Chlorophyll Pathways Research Articles

Chlorophyll biosynthesis in Chlamydomonas starts with the formation of glutamyl-tRNA.

An RNA moiety has been shown to be involved in the conversion of Glu to delta-aminolevulinic acid (ALA), the first committed intermediate of the chlorophyll pathway. We now have evidence suggesting that in Chlamydomonas, the first reaction for converting Glu to ALA is the aminoacylation of Glu to a Glu-specific tRNA. The Glu-tRNA thus formed could be the substrate for Glu-1-semialdehyde synthesis catalyzed by a postulated dehydrogenase. Glu-1-semialdehyde can be converted to ALA by an aminotransferase. Of the three reactions converting Glu to ALA, only the second reaction, catalyzed by a postulated dehydrogenase, is sensitive to inhibition by heme (a known inhibitor of ALA synthesis). We think the regulated enzyme of ALA synthesis is the postulated dehydrogenase. It is postulated that in the chloroplast of Chlamydomonas, the synthesis of ALA and the synthesis of proteins may share a common pool of glutamyl-tRNA.

Pigment protein complexes and functional properties of tetratype resulting from crosses between CP1 and CP2 less Chlamydomonas mutants

A chlorophyll b-less mutant of Chlamydomonas reinhardtii (Pg 27) was isolated after UV irradiation of the wild type cells. This photosynthetically competent mutant totally lacks chlorophyll b and the CP2 chlorophyll-protein complex. However, SDS-PAGE, proteolytic digestions and immunodetections demonstrated that the 24-25 Kd apoproteins of the lacking CP2 complex are still present in thylakoids of the Pg27 mutant. It is concluded that this CP2-less mutant is affected in the biosynthesis pathway of chlorophyll b.This CP2-less mutant was crossed with a CP1-less mutant (Fl5) Fluorescence emission spectra and fluorescence inductions in the presence of DCMU were analysed in the resulting (cp 2 (-) , cp 1 (+) ), (cp 2 (+) , cp 1 (-) ), (cp 2 (+) , cp 1 (+) ), cp 2 (-) , cp 1 (-) )tetratype. Differences in PS 2 optical cross section and in the relative amplitude or localisation of fluorescence emission peaks fit well with a quadripartite model where PS1 and PS2 would each correspond to a reaction centre core complex (CP1 and CP2 respectively) associated to a light harvesting antenna (LHC1 and LHC2 respectively). The occurrence of energy transfers from PS1 peripheral antenna to PS2 in the Fl 5 mutant shows that, in absence of CP1, at least a part of its associated PS1 light harvesting antenna migrates in the PS2 containing appressed thylakoids.

A model for the origin of photosynthesis--III. The ultraviolet photochemistry of uroporphyrinogen.

The photochemical ramifications of the high ultraviolet flux on the primordial earth prior to the formation of the ozone layer have been considered in a study of the ultraviolet photochemistry of uroporphyrinogen (urohexahydroporphyrin), a colorless compound which absorbs strongly at wavelengths less than 220 nanometers. Urohexahydroporphyrin was investigated since it is the first macrocycle formed on the biosynthetic pathway of chlorophyll and can be used to test the hypothesis that the biosynthetic pathway to chlorophyll recapitulates the evolutionary history of photosynthesis. When urohexahydroporphyrin is illuminated in aqueous anaerobic solution, hydrogen gas is produced. More hydrogen gas is produced in the presence of a colloidal platinum catalyst. The products of the photooxidation of urohexahydroporphyrin are urotetrahydroporphyrin (uroporphomethene) and uroporphyrin. This research shows how the oxidation of uroporphyrinogen to uroporphyrin, the first biogenetic porphyrin, could have occurred anaerobically and abiotically on the primordial earth.

Analysis of chlorophyll diagenesis—II. Solvent effects on metal incorporation into chlorophyll diagenetic products

We are attempting to quantify selected transitions in the diagenetic pathway of chlorophyll in deep ocean sediments. To analyze the aromatization from phorbides (less stable) to porphyrins (more stable), we used a model system which closely resembles the compounds identified in oceanic sediments. Kinetic parameters were determined for the incorporation of copper into deoxomesopyropheophorbide a methyl ester (DOMPP) and deoxophylloerythrin methyl ester (DPE) in a series of solvents ranging from highly polar and protic to non-polar and aprotic, representing varying sedimentary environments. In the reaction of copper with these two compounds, the activation parameters for the reaction with DOMPP indicate that DOMPP is more sensitive to the environment than DPE. Also, over the temperature range studied, DOMPP always reacted faster than DPE under similar conditions. These two observations lend support to our previous hypothesis that the phorbide to porphyrin transition includes the metallophorbide as a short-lived intermediate in the reaction sequence.

