PHYCOCYANOBILIN:CYSTEINE- 84 PHYCOBILIPROTEIN LYASE ACTIVITY OF

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 13, pp , March 31, by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Chromophore Attachment to Phycobiliprotein -Subunits PHYCOCYANOBILIN:CYSTEINE- 84 PHYCOBILIPROTEIN LYASE ACTIVITY OF CpeS-LIKE PROTEIN FROM ANABAENA Sp. PCC7120 * S Received for publication, December 27, 2005, and in revised form, January 30, 2006 Published, JBC Papers in Press, February 1, 2006, DOI /jbc.M Kai-Hong Zhao 1, Ping Su, Jian Li, Jun-Ming Tu, Ming Zhou 2, Claudia Bubenzer, and Hugo Scheer 1,3 From the Colleges of Life Science and Technology and of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan , Hubei, P.R. China and the Department Biologie I, UniversitätMünchen, Menzinger Strasse 67, D München, Germany The gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpes from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcs from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys- 84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine- 84 of the apo- -subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mm NaCl and 500 mm potassium phosphate, 37 C, and ph 7.5), no cofactors are required, and the lyase had a K m (PCB) 2.7 and 2.3 M, and a k cat and s 1 for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpes was introduced into E. coli with cpcb(c155i) or pecb(c155i), respectively, together with genes ho1 (encoding heme oxygenase) and pcya (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, are supramolecular complexes of phycobiliproteins and linkers (1 6). The phycobilin chromophores of the different phycobiliproteins span an absorption range from 460 to 670 nm and transfer excitation energy with high quantum efficiency to the photosynthetic reaction centers. The last step in phycobiliprotein biosynthesis is the covalent chromophore attachment to the apoprotein. Phycobilin chromophores are generally bound to the polypeptide, via thioether bonds, to conserved cysteine residues: phycocyanobilin (PCB) 4 and phycoviolobilin (PVB) * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S The on-line version of this article (available at contains supplemental Table S1 and supplemental Figs. S1 S3. are singly bound to C-3 at ring A of the tetrapyrroles, whereas phycoerythrobilin (PEB) and phycourobilin often have an additional bond to C-18 at ring D (7 10). Autocatalytic chromophore binding has been demonstrated for the core-membrane linker of the phycobilisome, ApcE (11), and for another group of biliproteins, the phytochromes (12 15). In contrast, chromophore binding to other phycobiliproteins is more complex. Spontaneous addition of PCB has been observed to all binding sites (Cys- 84, Cys- 84, and Cys- 155) of CPC, but it is slow and generally leads to a mixture of products (16, 17). Lyases, specific for attaching PCB to Cys- 84 of CpcA, and PecA, have been identified in several cyanobacteria (9). The PCB: -CPC lyase reversibly binds PCB to cysteine-84 of the -subunit of cyanobacterial phycocyanin (CpcA); it is composed of two subunits, CpcE and CpcF, which, in several species, are coded by genes on the respective biliprotein operon (18, 19). Similar lyases probably catalyze the attachment of PEB to cysteine- 84 (20, 21). The phylogenetically related PVB: -PEC lyases (EC ), PecE/F (22, 24) 5 catalyze not only the addition of PCB to cysteine-84 of the -subunit of phycoerythrocyanin, PecA, but also an isomerization that generates the photoactive PVB chromophore that is characteristic for -PEC (25, 26 28). A similar reaction sequence would lead from PEB to the protein-bound phycourobilin, which is present in many marine cyanobacteria and red algae; a first example for such an enzyme may be the RpeE/F fusion protein from Synechococcus sp. WH8102 (29). Two chromophores, PCB and PEB, are