg-protoporphyrin IX monomethyl ester to Mg-protoporphyrin IX.

NADP and NADPH stimulated the conversion of Mg-protoporphyrin IX to Mg-2,4-divinyl pheoporphyrin a₅. The conversion required oxygen and was half saturated at 50 micromolar dissolved O₂. The conversion was insensitive to inhibitors of iron-sulfur and heme-containing proteins, to Cu chelators, H₂O₂, and peroxide scavengers. However, the conversion was extremely sensitive to phenazine methosulfate, methylene blue, and methyl viologen.

A decrease of the plastids' ability to convert Mg-protoporphyrin IX to Mg-2,4-divinyl pheoporphyrin a₅ after lysis in 0.1 molar NaCl suggested a requirement for plastid integrity.

     

The reduction of vinyl side-chains of Mg-protoporphyrin IX monomethyl ester in vitro

Portions of crude homogenates of etiolated wheat seedlings incubated with Mg-protoporphyrin IX and $\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine}$ and then added to other portions of the same crude homogenates that were pretreated with [1-³H]ethanol and yeast alcohol dehydrogenase provided, after a short reaction period, ³H-labeled Mg-protoporphyrin IX monomethyl ester. The ³H-labeled Mg-protoporphyrin IX monomethyl ester thus obtained was shown to contain the ³H in one reduced (to ethyl) vinyl side-chain. Subsequently, ³H-labeled Mg-monoethyl-(monodivinyl)-protoporphyrin IX monomethyl ester was obtained when Mg-protoporphyrin IX monomethyl ester and [³H]NADH were added to dialyzed crude homogenates of etiolated wheat seedlings. Insignificant amounts of ³H were incorporated into poprhyrin substrates when Mg-2,4-divinylpheoporphyrin a₅ or [³H]NADPH were substituted in reaction mixtures for Mg-protoporphyrin IX monomethyl ester or [³H]NADPH, respectively. The results of these and further experiments suggest that an NADPH-dependent enzyme in the crude homogenates of etiolated wheat seedlings was capable of catalyzing the reduction to ethyl of one vinyl side-chain of Mg-protoporphyrin IX monomethyl ester. These findings suggest that the 4-vinyl side-chain reductive reaction likely occurs after the biosynthesis IX monomethyl ester, but before isocyclic ring formation in the pathway to chlorophyll a.     

SEPARATION OF CHLOROPHYLLS c1c2, AND c3 OF MARINE PHYTOPLANKTON BY REVERSED-PHASE-C18-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY1

We separated chlorophylls c₁ c₂, and c₃ of marine phytoplankton together with other pigments by a modification of the commonly applied reversed-phase-C18-high-performance liquid chromatography (RP-C18-HPLC) method. However, the chlorophyll c-like pigment 2,4, Mg-divinylpheoporphyrin as monomethyl ester, co-eluted with chlorophyll c₁. The method involves optimization of the mobile phase by using a very high ion strength solvent in combination with a high carbon loaded RP-C18 column. Fingerprints of the various taxonomic groups of algae can thus be developed in a single run, including separation of the carotenoids lutein and zeaxanthin.     

Intermediates in the formation of the chlorophyll isocyclic ring

Cell-free, organelle-free synthesis of Mg-2,4-divinylpheoporphyrin a₅ (MgDVP) from Mg-protoporphyrin IX monomethyl ester (Mg-Proto Me) has been described (Wong and Castelfranco 1984 Plant Physiol 75: 658-661). This system consists of plastid membrane and stromal fractions and requires O₂, NAD(P)H and S-adenosylmethionine (SAM). The synthetic 6-methyl-β-ketopropionate analog of Mg-Proto Me was converted to MgDVP by the same catalytic system in the presence of O₂ and NADPH. SAM was not required. A compound (X) displaying the kinetic behavior of an intermediate was isolated from reaction mixtures with Mg-Proto Me as the substrate, but not with the 6-methyl-β-ketopropionate analog as the substrate. X was identified as the 6-methyl-β-hydroxypropionate analog of Mg-Proto Me by conversion to the dimethyl ester with CH₂N₂ and comparison with authentic 6-β-hydroxydimethyl ester. X was converted to MgDVP by the same catalytic system in the presence of O₂ and NADPH. We conclude that the conversion of Mg-Proto Me to MgDVP proceeds through the 6-β-hydroxy and the 6-β-ketopropionate esters in agreement with earlier suggestions.     

