Biosynthesis of phycobilins. Ferredoxin-mediated reduction of biliverdin catalyzed by extracts of Cyanidium caldarium

Cell-free extract of the unicellular rhodophyte, Cyanidium caldarium catalyzes enzymatic reduction of biliverdin IX alpha to phycocyanobilin, the chromophore of the light-harvesting phycobiliprotein, phycocyanin. The enzyme activity is soluble, and the required reductant is NADPH. The extract has been separated into three protein fractions, all of which are required to reconstitute biliverdin reduction. One fraction contains ferredoxin, which was identified by its absorption spectrum. This fraction could be replaced with commercial ferredoxin derived from spinach or the red alga, Porphyra umbilicalis. The second protein fraction contains ferredoxin-NADP+ reductase, which was identified by the ability to catalyze ferredoxin-dependent reduction of cytochrome c in the presence of NADPH. This fraction could be replaced with commercial spinach ferredoxin-NADP+ reductase. These two components appear to be identical to previously described components of the algal heme oxygenase system that catalyzes biliverdin IX alpha formation from protoheme in C. caldarium extracts. The third protein fraction, in the presence of the first two (or their commercial counterparts) plus NADPH, catalyzes the reduction of biliverdin IX alpha to phycocyanobilin. The results indicate that the transformation of biliverdin to phycocyanobilin catalyzed by C. caldarium extracts is a ferredoxin-linked reduction process. The results also suggest the possibility that heme oxygenation and biliverdin reduction may occur in C. caldarium on associated enzyme systems.     

Biosynthesis of phycobilins. Ferredoxin-supported nadph-independent heme oxygenase and phycobilin-forming activities from Cyanidium caldarium.

The unicellular red alga, Cyanidium caldarium, synthesizes phycocyanobilin from protoheme via biliverdin IX alpha. In vitro transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins were previously shown to require NADPH, ferredoxin, and ferredoxin-NADP+ reductase, as well as specific heme oxygenase and phycobilin formation enzymes. The role of NADPH in these reactions was investigated in this study. The C. caldarium enzymatic activities that catalyze biliverdin IX alpha formation from protoheme, and phycobilin formation from biliverdin IX alpha, were partially purified by differential (NH4)2SO4 precipitation. The enzyme fractions, when supplemented with a light-driven ferredoxin-reducing photosystem I fraction derived from spinach leaves, catalyzed light-dependent transformation of protoheme to biliverdin IX alpha and biliverdin IX alpha to phycobilins, with or without the addition of NADPH and ferredoxin-NADP+ reductase. In the dark, neither reaction occurred unless NADPH and ferredoxin-NADP+ reductase were supplied. These results indicate that the only role of NADPH in both reactions of phycobilin biosynthesis, in vitro, is to reduce ferredoxin via ferredoxin-NADP+ reductase and that reduced ferredoxin can directly supply the electrons needed to drive both steps in the transformation of protoheme to phycocyanobilin.     

The biosynthesis of the chromophore of phycocyanin. Pathway of reduction of biliverdin to phycocyanobilin.

The later stages in the pathway of biosynthesis of phycocyanobilin, the chromophore of phycocyanin, were studied by using radiolabelled intermediates. Three possible pathways from biliverdin IX-alpha to phycocyanobilin were considered. 14C-labelled samples of key intermediates in two of the pathways, 3-vinyl-18-ethyl biliverdin IX-alpha and 3-ethyl-18-vinyl biliverdin IX-alpha, were synthesized chemically and were administered to cultures of Cyanidium caldarium that were actively synthesizing photosynthetic pigments in the light. Neither of these two compounds was apparently incorporated into the phycobiliprotein chromophore, suggesting that two of the three pathways were not operative. By elimination, the results imply that the third possible pathway, which involves phytochromobilin, the chromophore of phytochrome, represents the route for biosynthesis of phycocyanobilin. Unfortunately, since 14C-labelled phytochromobilin is not available, no direct proof of this pathway could be obtained. However, if correct, the present interpretation represents a unified pathway for biosynthesis of all plant bilins, via the intermediacy of phytochromobilin.     