biosynthesized from heme by ring opening at C-5 of the tetrapyrrole, followed by reduction and isomerization steps (30, 31). Heterologous in vivo synthesis of PCB, e.g. in Escherichia coli, is possible by the expression of two genes encoding heme oxygenase 1 (ho1) and PCB:ferredoxin oxidoreductase (pcya). When these were co-expressed in E. coli, together with the apophytochrome gene, cph1, by a dual plasmid system, photoreversibly active holophytochrome was produced (32, 33). Similarly, the entire pathway for the synthesis of -CPC or -PEC could be reconstituted in E. coli, when ho1 and pcya were co-expressed with cpca, cpce, and cpcf or with peca, pece, and pecf, respectively (34, 35). Recently, a group of four genes (cpcs, cpct, cpcu, and cpcv) has been identified in Synechococcus sp. PCC7002 that code for lyases that attach PCB to cysteine-84 (consensus numbering) of the -subunits of CPC and possibly allophycocyanin; homologous genes are also found in other cyanobacteria (36). Open reading frame alr0617 in Anabaena sp. PCC7120 (22) is homologous to this new type of lyase, in particular, to cpes of Fremyella diplosiphon (Calothrix PCC7601) (37), Gloeobacter violaceus PCC 7421 (38), and Synechococcus sp. WH8102 (39). Using 1 Supported by a Volkswagen Stiftung Partnership (I/77900). 2 Supported by the National Natural Science Foundation of China (Grant ). To whom correspondence may be addressed. Tel./Fax: ; khzhao@163.com. 3 Supported by the Deutsche Forschungsgemeinschaft (Grant SFB 533, TPA1). To whom correspondence may be addressed. Tel.: ; Fax: ; hugo.scheer@lmu.de. 4 The abbreviations used are: PCB, phycocyanobilin; Cpes, PCB:Cys- 84 phycobiliprotein lyase for cysteine- 84 of phycobiliproteins; CPC, cyanobacterial phycocyanin; CpcA, apoprotein of -CPC; CpcB, apoprotein of -CPC; CpcE and CpcF, subunits of PCB: - CPC lyase; HPLC, high performance liquid chromatography; KPP, potassium phosphate buffer; ME, mercaptoethanol; IPEB, phycoerythrobilin; PEC, phycoerythrocyanin; PecA, apoprotein of -PEC; PecB, apoprotein of -PEC; PecE and PecF, subunits of PVB: -PEC isomerase-lyase; PVB, phycoviolobilin; TX100, Triton X-100; CPE, C-phycoerythrin. 5 W. Kufer, A. Högner, M. Eberlein, K. Mayer, A. Buchner, and L. Gottschalk (1991) Gen- Bank TM accession number M MARCH 31, 2006 VOLUME 281 NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8573

2 biochemical, enzymatic, and molecular biological methods, we now show that alr0617 codes for a PCB:phycobiliprotein lyase that catalyzes the covalent attachment of PCB to cysteine-84 of the -subunits of CPC (CpcB) and PEC (PecB). In analogy to cpes from Calothrix PCC7601 (37), we therefore annotate alr0617 as cpes. 6 The enzymatic function of CpeS from Anabaena sp. PCC7120 was further demonstrated by coexpression in E. coli of dual plasmids containing cpcb(c155i) or pecb(c155i), with cpes (i.e. alr0617), ho1 and pcya, resulting in biosynthesis of the respective -subunits singly chromophorylated at cysteine- 84. MATERIALS AND METHODS Proteins Cloning and expression generally followed the standard procedures of Sambrook et al. (40). The integral genes, cpce and cpcf, were PCR-amplified as described previously from Anabaena sp., peca, pece, and pecf from Anabaena sp. PCC 7120 and Mastigocladus laminosus (Fischerella PCC7603) (15, 25). Mutants cpcb(c155i), cpcb(c84s), pecb(c155i), and pecb(c84s) were generated from cpcb and pecb of M. laminosus (41). The plasmids containing cpes ( alr0617), cpcb, pecb, and nblb (coding for a degrading lyase (42) from Anabaena sp. PCC 7120) were constructed in this work using the primers P1 P8 shown below. They were cloned first into pbluescript (Stratagene), and then subcloned into pet30a (Novagen). CpeS, without an His tag, was obtained by expressing pgemex (Promega) containing alr0617 (26). For the construction of dual plasmids, the following