Phycoerythrobilin synthase (PebS) of a marine virus. Crystal structures of the biliverdin complex and the substrate-free form

The reddish purple open chain tetrapyrrole pigment phycoerythrobilin (PEB; A(lambdamax) approximately 550 nm) is an essential chromophore of the light-harvesting phycobiliproteins of most cyanobacteria, red algae, and cryptomonads. The enzyme phycoerythrobilin synthase (PebS), recently discovered in a marine virus infecting oceanic cyanobacteria of the genus Prochlorococcus (cyanophage PSSM-2), is a new member of the ferredoxin-dependent bilin reductase (FDBR) family. In a formal four-electron reduction, the substrate biliverdin IXalpha is reduced to yield 3Z-PEB, a reaction that commonly requires the action of two individual FDBRs. The first reaction catalyzed by PebS is the reduction of the 15,16-methine bridge of the biliverdin IXalpha tetrapyrrole system. This reaction is exclusive to PEB biosynthetic enzymes. The second reduction site is the A-ring 2,3,3(1),3(2)-diene system, the most common target of FDBRs. Here, we present the first crystal structures of a PEB biosynthetic enzyme. Structures of the substrate complex were solved at 1.8- and 2.1-A resolution and of the substrate-free form at 1.55-A resolution. The overall folding revealed an alpha/beta/alpha-sandwich with similarity to the structure of phycocyanobilin:ferredoxin oxidoreductase (PcyA). The substrate-binding site is located between the central beta-sheet and C-terminal alpha-helices. Eight refined molecules with bound substrate, from two different crystal forms, revealed a high flexibility of the substrate-binding pocket. The substrate was found to be either in a planar porphyrin-like conformation or in a helical conformation and is coordinated by a conserved aspartate/asparagine pair from the beta-sheet side. From the alpha-helix side, a conserved highly flexible aspartate/proline pair is involved in substrate binding and presumably catalysis.     

Phycocyanobilin:ferredoxin oxidoreductase of Anabaena sp. PCC 7120. Biochemical and spectroscopic

In cyanobacteria, the biosynthesis of the phycobiliprotein and phytochrome chromophore precursor phycocyanobilin is catalyzed by the ferredoxin-dependent enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA), which mediates an atypical four-electron reduction of biliverdin IXalpha. Here we describe the expression, affinity purification, and biochemical characterization of recombinant PcyA from Anabaena sp. PCC 7120. A monomeric protein with a native M(r) of 30,400 +/- 5,000, recombinant PcyA forms a tight and stable stoichiometric complex with its substrate biliverdin IXalpha. The enzyme exhibits a strong preference for plant type [2Fe-2S] ferredoxins; however, flavodoxin can also serve as an electron donor. HPLC analyses establish that catalysis proceeds via the two electron-reduced intermediate 18(1),18(2)-dihydrobiliverdin, indicating that exovinyl reduction precedes A-ring (endovinyl) reduction. Substrate specificity studies indicate that the arrangement of the A- and D-ring substituents alters the positioning of the bilin substrate within the enzyme, profoundly influencing the course of catalysis. Based on these observations and the apparent lack of a metal or small molecule cofactor, a radical mechanism for biliverdin IXalpha reduction by phycocyanobilin:ferredoxin oxidoreductase is envisaged.     

A conserved histidine-aspartate pair is required for exovinyl reduction of biliverdin by a cyanobacterial phycocyanobilin:ferredoxin oxidoreductase

Phycocyanobilin:ferredoxin oxidoreductase is a member of the ferredoxin-dependent bilin reductase family and catalyzes two vinyl reductions of biliverdin IXalpha to produce phycocyanobilin, the pigment precursor of both phytochrome and phycobiliprotein chromophores in cyanobacteria. Atypically for ferredoxin-dependent enzymes, phycocyanobilin:ferredoxin oxidoreductase mediates direct electron transfers from reduced ferredoxin to its tetrapyrrole substrate without metal ion or organic cofactors. We previously showed that bound bilin radical intermediates could be detected by low temperature electron paramagnetic resonance and absorption spectroscopies (Tu, S., Gunn, A., Toney, M. D., Britt, R. D., and Lagarias, J. C. (2004) J. Am. Chem. Soc. 126, 8682-8693). On the basis of these studies, a mechanism involving sequential electron-coupled proton transfers to protonated bilin substrates buried within the phycocyanobilin:ferredoxin oxidoreductase protein scaffold was proposed. The present investigation was undertaken to identify catalytic residues in phycocyanobilin:ferredoxin oxidoreductase from the cyanobacterium Nostoc sp. PCC7120 through site-specific chemical modification and mutagenesis of candidate proton-donating residues. These studies identified conserved histidine and aspartate residues essential for the catalytic activity of phycocyanobilin:ferredoxin oxidoreductase. Spectroscopic evidence for the formation of stable enzyme-bound biliverdin radicals for the H85Q and D102N mutants support their role as a "coupled" proton-donating pair during the reduction of the biliverdin exovinyl group.     