Biosynthesis of the chromophore of phycobiliproteins. A study of mesohaem and mesobiliverdin as possible intermediates and further evidence for an algal haem oxygenase.

The possible roles of mesohaem and mesobiliverdin as metabolic precursors of phycocyanobilin, the chromophore of phycocyanin, were studied in the unicellular rhodophyte Cyanidium caldarium. Dark-grown cells of this organism, which had been exposed to mesohaem, were either incubated in the dark with 5-aminolaevulinate, which results in excretion of bilins into the suspending medium, or incubated in the light, which results in synthesis of phycocyanin within the cells. By using 14C-labelling, either in the mesohaem or in the 5-aminolaevulinate administered, it was shown that mesohaem is not a precursor of phycocyanobilin in either dark or light systems. However, mesohaem was converted into mesobiliverdin in both systems, a phenomenon that is further evidence for the existence of an algal haem oxygenase. The data also showed that mesobiliverdin is not a precursor of phycocyanobilin. These results suggest that algal bilins are formed via haem degradation to biliverdin in the same way as mammalian bile pigments.     

Biosynthesis of phycocyanobilin from exogenous labeled biliverdin in Cyanidium caldarium

Phycocyanin is a major light-harvesting pigment in bluegreen, red, and cryptomonad algae. This pigment is composed of phycocyanobilin chromophores covalently attached to protein. Phycocyanobilin is an open-chain tetrapyrrole structurally close to biliverdin. Biliverdin is formed in animals by oxidative ring-opening of protoheme. Recent evidence indicates that protoheme is a precursor of phycocyanobilin in the unicellular rhodophyte, Cyanidium caldarium. To find out if biliverdin is an intermediate in the conversion of protoheme to phycocyanobilin, [14C]biliverdin was administered along with N-methylmesoporphyrin IX (which blocks endogenous protoheme formation) to growing cells of C. caldarium. To avoid phototoxic effects due to the porphyrin, a mutant strain was used that forms large amounts of both chlorophyll and phycocyanin in the dark. After 12 or 24 h in the dark, cells were harvested and exhaustively extracted to remove free pigments. Next, protoheme was extracted. Phycocyanobilin was then cleaved from the apoprotein by methanolysis. Protoheme and phycocyanobilin were purified by solvent partition, DEAE-Sepharose chromatography, and preparative reverse-phase high-pressure liquid chromatography. Absorption was monitored continuously and fractions were collected for radioactivity determination. Negligible amounts of label appeared in the protoheme-containing fractions. A major portion of label in the eluates of the phycocyanobilin-containing samples coincided with the absorption peak at 22 min due to phycocyanobilin. In a control experiment, [14C]biliverdin was added to the cells after incubation and just before the phycocyanobilin-apoprotein cleavage step. The major peak of label then eluted with the absorption peak at 12 min due to biliverdin, indicating that during the isolation biliverdin is not converted to compounds coeluting with phycocyanobilin. It thus appears that exogenous biliverdin can serve as a precursor to phycocyanobilin in C. caldarium, and that the route of incorporation is direct rather than by degradation and reincorporation of 14C through protoheme.     

Phycobilin biosynthetic reactions in extracts of cyanobacteria

Phycobilins are the chromophores of phycobiliproteins, the light-harvesting pigments of cyanobacteria, red algae and cryptophytes. Phycobilins are biosynthesized from heme by the action of heme oxygenase, which converts heme to biliverdin, followed by the action of other enzymes that convert biliverdin to the phycobilins. We previously reported on the enzymes and biosynthetic intermediates of phycobilin formation in extracts of the unicellular red alga Cyanidium caldarium. Heme oxygenase activity has now been obtained from extracts of the cyanobacterium Synechocystis sp. PCC 6701. The reaction requirements are similar to those for the C. caldarium enzyme: heme substrate, reduced ferredoxin, and a second reductant such as ascorbate or Trolox. The enzymatic nature of the reaction was verified by two criteria in addition to the requirement for cell extract: production of only the IX isomer of the bilin product and inhibition by the substrate analog Sn-protoporphyrin IX. The enzyme was partially purified by high-speed centrifugation, 35–75% differential (NH4)2SO4 precipitation, and DEAE-cellulose anion exchange chromatography. In addition, extract capable of converting biliverdin IX to phycobilins has been obtained from Synechocystis sp. PCC 6701 and another cyanobacterium, Synechocystis sp. PCC 6803. Only the (3Z) isomers of the phycobilins accumulated in the incubations containing unfractionated cell extracts, in contrast to incubations with unfractionated C. caldarium extracts which produce both the (3Z) and (3E) isomers. Phycocyanobilin and phycoerythrobilin were produced in comparable amounts by Synechocystis sp. PCC 6701 extracts, but only phycocyanobilin accumulated in Synechocystis sp. PCC 6803 extracts. This difference in in vitro product accumulation correlates with the phycobilins that are found in vivo in these two cell types.     