genes from Anabaena sp. PCC 7120 were PCR-amplified via primers P8 P13: cpes, ho1 ( all1897, annotated according to its high homology with ho1 from Synechococcus PCC 6803 (43) (encoding heme oxygenase 1) and pcya ( alr3707), annotated according to its homology with pcya from Synechococcus PCC 6803 (43) (encoding PCB:ferredoxin oxidoreductase). The PCR-amplified ho1 and pcya were cloned together in pacy- CDuet-1 (Novagen) to produce pho-pcya, and alr0617 was cloned in pcdfduet-1 (Novagen) to produce pcdf-cpes. All molecular constructions were verified by sequencing as follows. P1: 5 -GGACCCGG- GATGACATTAGACGTATTTAC-3, upstream; P2: 5 -ATTCTCG- AGTTAACCTACAGCAGCAGCAG-3, downstream; P3: 5 -ATA- CCCGGGATGCTCGATGCTTTTTC-3, upstream; P4: 5 -GTGCT- CGAGTTAAACAACCGCAGAAGC-3, downstream; P5: 5 -GTAC- CCGGGATGAGTATTACACCTGAG-3, upstream; P6: 5 -CGC- CTCGAGCTAAACTGATTGTAAAGACT-3, downstream; P7: 5 -GCGCCCGGGATGAATATCGAAGAGTTTTTT-3, upstream; P8: 5 -GGGCTCGAGGTTTTAACTTGACGCAGAATT-3, downstream; P9: 5 -GGCGATATCCGGGATGAATATTGAAGAGTTT- 3, upstream; P10: 5 -CTACCATGGCGATGAGCAGCAATTTA- GCA-3, upstream; P11: 5 -ATCCTGCAGTTACTCAGCCGTG- GCAAGTT-3, downstream; P12: 5 -CGCGATATCGATGTCACT- TACTTCCATTC-3, upstream; and P13: 5 -TACCTCGAGCCGT- TATTCTGGGAGATC-3, downstream. For P1 P8, all upstream primers have a SmaI site (CCCGGG) and the downstream primers have a XhoI site (CTCGAG) (both marked in bold), which ensure correct ligation of the fragments to pbluescript and then to pet30. P1 and P2 were used for cpcb, P3 and P4 for pecb,p5and P6 for nblb, and P7 and P8 for alr0617. For P8 P13, the upstream primers for pcya and alr0617 have an EcoRV site (GATATC), and the downstream primers have an XhoI site (CTCGAG); the upstream primer for ho1 has an NcoI site (CCATTG), and the downstream 6 Anabaena sp. PCC7120 does not produce CPE. The name cpes was nonetheless retained for alr0617, at least for the time being, in view of the comparably broad substrate specificity of the lyase. primer has a PstI site (CTGCAG). P8 and P9 were used for alr0617, P10 and P11 for ho1, and P12 and P13 for pcya. Expressions The pet-based plasmids were used to transform E. coli BL21(DE3). Cells were grown in Luria-Bertani (LB) medium containing kanamycin (30 g ml 1 ) at 37 C for genes from M. laminosus or at 20 C for genes from Anabaena sp. PCC When the cell density reached A , isopropyl 1-thio- -D-galactopyranoside (1 mm) was added. Cells were collected by centrifugation 5 h after induction (for genes from M. laminosus) or after 6 h (for genes from Anabaena sp.), then washed twice with doubly distilled water and stored at 20 C until use (15, 27). The dual plasmids were transformed together into BL21(DE3) cells under the respective antibiotic selections (chloromycetin for pho- PcyA and streptomycin for pcdf-cpes). One of the four plasmids pet- CpcB(C155I), pet-cpcb, pet-pecb(c155i), or pet-pecb, was used together with pcdf-cpes and pho-pcya to produce PCB-CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), or PCB-PecB, respectively. In the control experiment, pcdf-cpes was omitted from the transformations. Cells were grown at 20 C in LB-medium containing kanamycin (20 g ml 1 ), chloromycetin (17 g ml 1 ), and streptomycin (25 g ml 1 ). When the cell density reached A , isopropyl 1-thio- -D-galactopyranoside (1 mm) was added. Cells were collected by centrifugation 12 h after induction, washed twice with doubly distilled water, and stored at 20 C until use. Cell pellets were resuspended in ice-cold potassium phosphate buffer (KPP, 20 mm, ph 7.2) containing NaCl (0.5 M) and disrupted by sonication (5 min, 45 W, Branson model 450W). The suspension was centrifuged at 12,000 g for 15 min at 4 C. The supernatant containing the crude proteins was purified via Ni 2 -affinity chromatography on chelating Sepharose (Amersham Biosciences). The initial buffer was