A new form of chlorophyll C involved in light-harvesting

A new form of chlorophyll c has been isolated from the pyrmnesiophyte Pavlova gyrans Butcher. This pigment is spectrally similar to chlorophyll c₂, but all the absorption maxima (454, 583, and 630 nm in diethyl ether) are shifted 4 to 6 nanometers to longer wavelengths. The new pigment can be separated from other chlorophyll c-type pigments by reversed-phase high performance liquid chromatography and thin layer chromatography. Both chlorophylls c₁ and c₂ are found with the new chlorophyll c pigment in P. gyrans, and it has also been detected in the chrysophyte Synura petersenii Korsh. The light-harvesting function of the new chlorophyll c pigment is indicated by its presence along with chlorophyll c₁ and c₂ in a light-harvesting pigment-protein complex isolated from P. gyrans in which chlorophyll c pigments efficiently transfer absorbed light energy to chlorophyll a.     

OCCURRENCE OF PHYIWATED CHLOROPHYLL c IN ISOCHRYSIS GALBANA AND ISOCHRYSIS SP. (CLONE T-ISO) (PRYMNESIOPHYCEAE)1

The pigment composition of two clones of Isochrysis galbana Parke (CCMP 1323 and CCAP 927/1), and Isochrysis sp. (clone T-ISO) was analyzed by high-performance liquid chromatography using a polymeric octadecylsilica column. Fluorescent peaks with retention times higher than chlorophyll a were detected for all three clones. The corresponding pigments were isolated and characterized in terms of their visible absorbance and fluorescence spectra. The pigments were similar to phytol-substituted chlorophyll c, previously isolated from Emiliania huxleyi (Lohm.) Hay and Mohler and other species containing chlmophyll c₃. The presence of phytol-substituted chlorophyll c in I. galbana which lacked chlorophyll c₃, increases the diversity of chlorophyll patterns for the Haptophyta, which can be grouped, at present, into six different pigment types. This is the jrst observation of a haptophyte containing the apolar phytylated chlorophyll c-like pigment but lacking chlorophyll c₃.     

Energy transfer and pigment composition in three chlorophyll b-containing light-harvesting complexes isolated from Mantoniella squamata (Prasinophyceae), Chlorella fusca (Chlorophyceae) and Sinapis alba

Light-harvesting Chl a/b protein complexes were isolated from the higher plant Sinapis alba, the green alga Chlorella fusca, and the prasinophycean alga Mantoniella squamata by mild gel electrophoresis. The energy transfer from chlorophyll b and the accessory xanthophyll was measured by means of fluoresence spectroscopy at 77 K. The pigment composition of the isolated antenna complexes was determined by high performance liquid chromatography in order to calculate the number of light absorbing molecules per chlorophyll a in the different light-harvesting complexes. These results were complemented by the quantitation of the pigments in total thylakoids as well as in the different electrophoretic fractions. On the basis of these data the in vivo ratios of xanthophylls per chlorophyll a could be estimated. The results show that the light-harvesting complexes from Chlorella and from Sinapis exhibit identical ratios of total xanthophylls per chlorophyll a. By contrast, in the prasinophycean alga Mantoniella, the light-harvesting complex markedly differs from the other chlorophyll b containing proteins. It contains, in addition to neoxanthin and violaxanthin, high amounts of prasinoxanthin and its epoxide, which contribute significantly to light absorption. The concentration of chlorophyll b in the complex is very much higher in the antenna of Mantoniella than in those of Chlorella and Sinapis. Furthermore, it must be emphasized that in addition to chlorophyll b, a third chlorophyll species acts in the energy transfer to chlorophyll a. This chlorophyll c-like pigment is found to be present in a concentration which improves very efficiently the absorption in blue light. In light of these results it can be concluded that the absorption cross section in Mantoniella is higher not only because of an enhanced number of light-harvesting particles in the membrane, but also because of a higher ratio of accessory pigments to chlorophyll a.Abbreviations Chl Chlorophyll - FP Free Pigments - HPLC High Performance Liquid Chromatography - LHC Light-harvesting Chlorophyll protein complex - PAGE Polyacrylamide Gel Electrophoresis - PS Photosystem     