Enzymic Transformation of Biliverdin to Phycocyanobilin by Extracts of the Unicellular Red Alga Cyanidium caldarium

Cell-free extracts of the unicellular red alga Cyanidium caldarium catalyze the transformation of biliverdin to a product indistinguishable from phycocyanobilin, the free bilin derived from phycocyanin by methanolysis. Crude cell-free extract requires biliverdin as the only substrate, but after removal of low molecular weight components by gel filtration, the reaction shows an additional requirement for a reduced pyridine nucleotide. Boiled extract is enzymically inactive, activity is not sedimented by high-speed centrifugation, and mesobiliverdin cannot serve as a substrate.

Incubation of cell extracts with biliverdin yields two products with very similar spectrophotometric properties in acidic methanol, but which are separable by reverse-phase high pressure liquid chromatography. The same two products are formed by methanolysis of protein-bound phycocyanin chromophore, with the late-eluting one predominating. The two products derived from either phycocyanin methanolysis or cell extract incubation with biliverdin are partially interconvertible and they form the same ethylidine-free isomeric derivative, mesobiliverdin. Their absorption spectra correspond to those of the Z- and E-ethylidine isomers of phycocyanobilin. Based on previous work showing that the major methanolysis product has the E-ethylidine configuration, the other product of methanolysis and enzymic biliverdin transformation is therefore the Z-ethylidine isomer. The time course for formation of the two products during incubation suggests that the early-eluting product is the precursor of the late-eluting one. These results suggest that Z-ethylidine phycocyanobilin is the precursor of the E-ethylidine isomer, and that the latter may be a normal cellular precursor to protein-bound phycocyanin chromophore.

     

Chromophore attachment to biliproteins: specificity of PecE/PecF, a lyase-isomerase for the photoactive 3(1)-cys-alpha 84-phycoviolobilin chromophore of phycoerythrocyanin

PecE and PecF, the products of two phycoerythrocyanin lyase genes (pecE and pecF) of Mastigocladus laminosus (Fischerella), catalyze two reactions: (1) the regiospecific addition of phycocyanobilin (PCB) to Cys-alpha 84 of the phycoerythrocyanin alpha-subunit (PecA), and (2) the Delta 4-->Delta 2 isomerization of the PCB to the phycoviolobilin (PVB)-chromophore [Zhao et al. (2000) FEBS Lett. 469, 9-13]. The alpha-apoprotein (PecA) as well PecE and PecF were overexpressed from two strains of M. laminosus, with and without His-tags. The products of the spontaneous addition of PCB to PecA, and that of the reaction catalyzed by PecE/F, were characterized by their photochemistry and by absorption, fluorescence, circular dichroism of the four states obtained by irradiation with light (15-Z/E isomers of the chromophore) and/or modification of Cys-alpha 98/99 with thiol-directed reagents. The spontaneous addition leads to a 3(1)-Cys-PCB adduct, which is characteristic of allophycocyanins and phycocyanins, while the addition catalyzed by PecE and PecF leads to a 3(1)-Cys-PVB adduct which after purification was identical to alpha-PEC. The specificity and kinetics of the chromophore additions were investigated with respect to the structure of the bilin substrate: The 3-ethylidene-bilins, viz., PCB, its 18-vinyl analogue phytochromobilin, phycoerythrobilin and its dimethylester, react spontaneously to yield the conventional addition products (3-H, 3(1)-Cys), while the 3-vinyl-substituted bilins, viz., bilirubin and biliverdin, were inactive. Only phycocyanobilin and phytochromobilin are substrates to the addition-isomerization reaction catalyzed by PecE/F. The slow spontaneous addition of phycoerythrobilin is not influenced, and there is in particular no catalyzed isomerization to urobilin.     