Radical mechanism of cyanophage phycoerythrobilin synthase (PebS)

PEB (phycoerythrobilin) is a pink-coloured open-chain tetrapyrrole molecule found in the cyanobacterial light-harvesting phycobilisome. Within the phycobilisome, PEB is covalently bound via thioether bonds to conserved cysteine residues of the phycobiliprotein subunits. In cyanobacteria, biosynthesis of PEB proceeds via two subsequent two-electron reductions catalysed by the FDBRs (ferredoxin-dependent bilin reductases) PebA and PebB starting from the open-chain tetrapyrrole biliverdin IXα. A new member of the FDBR family has been identified in the genome of a marine cyanophage. In contrast with the cyanobacterial enzymes, PebS (PEB synthase) from cyanophages combines both two-electron reductions for PEB synthesis. In the present study we show that PebS acts via a substrate radical mechanism and that two conserved aspartate residues at position 105 and 206 are critical for stereospecific substrate protonation and conversion. On the basis of the crystal structures of both PebS mutants and presented biochemical and biophysical data, a mechanism for biliverdin IXα conversion to PEB is postulated and discussed with respect to other FDBR family members.     

Function and distribution of bilin biosynthesis enzymes in photosynthetic organisms

Bilins are open-chain tetrapyrrole molecules essential for light-harvesting and/or sensing in many photosynthetic organisms. While they serve as chromophores in phytochrome-mediated light-sensing in plants, they additionally function in light-harvesting in cyanobacteria, red algae and cryptomonads. Associated to phycobiliproteins a variety of bile pigments is responsible for the specific light-absorbance properties of the organisms enabling efficient photosynthesis under different light conditions. The initial step of bilin biosynthesis is the cleavage of heme by heme oxygenases (HO) to afford the first linear molecule biliverdin. This reaction is ubiquitously found also in non-photosynthetic organisms. Biliverdin is then further reduced by site specific reductases most of them belonging to the interesting family of ferredoxin-dependent bilin reductases (FDBRs)-a new family of radical oxidoreductases. In recent years much progress has been made in the field of heme oxygenases but even more in the widespread family of FDBRs, revealing novel biochemical FDBR activities, new crystal structures and new ecological aspects, including the discovery of bilin biosynthesis genes in wild marine phage populations. The aim of this review is to summarize and discuss the recent progress in this field and to highlight the new and remaining questions.     

Efficient phage-mediated pigment biosynthesis in oceanic cyanobacteria

Although the oceanic cyanobacterium Prochlorococcus harvests light with a chlorophyll antenna [1-3] rather than with the phycobilisomes that are typical of cyanobacteria, some strains express genes that are remnants of the ancestral Synechococcus phycobilisomes [4]. Similarly, some Prochlorococcus cyanophages, which often harbor photosynthesis-related genes [5], also carry homologs of phycobilisome pigment biosynthesis genes [6, 7]. Here, we investigate four such genes in two cyanophages that both infect abundant Prochlorococcus strains [8]: homologs of heme oxygenase (ho1), 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (pebA), ferredoxin (petF) in the myovirus P-SSM2, and a phycocyanobilin:ferredoxin oxidoreductase (pcyA) homolog in the myovirus P-SSM4. We demonstrate that the phage homologs mimic the respective host activities, with the exception of the divergent phage PebA homolog. In this case, the phage PebA single-handedly catalyzes a reaction for which uninfected host cells require two consecutive enzymes, PebA and PebB. We thus renamed the phage enzyme phycoerythrobilin synthase (PebS). This gene, and other pigment biosynthesis genes encoded by P-SSM2 (petF and ho1), are transcribed during infection, suggesting that they can improve phage fitness. Analyses of global ocean metagenomes show that PcyA and Ho1 occur in both cyanobacteria and their phages, whereas the novel PebS-encoding gene is exclusive to phages.     

Phycobilins of cryptophycean algae. Occurrence of dihydrobiliverdin and mesobiliverdin in cryptomonad biliproteins.