Polypeptide sequence of the chlorophyll a/b/c-binding protein of the prasinophycean alga Mantoniella squamata

The primary structure of the Chla/b/c-binding protein from Mantoniella squamata is determined. This is the first report that protein sequencing reveals one modified amino acid resulting in a LHCP-specific TFA-cleavage site. The comparison of the sequence of Mantoniella with other Chla/b-and Chla/c-binding proteins shows that the modified amino acid is located in a region which is highly conserved in all these proteins. The alignment also reveals that the LHCP of Mantoniella is related to the Chla/b-binding proteins. Finally, possible Chl-binding regions are discussed.Abbreviations a.m.u. atomic mass unit - LHC light-harvesting complex - LHC II major LHC of Photosystem II - LHCP light-harvesting chlorophyll-binding protein - LSIMS liquid secondary ion mass spectrometry - TFA trifluoroacetic acid     

The Chlorophyll Biosynthetic Enzyme Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase (Characterization and Partial Purification from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803)

A universal structural feature of chlorophyll molecules is the isocyclic ring. This ring is formed by the action of the enzyme Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, which catalyzes a complex reaction in which Mg-protoporphyrin IX monomethyl ester is converted to divinyl protochlorophyllide (also called Mg-2,4-divinylpheoporphyrin a5), with the participation of NADPH and O2. Cyclase activity was demonstrated in lysed Chlamydomonas reinhardtii chloroplasts and extracts of Synechocystis sp. PCC 6803. The yield of the reaction product was increased by the addition of catalase and ascorbate or isoascorbate to the incubation mixture. These compounds may act by preventing degradation of the tetrapyrroles by reactive oxygen species. Cyclase activity from C. reinhardtii was not inhibited by the flavoprotein inhibitor quinacrine or by the hemoprotein inhibitors CO, KCN, or NaN3. In contrast, cyclase activity in extracts of C. reinhardtii and Synechocystis sp. PCC 6803 was inhibited by chelators of Fe, suggesting that nonheme Fe is involved in the reaction. Cyclase in lysed C. reinhardtii chloroplasts was associated with the membranes, and attempts to further fractionate or solubilize the activity were unsuccessful. In contrast, cyclase in Synechocystis sp. PCC 6803 extracts could be separated into soluble and membrane components, both of which were required for reconstitution of activity. The membrane component retained activity after it was solubilized by the detergent n-octyl-[beta]-D-glucopyranoside in the presence of glycerol and Mg2+. The solubilized membrane component was purified further by dye-affinity and ion-exchange chromatography.     

Comparative analysis of the composition of two chlorophyll-b-containing light-harvesting complexes

The major light-harvesting complexes from Mantoniella squamata (Prasinophyceae) and from Chlorella fusca (Chlorophyceae) were analyzed with respect to polypeptide composition and pigmentation. It was found that the polypeptides of Mantoniella are smaller than those of Chlorella and bind twice the amount of pigment. We assume that the amount of pigment per polypeptide is of ecological as well as of taxonomical importance.Abbreviations Chl chlorophyll - LHC light-harvesting complex - Xan xanthophyll We thank the support by the Deutsche Forschungsgemeinschaft.     