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.     

Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli

Many cyanobacteria use brilliantly pigmented, multisubunit macromolecular structures known as phycobilisomes as antenna to enhance light harvesting for photosynthesis. Recent studies have defined the enzymes that synthesize phycobilin chromophores as well as many of the phycobilin lyase enzymes that attach these chromophores to their cognate apoproteins. The ability of the phycocyanin α-subunit (CpcA) to bind alternative linear tetrapyrrole chromophores was examined through the use of a heterologous expression system in Escherichia coli. E. coli strains produced phycocyanobilin, phytochromobilin, or phycoerythrobilin when they expressed 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), 3Z-phytochromobilin:ferredoxin oxidoreductase (HY2) from Arabidopsis thaliana, or phycoerythrobilin synthase (PebS) from the myovirus P-SSM4, respectively. CpcA from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 was coexpressed in these strains with the phycocyanin α-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin α-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Both lyases were capable of attaching three different linear tetrapyrrole chromophores to CpcA; thus, up to six different CpcA variants, each with a unique chromophore, could be produced with this system. One of these chromophores, denoted phytoviolobilin, has not yet been observed naturally. The recombinant proteins had unexpected and potentially useful properties, which included very high fluorescence quantum yields and photochemical activity. Chimeric lyases PecE/CpcF and CpcE/PecF were used to show that the isomerizing activity that converts phycocyanobilin to phycoviolobilin resides with PecF and not PecE. Finally, spectroscopic properties of recombinant phycocyanin R-PCIII, in which the CpcA subunits carry a phycoerythrobilin chromophore, are described.     

Enzymatic heme oxygenase activity in soluble extracts of the unicellular red alga, Cyanidium caldarium

Extracts of the phycocyanin-containing unicellular red alga, Cyanidium caldarium, catalyzed enzymatic cleavage of the heme macrocycle to form the linear tetrapyrrole bilin structure. This is the key first step in the branch of the tetrapyrrole biosynthetic pathway leading to phycobilin photosynthetic accessory pigments. A mixed-function oxidase mechanism, similar to the biliverdin-forming reaction catalyzed by animal cell-derived microsomal heme oxygenase, was indicated by requirements for O2 and a reduced pyridine nucleotide. To avoid enzymatic conversion of the bilin product to phycocyanobilins and subsequent degradation during incubation, mesoheme IX was substituted for the normal physiological substrate, protoheme IX. Mesobiliverdin IX alpha was identified as the primary incubation product by comparative reverse-phase high-pressure liquid chromatography and absorption spectrophotometry. The enzymatic nature of the reaction was indicated by the requirement for cell extract, absence of activity in boiled cell extract, high specificity for NADPH as cosubstrate, formation of the physiologically relevant IX alpha bilin isomer, and over 75% inhibition by 1 microM Sn-protoporphyrin, which has been reported to be a competitive inhibitor of animal microsomal heme oxygenase. On the other hand, coupled oxidation of mesoheme, catalyzed by ascorbate plus pyridine or myoglobin, yielded a mixture of ring-opening mesobiliverdin IX isomers, was not inhibited by Sn-protoporphyrin, and could not use NADPH as the reductant. Unlike the animal microsomal heme oxygenase, the algal reaction appeared to be catalyzed by a soluble enzyme that was not sedimentable by centrifugation for 1 h at 200,000g. Although NADPH was the preferred reductant, small amounts of activity were obtained with NADH or ascorbate. A portion of the activity was retained after gel filtration of the cell extract to remove low-molecular-weight components. Considerable stimulation of activity, particularly in preparations that had been subjected to gel filtration, was obtained by addition of ascorbate to the incubation mixture containing NADPH. The results indicate that C. caldarium possesses a true heme oxygenase system, with properties somewhat different from that catalyzing heme degradation in animals. Taken together with previous results indicating that biliverdin is a precursor to phycocyanobilin, the results suggest that algal heme oxygenase is a component of the phycobilin biosynthetic pathway.     