Analysis of chlorophyll diagenesis—I. Kinetics of the phorbide to porphyrin transition

The objective of this project is to quantify selected transitions in the diagenetic pathways of chlorophyll in deep ocean sediments, initially the aromatization from phorbides (unstable) to porphyrins (stable), using a model system which closely resembles the compounds identified in oceanic sediments. We determined the kinetic parameters for the aromatization of deoxomesopyropheophorbide a methyl ester (DOMPP) to deoxophylloerythrin methyl ester (DPE) in the presence of Cu(II) in a series of solvents ranging from highly polar and protic to nonpolar and aprotic, representing varying sedimentary environments. With Cu(II) in ethanol, the aromatization occurs through a pathway which involves a relatively slow initial formation of CuDOMPP, rapid aromatization to CuDPE, and finally an equilibrium reaction in the presence of clay which induces demetalation to form the free base DPE. Based on our model studies, only DOMPP, CuDPE and some DPE would be detected in significant quantities in more polar, protic sedimentary environments on a geologic time scale, although the story might be different with changed environment polarity. Our model studies may ultimately reconcile laboratory observations with those based on DSDP cores which appear to indicate that the diagenetic sequence proceeds from phorbide to free base porphyrin, followed by metalation of the porphyrin.

Input and early diagenesis of chlorophylls in a temperate intertidal sediment

This paper reports the initial steps in the degradation of chlorophyll- a in a temperate, sandy intertidal sediment from a low-energy coastline at Corner Inlet, SE. Victoria. The chlorophyll- a is shown to be derived primarily from diatom input into the surface layers and it is possible to show that in this environment, relative stabilities are: chlorophyll- a > chlorophyllide-C 2 > chlorophyllide-C 1. Traces only of chlorophyll- b are present, and little or no degradation of this pigment with depth was observed. Bacteriochlorophyll- a, bacteriopheophytin- a concentrations are inversely proportional to one another in the upper sedimentary layers and the depth profile suggests a stabilised environment. A depth profile of a range of chlorophyll- a derivatives, including pheophytin- a, provides no basis for accepting that they are intermediates on the direct degradative pathway of chlorophyll- a in this environment. Rather, the evidence favours the view that chlorophyll- a degrades primarily to colourless compounds.

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4- 14C]levulinic acid into [ 14C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp ( E 430- F 631)] exhibited a soret excitation maximum at 430 nm ( E 430) and a fluorescence emission maximum at 631 nm ( F 631) in methanol/acetone (4 : 1, v/v). Cp ( E 430- F 631) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.

Introductory remarks

Our present knowledge of the mode of formation in living cells of the porphyrins dates from the classic paper by Shemin & Rittenberg, who showed that the nitrogen atom of glycine was specifically used in the biosynthesis of protoporphyrin. In the following five years it was demonstrated that glycine and succinyl-coenzyme A combine under the influence of a specific enzyme, and in the presence of pyridoxal phosphate, to give 5-aminolaevulinic acid, and the carbon and nitrogen atoms of this compound account for all the carbon and nitrogen atoms in protoporphyrin. The main outlines of the reactions leading to haem are now established. We have also good reasons to believe that chlorophylls a and b and others are derived from protoporphyrin. It is also well established that the insertion of magnesium is a first step in the specific biosynthetic pathway of chlorophyll, and this insertion is followed or associated with the esterification of one of the carboxyl groups of one of the two propionic acid side-chains. Full details of these reactions and particularly the sequence of the various steps are still a matter for further investigation.

Chlorophyll and photosynthesis

Photosynthesis is the prerequisite of all life on earth. Chlorophyll fulfils the requirements for photosynthesis: the absorption of visible light, the photochemical capabilities, a rich supply of redox levels and chemical stability. The biosynthetic pathway of chlorophyll can be read as the evolutionary history of photosynthesis. Our exegesis is that the primary porphyrins served for early photosynthesis. The porphyrins readily photo-oxidize organic compounds under the reducing, aqueous conditions of this early era. The formation of oxidized substances in a reducing atmosphere supplied the thermodynamic gradient necessary for organized life processes. Conversely, the closed-shell metalloporphyrins, notable magnesium porphyrins, are powerful photoreducing agents. When coupled to the ultimate electron source, water, oxygen was produced and the modern era of photosynthesis was born. At the same time, the efficiency and usefulness of the photopigments was increased by incorporating them into the organized cellular system of membranes. The clear gradient of ionic to hydrophophic structures along the biosynthetic pathway from porphyrins to chlorophyll supports this view. Experimental evidence on the photochemistry of porphyrins pigments in solution and in lipid bilayers form the basis for these arguments. In this way we can relate the structure of chlorophyll to its function in photosynthesis.