KPP (20 mm, ph 7.2) containing NaCl (1 M), the elution buffer also contained imidazole (0.5 M) or EDTA (100 mm). After collection, the samples were dialyzed twice against the initial buffer and kept at 20 C until use. Quantification of Phycocyanobilin and Protein PCB was prepared as described before (25). PCB concentrations were determined spectroscopically using an extinction coefficient ,900 M 1 cm 1 in methanol/2% HCl (44). Protein concentrations were determined with the protein assay reagent (Bio-Rad) according to the instructions given by the supplier, using bovine serum albumin as the standard (45). SDS-PAGE SDS-PAGE was performed with the buffer system of Laemmli (46). The gels were stained with Coomassie Brilliant Blue R for the protein, and with ZnCl 2 for bilin chromophores (47). The amounts of bilins in reconstituted phycobiliproteins were quantified by comparing their fluorescence intensity on the same SDS-PAGE. Gels were photographed, the negatives were scanned (model NUSCAN 700, Shanghai Zhongjing Computer Ltd., China), and the intensity was evaluated with Photoshop 6.0 (Adobe). Care was taken to avoid saturation. Complex Formation of CpeS with Other Proteins CpeS with no His tag was incubated with His-tagged apo-proteins (CpcB(C155I) or PecB(C155I)), 84 lyases (CpcE, CpcF, PecE, and PecF), or CpeS at 4 C overnight. The mixtures were then loaded on a Ni 2 affinity column, washed three times with 5 column volumes of initial buffer (see above), once with the same buffer but also containing 50 mm imidazole, and, finally, with the same buffer containing 0.5 M imidazole. The eluate from the last wash was analyzed by SDS-PAGE. Spectroscopy UV-visible absorption spectra were recorded by a Lamda 25 spectrometer (PerkinElmer Life Sciences). Formation of the photochromic PVB-PecA (i.e. -PEC) in the lyase reaction was monitored by the absorption at 570 nm and by double-difference spectroscopy of the reversible photoreaction of the PVB chromophore as previously described (48). Fluorescence spectra were recorded with a 8574 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 13 MARCH 31, 2006

3 spectrofluorometer (PerkinElmer Life Sciences model LS 45), and were not corrected. Excitation was done on the blue side of the visible absorption maxima ( nm). The extinction coefficients of the reconstituted and biosynthesized PCB-CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), and PCB-PecB were determined based on the extinction coefficient of PCB in CPC in 8 M acidic urea (49) ( ,500 M 1 cm 1 ). Fluorescence yields F of the reconstituted and biosynthesized PCB-CpcB(C155I), PCB-CpcB, PCB- PecB(C155I), and PCB-PecB were determined in KPP buffer (20 mm, ph 7.2), using the known fluorescence quantum yield ( F 0.27) of CPC from Anabaena sp. PCC 7120 (50) as standard. Lyase Activity Assay: Optimization of Reconstitution PCB was reconstituted by incubating the desired apoprotein (CpcB(C155I), CpcB(C84S), CpcB, PecB(C155I), PecB(C84S), or PecB, M), with CpeS (12 40 M) in KPP ( mm, ph ) containing one or more of the following: Tris HCl (0 100 mm), NaCl (0 500 mm), mercaptoethanol (ME, 0 5 mm), various metal ions (0 5 mm), EDTA (0 20 mm), Triton X-100 (TX100) or reduced TX100 (0 1% v/v) and glycerol (0 25% v/v). Then PCB (final concentration 3 10 M) was added as a concentrated solution ín Me 2 SO (0.3 1 mm), such that the final concentration of Me 2 SO was 1% (v/v). The mixture was incubated at C for 1 3 h in the dark. Reconstitution was assayed via the absorption and fluorescence of the mixture. Optimum conditions were as follows: KPP (500 mm, ph 7.5), PCB (5 M), NaCl (150 mm), EDTA (1 mm), no Tris HCl, ME, metal ions, TX100, or glycerol, incubation for1hat37 C.After centrifugation at 12,000 g for 15 min, the supernatant was inspected by absorption and fluorescence spectroscopy. The sample was then purified