Insights into phycoerythrobilin biosynthesis point toward metabolic channeling

Phycoerythrobilin is a linear tetrapyrrole molecule found in cyanobacteria, red algae, and cryptomonads. Together with other bilins such as phycocyanobilin it serves as a light-harvesting pigment in the photosynthetic light-harvesting structures of cyanobacteria called phycobilisomes. The biosynthesis of both pigments starts with the cleavage of heme by heme oxygenases to yield biliverdin IXalpha, which is further reduced at specific positions by ferredoxin-dependent bilin reductases (FDBRs), a new family of radical enzymes. The biosynthesis of phycoerythrobilin requires two subsequent two-electron reductions, each step being catalyzed by one FDBR. This is in contrast to the biosynthesis of phycocyanobilin, where the FDBR phycocyanobilin: ferredoxin oxidoreductase (PcyA) catalyzes a four-electron reduction. The first reaction in phycoerythrobilin biosynthesis is the reduction of the 15,16-double bond of biliverdin IXalpha by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated pi -electron system thereby blue-shifting the absorbance properties of the linear tetrapyrrole. The second FDBR, phycoerythrobilin:ferredoxin oxidoreductase (PebB), then reduces the A-ring 2,3,3(1),3(2)-diene structure of 15,16-dihydrobiliverdin to yield phycoerythrobilin. Both FDBRs from the limnic filamentous cyanobacterium Fremyella diplosiphon and the marine cyanobacterium Synechococcus sp. WH8020 were recombinantly produced in Escherichia coli and purified, and their enzymatic activities were determined. By using various natural bilins, the substrate specificity of each FDBR was established, revealing conformational preconditions for their unique specificity. Preparation of the semi-reduced intermediate, 15,16-dihydrobiliverdin, enabled us to perform steady state binding experiments indicating distinct spectroscopic and fluorescent properties of enzyme.bilin complexes. A combination of substrate/product binding analyses and gel permeation chromatography revealed evidence for metabolic channeling.     

Holophytochrome assembly. Coupled assay for phytochromobilin synthase in organello

Utilizing an in vitro coupled assay system, we show that isolated plastids from cucumber cotyledons convert the linear tetrapyrrole biliverdin IX alpha to the free phytochrome chromophore, phytochromobilin, which assembles with oat apophytochrome to yield photoactive holoprotein. The spectral properties of this synthetic phytochrome are indistinguishable from those of the natural photoreceptor. The plastid-dependent biliverdin conversion activity is strongly stimulated by both NADPH and ATP. Substitution of the nonnatural XIII alpha isomer of biliverdin for the IX alpha isomer affords a synthetic holophytochrome adduct with blue-shifted difference spectra. These results, together with experiments using boiled plastids, indicate that phytochromobilin synthesis from biliverdin is enzyme-mediated. Experiments where NADPH (and ATP) levels in intact developing chloroplasts are manipulated by feeding the metabolites 3-phosphoglycerate, dihydroxyacetone phosphate, and glucose 6-phosphate or by illumination with white light, support the hypothesis that the enzyme that accomplishes this conversion, phytochromobilin synthase, is plastid-localized. It is therefore likely that all of the enzymes of the phytochrome chromophore biosynthetic pathway reside in the plastid.     

Biosynthesis of phycobilins. 15,16-Dihydrobiliverdin IX alpha is a partially reduced intermediate in the formation of phycobilins from biliverdin IX alpha

A partially purified protein fraction from the phycocyanin-containing unicellular rhodophyte, Cyanidium caldarium, reductively transforms biliverdin IX alpha to a violet colored bilin in the presence of NADPH, ferredoxin, and ferredoxin-NADP+ reductase. This bilin has a violin-like absorption spectrum with maxima at 335 and 560 nm in methanolic HCl and at 337, 567, and 603-604 nm in CHCl3. The bilin has been determined to be 15,16-dihydrobiliverdin IX alpha by comparative spectrophotometry and 1H NMR spectroscopy. This product of biliverdin IX alpha reduction is converted enzymatically to phycobilins by further reduction. A general biosynthetic pathway is proposed which accounts for the formation of the phycobilins from biliverdin IX alpha by a two-step reduction process followed by isomerization.     