The Biosynthesis of δ-Aminolevulinic Acid in Higher Plants

delta-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various (14)C-labeled precursors and levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydrase. The radioactivity in the accumulated delta-aminolevulinic acid was measured.The most effective labeled precursors were the 5 carbon dicarboxylic compounds glutamate, glutamine, and alpha-ketoglutarate. (14)C-Labeled glycine and succinate were relatively poor. The carboxyl and the methylene carbons of glycine were incorporated into delta-aminolevulinic acid to about equal extent. The carboxyl carbon of glutamate was incorporated almost as well as the internal carbons of the same compound. These results are inconsistent with the succinyl CoA-glycine succinyl transferase (delta-aminolevulinic acid synthetase) mode of delta-aminolevulinic acid production.When the same experiments were performed on turkey blood (which, as avian blood in general, possesses delta-aminolevulinic acid synthetase), delta-aminolevulinic acid was labeled most effectively from glycine-2-(14)C, moderately well from glycine-1-(14)C and glutamate-3,4-(14)C and not at all from glutamate-1-(14)C.It appears probable that greening higher plant tissues possess an alternate route to delta-aminolevulinic acid in which the carbon skeleton of glutamate (and alpha-ketoglutarate) is incorporated intact into the first committed metabolite of the chlorophyll pathway.

Thermal Alteration of Blue-Green Algae and Blue-Green Algal Chlorophyll

The thermal alteration of blue-green algae (Lyngbya) and blue-green algal chlorophyll has been studied at temperatures ranging from 25 to 300°C for periods ranging from ½ hour to 2,000 hours under both oxic and anoxic conditions. An irreversible series of time- and temperature-dependent color changes was observed in the cell walls, intracellular pigments, and extracellular sheaths of the heated algae. These sequential color changes are comparable to changes during eometamorphism of plant microfossils, especially palynomorphs, and seem similar to pigment changes during maturation of Tertiary organic matter. Inferred degradation pathways of intracellular chlorophyll differ from those reported for extracellular chlorophyll and its derivatives; intracellular pigment apparently become grafted onto cellular macromolecules, a process that presumably occurs also during natural diagenesis. Formation of such complexes could be an important diagenetic process affecting the types and distribution of pigments in sediments and sedimentary rocks.

Chloroplast ultrastructure in pigment-deficient mutants of Zea mays under reduced light

The fine structure of normal and mutant seedlings of Zea mays when grown under weak light conditions was compared with their ability to produce precursors and final products along the biosynthetic pathways of chlorophyll and carotenoid pigments to determine the effect of each on plastid structure. Seedlings of normal, pale green, yellow-green, yellow, albino, and green mosaic mutants were grown in 2 000 footcandles of light and also in 0.5 to 1 footcandle for 11–14 days at 27°C. Samples of leaf tissue were taken at the same time for electron microscopic studies and for spectrophotometrical analyses of pigment content. Samples from dark-grown seedlings were analyzed for protochlorophyllide. Growth of these mutants in dim light conditions in which the chlorophyll was not photooxidized, and thus could accumulate, was useful in determining the potential development of the mutants. Different levels of pigment and precursor accumulation and protochlorophyllide conversion correlated well with plastid structural differences, providing information regarding the location of the genetic blocks.

Investigations on the biogenesis of chlorophyll a: III. Biosynthesis of Mg-vinylpheoporphine a 5 methylester from Mg-protoporphine IX monomethylester as observed in Chlorella mutants

Ultraviolet Chlorella mutants are described. Structures are suggested for the porphyrins which they accumulate and which appear to complete the gap in the proposed biosynthetic pathway of chlorophyll a between Mg-protoporphine IX monomethyl ester and vinylpheoporphine a 5 methyl ester. In this sequence, formation of the isocyclic ring is preceded by reduction of the 4-vinyl side chain. Structure assignments were made primarily by mass spectrometry of a mixture of the esterified porphyrins, oxidation of the mixtures, and identification of the products (maleimides).

(Minus) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, an enzyme in the biosynthetic pathway of chlorophyll in Zea mays.

The enzyme (-) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, which catalyzes the transfer of the methyl group from (-) S-adenosyl-L-methionine to magnesium protoporphyrin to form magnesium protoporphyrin monomethyl ester, has been detected in chloroplasts isolated from Zea mays. Zinc protoporphyrin and free protoporphyrin also act as substrates in the system, although neither one is as active as magnesium protoporphyrin. THE FOLLOWING SCHEME OF CHLOROPHYLL SYNTHESIS IN HIGHER PLANTS IS PROPOSED: delta-aminolevulinic acid --> --> --> protoporphyrin --> magnesium protoporphyrin --> magnesium protoporphyrin monomethyl ester --> --> --> chlorophyll a.

Chlorophyllase of tobacco plants. II. Enzymic phytylation of chlorophyllide and pheophorbide in vitro

The breakdown of chlorophyll by the water-soluble chlorophyllase prepared from tobacco chloroplasts was decreased by a comparatively low concentration of phytol. The same enzyme preparation could phytylate chlorophyllide to give chlorophyll. It also synthesized pheophytin from pheophorbide and phytol. Chlorophyllide and pheophorbide were prepared by a simple and quick method. They were identified by their mobility on chromatograms, HCl numbers, and absorption spectra. The possibility that chlorophyllase acts in the biosynthetic pathway of chlorophyll in higher plants as well as in vitro systems was discussed.