by Ni 2 -affinity chromatography (see above) and the imidazole (500 mm) eluate was dialyzed to remove imidazole. The dialysate was then inspected again by absorption and fluorescence spectroscopy. For kinetic tests, only purified proteins were used. Reconstitution of PCB with CpcB and PecB was followed by fluorescence (emission at 645 and 630 nm and excitation at 620 and 600 nm, respectively). The purified apo-proteins (CpcB(C155I) or PecB(C155I), 25 M) and different amounts of PCB were incubated at 37 C in the optimum reconstitution system (see above). At regular time intervals, the reaction was terminated by rapidly cooling the samples on ice to 0 C, and then the product was analyzed spectroscopically without delay (11, 27). K m, v max, and k cat were calculated from Lineweaver-Burk plots, using Origin V7 (Origin Lab Corp.). Chromophore Cleavage Assay CpeS (2 M) was incubated for3hat 37 C with donor chromoprotein (reconstituted and purified PCB- CpcB(C155I) or PCB-PecB(C155I), 1.0 M)), PecA (4 M), and PecE/F (4 M). In the control experiments, CpeS was omitted under otherwise identical conditions. Chromophore transfer was assayed by the reversible photochemistry of reconstituted PVB-PecA (i.e. -PEC (48)). Chromopeptides from PCB-CpcB(C155I) and PCB-PecB(C155I) Reconstituted chromoproteins, CpcB(C155I) or PecB(C155I), were purified by Ni 2 chromatography (see above) and dialyzed against KPP (20 mm, ph 7.2). The desired chromoprotein solution was acidified with HCl to ph 1.5, and pepsin was added to the sample (1:1, w/w). The mixture was incubated for 3 h at 37 C and then fractionated on Bio-Gel P-60 (Bio-Rad) equilibrated with dilute HCl (ph 2.5). Colorless peptides and salts were eluted with the same solvent (51), and the adsorbed chromopeptides were eluted with acetic acid (30%, v/v) in dilute HCl (ph 2.5). The collected samples were dried with a rotary evaporator and subjected to HPLC (Waters 2695 system with model 2487 variable wavelength detector) on a Zorbax 300SB-C18 column (Agilent Technologies) using a gradient of KPP (100 mm, ph 2.1) and acetonitrile (80:20 to 60:40), as described by Storf (52). For the controls, natural -CPC and -PEC were isolated from M. laminosus via isoelectric focusing (48) and subjected to the same procedure. RESULTS Expression and Purification of the Apoproteins and CpeS Shen et al. (36) have recently identified lyase activities for the products of a family of genes, cpcs,-u, and -V, and for the more distantly related cpct from Synechococcus sp. PCC7002. The genome of Anabaena sp. PCC7120 contains a single gene homologous to cpcs, namely alr0617. This gene was expressed in E. coli, with and without N-terminal His and S tags. Incubation of PCB and CpcB with CpeS resulted in a rapid increase of absorption 619 nm and the emergence of a bright red fluorescence ( max 643 nm). Identical results were obtained with a mutant lacking the -155 binding site, namely CpcB(C155I) (Fig. 1A), however, there was no such reaction with mutant CpcB(C84S) lacking the binding cysteine- 84 (see below). The affinity-purified product has the intense visible absorption and fluorescence typical of native biliproteins, with the position of the bands red-shifted compared with holo- -CPC, peaking in the range associated with the cysteine- 84 chromophore of CPC (53, 54). The same reaction with PecB, the apo- -subunit of PEC, or the mutant PecB(C155I) (Fig. 1B), led to a product with blue-shifted absorption ( max 602 nm) and fluorescence bands ( max 629 nm) but otherwise similar characteristics. Again, the band-positions correspond to those assigned to the 84-chromophore of PEC from M. laminosus (55), which are blue-shifted compared with the ones of the respective CPC chromophore. There was, again, no such reaction with the mutant lacking cysteine- 84, PecB(C84S) (see below). In both cases, the binding