Bile pigment synthesis in plants. Incorporation of haem into phycocyanobilin and phycobiliproteins in Cyanidium caldarium.

A procedure was developed whereby haem was taken up by dark-grown cells of the unicellular rhodophyte Cyanidium caldarium. These cells were subsequently incubated either in the dark with 5-aminolaevulinate, which results in excretion of phycocyanobilin into the suspending medium or incubated in the light, which results in synthesis and accumulation of phycocyanin and chlorophyll a within the cells. Phycocyanobilin was isolated from phycocyanin by cleavage from apoprotein in methanol. Phycocyanobilin prepared from phycocyanin or excreted from cells given 5-aminolaevulinate was methylated and purified by t.l.c. By using 14C labelling either in the haem or in 5-aminolaevulinate administered, haem incorporation into phycocyanobilin was demonstrated in both dark and light systems. Since chlorophyll a synthesized in the light in the presence of labelled haem contained no radioactivity, it was clear that haem was directly incorporated into phycocyanobilin and not first converted into protoporphyrin IX. These results clearly demonstrate phycocyanobilin synthesis via haem and not via magnesium protoporphyrin IX as has also been postulated.     

Phytochrome chromophore biosynthesis. Treatment of tetrapyrrole-deficient Avena explants with natural and non-natural bilatrienes leads to formation of spectrally active holoproteins

Etiolated Avena seedlings grown in the presence of 4-amino-5-hexynoic acid, an inhibitor of 5-aminolevulinic acid synthesis in plants, contain less than 10% of the spectrally detectable levels of phytochrome found in untreated seedlings (Elich, T.D., and Lagarias, J.C. (1988) Plant Physiol. 88, 747-751). In this study, incubation of explants from such seedlings with [14C]biliverdin IX alpha led to rapid covalent incorporation of radiolabel into a single 124-kDa polypeptide in soluble protein extracts. Immunoprecipitation experiments confirmed that this protein was phytochrome. Parallel experiments were performed with four unlabeled linear tetrapyrroles, the naturally occurring biliverdin IX alpha isomer, two non-natural isomers, biliverdin XIII alpha and biliverdin III alpha, and phycocyanobilin-the cleaved prosthetic group of the light-harvesting antenna protein C-phycocyanin. In all cases, except for the III alpha isomer of biliverdin, a time-dependent recovery of photoreversible phytochrome was observed. The newly formed phytochrome obtained after incubation with biliverdin IX alpha exhibited spectral characteristics identical with those of the native protein. In contrast, the spectral properties of phytochromes formed during incubation with biliverdin XIII alpha and phycocyanobilin differed significantly from those of the native chromoprotein. These results indicate that biliverdin IX alpha is an intermediate in the biosynthesis of the phytochrome chromophore and that phytochromes with prosthetic groups derived from bilatrienes having non-natural D-ring substituents are photochromic.     

Phytochrome assembly. The structure and biological activity of 2(R),3(E)-phytochromobilin derived from phycobiliproteins.