Structures of the open-chain tetrapyrrole (bilin) prosthetic groups of the cryptophycean biliproteins phycocyanin 645 (Cr-PC 645; from strain UW374), phycoerythrin 566 (Cr-PE 566; from strain Bermani) and phycoerythrin 545 (Cr-PE 545; from Proteomonas sulcata Hill & Wetherbee) were examined by absorption, 1H NMR spectroscopy, and mass spectrometry. These biliproteins carry the following covalently attached bilins: Cr-PC 645 (alpha subunit) has one mesobiliverdin, (beta subunit), two phycocyanobilins and a doubly linked 15,16-dihydrobiliverdin; Cr-PC 566 (alpha), bilin 584, (beta), phycoerythrobilin and two bilin 584 chromophores (Wedemayer, G.J., Wemmer, D.E., and Glazer, A.N. (1991) J. Biol. Chem. 266, 4731-4741); Cr-PE 545 (alpha) has one 15,16-dihydrobiliverdin and (beta), only phycoerythrobilins. This is the first report of naturally occurring biliproteins carrying either 15,16-dihydrobiliverdin or mesobiliverdin chromophores. Native cryptomonad phycobiliproteins have been classified on the basis of the position of their long wavelength absorption maxima. However, comparison of the bilins of Cr-PE 566 from strain Bermani with those of Cr-PE 566 of strain CBD shows that the two proteins carry different bilins on the alpha subunit. Consequently, the identity of the bilin prosthetic groups on cryptophycean phycobiliproteins cannot be unambiguously inferred from simple inspection of the visible absorption spectra.     

Recombinant holophytochrome in Escherichia coli.

We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXalpha which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies.     

Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins

Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes, and cryptophytes for light-harvesting and consist of apoproteins covalently associated with open-chain tetrapyrrole chromophores. Although the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single type of phycobiliprotein that is localized in the thylakoid lumen. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. In this study, we identified and characterized enzymes involved in PEB biosynthesis. In addition, we present the first in-depth biochemical characterization of a eukaryotic phycobiliprotein lyase (GtCPES). Plastid-encoded HO (GtHo) was shown to convert heme into biliverdin IXα providing the substrate with a putative nucleus-encoded DHBV:ferredoxin oxidoreductase (GtPEBA). A PEB:ferredoxin oxidoreductase (GtPEBB) was found to convert DHBV to PEB, which is the substrate for the phycobiliprotein lyase GtCPES. The x-ray structure of GtCPES was solved at 2.0 Å revealing a 10-stranded β-barrel with a modified lipocalin fold. GtCPES is an S-type lyase specific for binding of phycobilins with reduced C15=C16 double bonds (DHBV and PEB). Site-directed mutagenesis identified residues Glu-136 and Arg-146 involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apophycobiliprotein CpeB is proposed and discussed.     

Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms

Phytobilins are linear tetrapyrrole precursors of the light-harvesting prosthetic groups of the phytochrome photoreceptors of plants and the phycobiliprotein photosynthetic antennae of cyanobacteria, red algae, and cryptomonads. Previous biochemical studies have established that phytobilins are synthesized from heme via the intermediacy of biliverdin IX alpha (BV), which is reduced subsequently by ferredoxin-dependent bilin reductases with different double-bond specificities. By exploiting the sequence of phytochromobilin synthase (HY2) of Arabidopsis, an enzyme that catalyzes the ferredoxin-dependent conversion of BV to the phytochrome chromophore precursor phytochromobilin, genes encoding putative bilin reductases were identified in the genomes of various cyanobacteria, oxyphotobacteria, and plants. Phylogenetic analyses resolved four classes of HY2-related genes, one of which encodes red chlorophyll catabolite reductases, which are bilin reductases involved in chlorophyll catabolism in plants. To test the catalytic activities of these putative enzymes, representative HY2-related genes from each class were amplified by the polymerase chain reaction and expressed in Escherichia coli. Using a coupled apophytochrome assembly assay and HPLC analysis, we examined the ability of the recombinant proteins to catalyze the ferredoxin-dependent reduction of BV to phytobilins. These investigations defined three new classes of bilin reductases with distinct substrate/product specificities that are involved in the biosynthesis of the phycobiliprotein chromophore precursors phycoerythrobilin and phycocyanobilin. Implications of these results are discussed with regard to the pathways of phytobilin biosynthesis and their evolution.     

Structural and mechanistic insight into the ferredoxin-mediated two-electron reduction of bilins

PEB (phycoerythrobilin) is one of the major open-chain tetrapyrrole molecules found in cyanobacterial light-harvesting phycobiliproteins. In these organisms, two enzymes of the ferredoxin-dependent bilin reductase family work in tandem to reduce BV (biliverdin IXα) to PEB. In contrast, a single cyanophage-encoded enzyme of the same family has been identified to catalyse the identical reaction. Using UV-visible and EPR spectroscopy we investigated the two individual cyanobacterial enzymes PebA [15,16-DHBV (dihydrobiliverdin):ferredoxin oxidoreductase] and PebB (PEB:ferredoxin oxidoreductase) showing that the two subsequent reactions catalysed by the phage enzyme PebS (PEB synthase) are clearly dissected in the cyanobacterial versions. Although a highly conserved aspartate residue is critical for both reductions, a second conserved aspartate residue is only involved in the A-ring reduction of the tetrapyrrole in PebB and PebS. The crystal structure of PebA from Synechococcus sp. WH8020 in complex with its substrate BV at a 1.55 ? (1 ?=0.1 nm) resolution revealed further insight into the understanding of enzyme evolution and function. Based on the structure it becomes obvious that in addition to the importance of certain catalytic residues, the shape of the active site and consequently the binding of the substrate highly determines the catalytic properties.     