reactions to cysteine- 84 catalyzed by CpeS are, under optimized conditions (see below), at least 10-fold increased compared with the spontaneous, non-enzymatic addition (17) of PCB to the respective subunits (Fig. 2). There was, likewise, no reaction with the -subunits of these proteins, CpcA and PecA (not shown). Reconstituted and purified PCB-CpcB(C155I) and PCB- PecB(C155I) were analyzed via SDS-PAGE (Fig. 3): in the presence of Zn 2 they showed the fluorescence that is characteristic for bilins covalently bound to the proteins. We therefore conclude that CpeS catalyzes the site-selective attachment of PCB to cysteine- 84 in both CpcB and PecB. This conclusion was further supported by the following heterologous in vivo experiments. Two genes from Anabaena sp. PCC 7120, ho1 and pcya (56) (coding for proteins required to synthesize PCB from heme), were co-expressed in E. coli with cpes and cpcb(c155i) or pecb(c155i). Absorption and fluorescence of the chromoproteins obtained from this system were virtually identical with the respective ones obtained in vitro (Fig. 1 (C F) and Table 1). Reconstitution Conditions Because of the pet30-derived plasmids used in this study, the proteins produced carry N-terminal extensions containing a His and a S tag, plus two protease sites for thrombin and enterokinase, thus facilitating their purification and also improving their solubility. Usually, these tags interfered neither with the reactivity of the apoproteins (e.g. PecA and ApcE) nor with the enzymatic functions of the lyase (e.g. PecE/F) (11, 15, 25, 26). There was also no difference in the lyase activity of CpeS, irrespective of the presence or absence of the tags (not shown). Several investigations were made to define cofactors and optimize reconstitution conditions: (a) Purified CpeS tends to precipitate in low ionic strength buffers, but shows good solubility and lyase activity in potassium phosphate buffer (KPP, 500 mm) (supplemental Fig. S1). Both were further improved by the addition of NaCl ( mm). Unlike the -84-lyases (20, 27), CpeS is more active in KPP than in Tris buffer MARCH 31, 2006 VOLUME 281 NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8575

4 FIGURE 1. Chromoprotein formation catalyzed by CpeS. A, absorption (heavy lines) and fluorescence spectra (thin lines) of PCB-CpcB(C155I) obtained by in vitro reconstitution. The purified sample containing 0.5 M imidazole from the elution buffer ( max,abs 620 nm, max,em 643 nm) is shown by solid lines, and the dialyzed sample free of imidazole by is represented by dashed lines ( max,abs 619 nm, max,em 643 nm). B, absorption (heavy lines) and fluorescence spectra (thin lines) of PCB-PecB(C155I) obtained by in vitro reconstitution. The purified sample containing 0.5 M imidazole from the elution buffer ( max,abs 603 nm, max,em 619 nm) is shown by solid lines, and the dialyzed sample free of imidazole by is represented by dashed lines ( max,abs 603 nm, max,em 629 nm). C, absorption (heavy lines) and fluorescence spectra (thin lines) of PCB-CpcB(C155I) (gene from 8576 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 13 MARCH 31, 2006

5 FIGURE 3. SDS-PAGE of His-tagged PCB-CpcB(C155I) (lanes B and b) and PCB- PecB(C155I) (lanes A and a) and CpeS (lanes C and c), detected by Coomassie staining (left) and Zn 2 -induced fluorescence (right). Lane M, protein markers (from top to bottom: 67, 45, 36, 29, 24, 20, and 14 kda). CpcB(C155I) (calculated molecular weight, including His and Ser tags, 24,209) and PecB(C155I) (calculated molecular weight, including tags, 24,306) are derived from M. laminosus, and CpeS from Anabaena sp. PCC7120 (calculated molecular mass, including tags, 25,936 Da), expressed in E. coli and purified by Ni 2 -affinity chromatography. Reconstitutions were done in vitro under optimum conditions (see text). FIGURE 2. Quantitative