The Existence of Chlorophyll c in the Chl b-Containing,Light-Harvesting Complex of the Green Alga Mantoniella squamata (Prasinophyceae)

The prasinophycean alga Mantoniella contains, in addition to Chl a and b, at least a third green pigment which is functionally active in the light-harvesting antenna. This third Chl was isolated in order to elucidate its chemical structure. The absorption and fluorescence spectra were measured not only from the purified pigment but also from its pheophytin and its methylpheophorbide. The spectra were compared with those of authentic Chl c-1 and c-2, which were isolated from the diatom Nitzschia sp. and with Mg-DVPP (purified from Rhodobacter). The results show that the pigment from Mantoniella compares best with Chl c-1. In order to clarify the spectral data, Chl c-1 and c-2, Mg-DVPP, and the pigment from Mantoniella were subjected to a chromatographic system that is able to separate these porphyrins. The chromatographic analysis clearly shows that the pigment from Mantoniella co-migrates with Chl c-1 and not with the bacterial pigment. Mantoniella is the first organism which has been demonstrated to contain Chl a, b, and authentic c.     

DETECTION OF CHLOROPHYLLS c1, c2 AND c3 IN PIGMENT EXTRACTS OF PRYMNESIUM PARWM (PRYMNESIOPHYCEAE)1

Three chlorophyll c-type pigments were separated by reversed-phase high Performance liquid chromatography and thin-layer chromatography from pigment extracts of the prymnesiophyte, Prymnesium parvum Carter. Based on spectral characteristics, retention times, and comparison with reference pigments isolated from the diatom Phaeodactylum tricornutum Bohlin, two of these pigments were identijied as chlorophyll c₁ and c₂. The other pigment was identified by its absorption spectrum and thin-layer chromatography retention times as the newly described chlorophyll c₃. However, in other prymnesiophytes so far examined, chlorophyll c₁ and chlorophyll c₃ were present with no chlorophyll c_l. The discovery of chlorophyll c₃ with chlorophyll c₁ and chlorophyll c₃ in Prymnesium parvum therefore represents the first report of this combination of pigments in prymnesiophytes.     

Preparation of chlorophyll a, chlorophyll b and bacteriochlorophyll a by means of column chromatography with diethylaminoethylcellulose

Chlorophyll a, chlorophyll b and bacteriochlorophyll a were prepared by means of column chromatography with Sephadex LH-20 and diethylaminoethylcellulose. This method provides purified preparations of chlorophylls in about 3 h.To prepare chlorophyll a, blue-green or red algae were used as the starting material. Chlorophyll a was extracted with 90% aqueous acetone from cells of blue-green algae, Anabaena variabilis, Anacystis nidulans and Tolypothrix tenuis, and with 90% aqueous methanol from thalli of a red alga, Porphyra yezoensis. Chlorophyll a was collected as precipitates by adding dioxane and water to the extract according to the method of Iriyama et al. [6]. The crude chlorophyll a preparation was applied to a Sephadex LH-20 column with chloroform as the eluent and then to a DEAE-cellulose column with a chloroform/methanol mixture (49 : 1, v/v) as the eluent. Analysis with thin layer chromatography revealed that the chlorophyll a preparation contained no detectable contaminants.Bacteriochlorophyll a was prepared in a similar manner from purple photosynthetic bacteria, Rhodopseudomonas spheroides and Chromatium vinosum.In order to prepare chlorophyll b, chloroplasts of spinach leaves were used as the starting material. A mixture of chlorophylls a and b was obtained in the same way as described for the preparation of chlorophyll a from the blue-green algae. To separate chlorophyll b from chlorophyll a, the mixture was applied to a diethylaminoethylcellulose column which was developed with a hexane/2-propanol mixture (5 : 2, v/v).     