Structural Insights into Vinyl Reduction Regiospecificity of Phycocyanobilin:Ferredoxin Oxidoreductase (PcyA)

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase family. Unlike other ferredoxin-dependent bilin reductases that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXα (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria, and cryptophytes. To address the structural basis for the reduction regiospecificity of PcyA, we report new high resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl reduction activity. These include the BV complex of the E76Q mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18¹,18²-dihydrobiliverdin IXα (18EtBV) and with biliverdin XIIIα (BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA.     

Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli

By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors.     

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.     

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.     

CpeS Is a Lyase Specific for Attachment of 3Z-PEB to Cys82 of β-phycoerythrin from Prochlorococcus marinus MED4

In contrast to the majority of cyanobacteria, the unicellular marine cyanobacterium Prochlorococcus marinus MED4 uses an intrinsic divinyl-chlorophyll-dependent light-harvesting system for photosynthesis. Despite the absence of phycobilisomes, this high-light adapted strain possesses β-phycoerythrin (CpeB), an S-type lyase (CpeS), and enzymes for the biosynthesis of phycoerythrobilin (PEB) and phycocyanobilin. Of all linear tetrapyrroles synthesized by Prochlorococcus including their 3Z- and 3E-isomers, CpeS binds both isomers of PEB and its biosynthetic precursor 15,16-dihydrobiliverdin (DHBV). However, dimerization of CpeS is independent of bilins, which are tightly bound in a complex at a ratio of 1:1. Although bilin binding by CpeS is fast, transfer to CpeB is rather slow. CpeS is able to attach 3E-PEB and 3Z-PEB to dimeric CpeB but not DHBV. CpeS transfer of 3Z-PEB exclusively yields correctly bound βCys⁸²-PEB, whereas βCys⁸²-DHBV is a side product of 3E-PEB transfer. Spontaneous 3E- and 3Z-PEB addition to CpeB is faulty, and products are in both cases βCys⁸²-DHBV and likely a PEB bound at βCys⁸² in a non-native configuration. Our data indicate that CpeS is specific for 3Z-PEB transfer to βCys⁸² of phycoerythrin and essential for the correct configuration of the attachment product.     

Induced-fitting and electrostatic potential change of PcyA upon substrate binding demonstrated by the crystal structure of the substrate-free form

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the sequential reduction of the vinyl group of the D-ring and the A-ring of biliverdin IXalpha (BV) using ferredoxin to produce phycocyanobilin, a pigment used for light-harvesting and light-sensing in red algae and cyanobacteria. We have determined the crystal structure of the substrate-free form of PcyA from Synechocystis sp. PCC 6803 at 2.5 A resolution. Structural comparison of the substrate-free form and the PcyA-BV complex shows major changes around the entrance of the BV binding pocket; upon BV binding, two alpha-helices and nearby side-chains move to produce tight BV binding. Unexpectedly, these movements localize the positive charges around the BV binding site, which may contribute to the proper binding of ferredoxin to PcyA. In the substrate-free form, the side-chain of Asp105 was located at a site that would be underneath the BV A-ring in the PcyA-BV complex and hydrogen-bonded with His88. We propose that BV is protonated by a mechanism involving conformational changes of these two residues before reduction.     

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.     

Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis

Phytochromobilin (PPhiB) is an open chain tetrapyrrole molecule that functions as the chromophore of light-sensing phytochromes in plants. Derived from heme, PPhiB is synthesized through an open chain tetrapyrrole intermediate, biliverdin IXalpha (BV), in the biosynthesis pathway. BV is subsequently reduced by the PPhiB synthase HY2 in plants. HY2 is a ferredoxin-dependent bilin reductase that catalyzes the reduction of the A-ring 2,3,3(1),3(2)-diene system to produce an ethylidene group for assembly with apophytochromes. In this study, we sought to determine the catalytic mechanism of HY2. Data from UV-visible and EPR spectroscopy showed that the HY2-catalyzed BV reaction proceeds via a transient radical intermediate. Site-directed mutagenesis showed several ionizable residues that are involved in the catalytic steps. Detailed analysis of these site-directed mutants highlighted a pair of aspartate residues central to proton donation and substrate positioning. A mechanistic prediction for the HY2 reaction is proposed. These results support the hypothesis that ferredoxin-dependent bilin reductases reduce BV through a radical mechanism, but their double bond specificity is decided by strategic placement of different proton-donating residues surrounding the bilin substrate in the active sites.     

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.