The unicellular rhodophyte, Porphyridium cruentum, and the filamentous cyanobacterium, Calothrix sp. PCC 7601, contain phycobiliproteins that have covalently bound phycobilin chromophores. Overnight incubation of solvent-extracted cells at 40 degrees C with methanol liberates free phycobilins that are derived from the protein-bound bilins by methanolytic cleavage of the thioether linkages between bilin and apoprotein. Two of the free bilins were identified as 3(E)-phycocyanobilin and 3(E)-phycoerythrombilin by comparative spectrophotometry and high pressure liquid chromatography. Methanolysis also yields a third bilin free acid whose absorption and 1H NMR spectra support the assignment of the 3(E)-phytochromobilin structure. This novel bilin is the major pigment isolated from cells that are pre-extracted with acetone-containing solvents. Since phytochrome- or phytochromobilin-containing proteins are not present in either organism, the 3(E)-phytochromobilin must arise by oxidation of phycobilin chromophores. This pigment is not obtained by similar treatment of a cyanobacterium and a rhodophyte that lack phycoerythrin. Therefore, 3(E)-phytochromobilin appears to be derived from phycoerythrobilin-containing proteins. Comparative CD spectroscopy of 3(E)-phytochrombilin and 3(E)-phycocyanobilin suggests that the two bilins share the R stereochemistry at the 2-position in the reduced pyrrole ring. Incubation of 2(R),3(E)-phytochromobilin with recombinant oat apophytochrome yields a covalent bilin adduct that is photoactive and spectrally indistinguishable from native oat phytochrome isolated from etiolated seedlings. These results establish that the phycobiliprotein-derived 2(R),3(E)-phytochromobilin is a biologically active phytochrome chromophore precursor.     

Biliprotein light-harvesting strategies, phycoerythrin 566

A series of experiments on the light-harvesting properties of the cryptomonad biliprotein phycoerythrin 566 has been carried out on purified protein isolated from Cryptomonas ovata. Although this pigment has an absorption maximum at 566 nm, a property very close to that of other phycoerythrins, it was found to have a totally unique set of chromophores. The chromophores (bilins) responsible for its absorption spectrum were analyzed by a number of approaches. Chromophore-containing peptides were produced by trypsin treatment and purified in order to isolate the individual peptide-bound bilins free of overlapping absorption. These chromopeptides, after comparison with appropriate controls, showed that three spectrally distinct bilins occurred on the purified oligomeric protein. Two of the bilins were the well-known phycoerythrobilin and cryptoviolin, but the third was previously undiscovered and had an absorption spectrum between that of cryptoviolin and phycocyanobilin. Since the spectral diversity of the three bilins was fully maintained in solvents that minimize the effects of apoprotein on the spectra of the bilins, it is likely that the three bilins are also structurally dissimilar. The alpha and beta subunits, which constitute the protein, were separated by ion-exchange chromatography, and the new bilin was found to be the sole chromophore on the alpha subunit. It was also found that at least two alpha subunits could be separated and they both had this unusual bilin (cryptobilin 596). The beta subunit, therefore, contained both phycoerythrobilin and cryptoviolin. On the basis of the spectra of the three chromopeptides, the absorption spectrum of the protein was modeled using the known absorptivities of cryptoviolin and phycoerythrobilin.     

The Phytochrome-Deficient pcd1 Mutant of Pea Is Unable to Convert Heme to Biliverdin IX[alpha

We isolated a new pea mutant that was selected on the basis of pale color and elongated internodes in a screen under white light. The mutant was designated pcd1 for phytochrome chromophore deficient. Light-grown pcd1 plants have yellow-green foliage with a reduced chlorophyll (Chl) content and an abnormally high Chl a/Chl b ratio. Etiolated pcd1 seedlings are developmentally insensitive to far-red light, show a reduced response to red light, and have no spectrophotometrically detectable phytochrome. The phytochrome A apoprotein is present at the wild-type level in etiolated pcd1 seedlings but is not depleted by red light treatment. Crude phytochrome preparations from etiolated pcd1 tissue also lack spectral activity but can be assembled with phycocyanobilin, an analog of the endogenous phytochrome chromophore phytochromobilin, to yield a difference spectrum characteristic of an apophytochrome-phycocyanobilin adduct. These results indicate that the pcd1-conferred phenotype results from a deficiency in phytochrome chromophore synthesis. Furthermore, etioplast preparations from pcd1 seedlings can metabolize biliverdin (BV) IX[alpha] but not heme to phytochromobilin, indicating that pcd1 plants are severely impaired in their ability to convert heme to BV IX[alpha]. This provides clear evidence that the conversion of heme to BV IX[alpha] is an enzymatic process in higher plants and that it is required for synthesis of the phytochrome chromophore and hence for normal photomorphogenesis.