Chlorophyll analysis by high-performance liquid chromatography

The separation and determination of chlorophylls by high-performance liquid chromatography (HPLC) is described. Chlorophylls and their derivatives were separated by reversed-phase HPLC based on hydrophobic interaction between solute and support, using an octadecyl silica column and elution with 100% methanol. Separated pigments were detected fluorometrically with a sensitivity in the picomole range: the fluorescence response was linear over a wide pigment concentration range. Resolution of five chlorophylls a and four protochlorophyll species esterified with different alcohols was achieved within 22 min in a single experiment. This method can be used for the determination of chlorophyll b, bacteriochlorophyll a esters and products synthesized from chlorophyll, but not for nonesterified pigments, i.e., chlorophyllide, protochlorophyllide and chlorophyll c. The chromatographic mobility of chlorophyll a esterified with different alcohols increases with increasing number of carbon atoms in the esterifying alcohols. The plots obtained from the logarithm of the capacity factor (k′) of these pigments versus the numbers of carbon atoms of the alcohol molecule gave a straight line, thus permitting the estimation of the chain length of unknown pigment esterifying alcohols. This HPLC separation technique did not cause the formation of artifacts. The deviation of the individual retention time for each pigment is less than ±0.5%, thus making this method suitable for the rapid identification and quantification of unknown pigments.     

Studies on phytoplankton pigments in Porto Novo waters (India). I. Mangrove

The annual variations of phytoplankton pigments were studied from January to December, 1971, at two stations of the local mangrove (Pichavaram) environment. At these two stations, chlorophyll a varied from 2.90 to 35.06; chlorophyll b from 0 to 10.02 and chlorophyll c from 0 to 18.12 μg/l. Plant carotenoids varied from 1.56 to 13.83 MSPU/m³ and phaeopigments from 0 to 12.28 μg/l. The main (primary) peak of chlorophyll a was recorded during March at Station 1, and during June at Station 2.Secondary maxima occurred during June and August at Station 1, and during September at Station 2. During the period studied chlorophyll a was the dominant pigment at both the stations, followed by chlorophyll c and chlorophyll b in that order. The increase in the concentration of pigments was mainly due to the presence of phytoplankton species belonging to the genera such as Coscinodiscus, Rhizosolenia, Thalassiothrix, Melosira, Chaetoceros and Biddulphia. During October, phytoplankton was less and the pigment concentration was also low.     

Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH⁻₄). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH⁻₄ in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13¹-deoxo-13¹-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo⁻ and P680⁺. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Q_y and Q_x transitions of this molecule were determined to be located at 5°C at 679.5–680 nm and 542 nm, respectively.     

Antenna replacement in the evolutionary origin of chloroplasts

The endosymbiotic origin of chloroplasts from unicellular cyanobacteria is presently beyond doubt. Oxygenic photosynthesis is based on coordinated action of the two photosystems (PS), PS I and PS II, cooperating with several variants of the pigment antenna. In cyanobacteria, red algae, and glaucophytes, phycobilisomes (PBS) act as antennae, while in terrestrial plants, as well as in most macro- and microalgae, antennae are formed by chlorophyll a/b- and chlorophyll a/c-containing proteins. Advantages and disadvantages of the PBS antenna compared to other light-harvesting complexes form the basis for adaptive variations of the antenna in the course of development of eukaryotic photosynthesis. During the evolution of the “green” and “chromophyte” lineages of the chloroplasts, PBS, in spite of their optimal features of light absorption, were replaced by chlorophyll a/b- and chlorophyll a/c-containing light-harvesting complexes. Development of the cell wall associated with the limitation of motility and tissue formation in photosynthetic eukaryotes were the factors responsible for the antenna shift. The subsequent redistribution of cell resources in favor of cellulose biosynthesis required for increased CO₂ consumption, higher PS II levels, and greater number and density of the thylakoids in the chloroplasts, was incompatible with the energy-consuming and overly large PBS antenna.