fluorescence of tagged PCB-CpcB(C155I) (A) and PCB- PecB(C155I) (B) from in vivo reconstitutions in E. coli and purified by Ni 2 -affinity chromatography. Solid lines represent chromoproteins from cells transformed with the full complement of genes, namely cpes, ho1 and pcya (all untagged) from Anabaena sp. PCC7120, and tagged cpcb or pecb from M. laminosus, and dashed lines represent chromoproteins from cells lacking cpes. Cells were grown under identical conditions. (not shown). (b) CpeS had good lyase activity at ph 7 9. The optimal ph was (Fig. S2), which is comparable to the 84-lyases, CpcE/F (21) and PecE/F (25). (c) Highest activity of CpeS was obtained at 45 C. However, the resulting chromoproteins tend to precipitate at this temperature, and best results were obtained at 37 C (Fig. S3). (d) The Influences of other factors known to affect 84-lyases (metal ions, EDTA, and thiols) on the activity of CpeS are summarized in Table S1. Among the tested metal ions, Ca 2 and Mg 2 inhibited slightly, Fe 2 and Mn 2 more strongly, and Co 2,Cu 2,Ni 2, and Zn 2 inhibited the lyase activity completely. Unlike the 84-lyases (27), CpeS therefore does not seem to require a divalent metal ion as cofactor. This was supported by experiments in the presence of EDTA, where concentrations as high as 20 mm were without any influence. Because EDTA can bind divalent metal ions in the reaction buffer and thereby reduce chromophore oxidation, but excessive EDTA may cause problems in the subsequent product purification over Ni 2 -affinity columns, 1 mm EDTA was routinely added to the reconstitution system. (e) ME inhibited the CpeScatalyzed reconstitution (Table S1). This is again different from the 84-lyases, which tolerate (and are in some cases activated by) ME (20, 27). ( f ) At concentrations 0.1%, TX100 inhibited the CpeS-catalyzed reconstitution. TX100 has previously been reported to inhibit, too, the spontaneous addition of PCB to cysteine- 84 of CpcB, while favoring that at cysteine- 155, which was rationalized by a conformational change of the chromophore induced by the detergent (41). (g) The -84 lyases (CpcE, CpcF, PecE, and PecF) slightly (5 17%) increased the activity of CpeS, whereas the protein involved in chromophore detachment, NblB (42), slightly decreased the activity (13%). Interaction of CpeS with each of the above lyases was tested by Ni 2 affinity chromatography using the respective immobilized His-tagged protein, and untagged CpeS as bait. SDS-PAGE of the eluted samples gave no indication for binding of CpeS with CpcE, CpcF, PecE, PecF, or NblB. Binding assays were also negative with His-tagged target subunits (CpcB and PecB) and with His-tagged CpeS. According to this assay, CpeS does not interact with any of the other proteins, nor with itself, in the absence of PCB. This is quite different from the 84-lyases, which act as 1:1 complexes of at least two subunits (21, 28). This conclusion is further proved by the biosyntheses of PCB-CpcB(C155I) and PCB-PecB(C155I) in E. coli (see below). The 84-lyase, CpcE/F, catalyzes not only the chromophore attachment to CpcA, but also its cleavage (15, 20), and it can also transfer PCB of -CPC to suitable apoproteins, including a cyanobacterial apophytochrome (15). The PecA/E/F system was used as an assay to check whether CpeS can catalyze the reverse reaction, too, and liberate the M. laminosus) biosynthesized in E. coli. The supernatant of the cell lysate ( max,abs 618 nm, max,em 644 nm) is shown by solid lines, and the purified and dialyzed sample free of imidazole is represented by dashed lines ( max,abs 619 nm, max,em 644 nm). D, absorption (heavy lines) and fluorescence spectra (thin lines) of PCB-PecB(C155I) (gene from M. laminosus) biosynthesized in E. coli. The supernatant of the cell lysate ( max,abs 601 nm, max,em 629 nm) is shown by solid lines, and the purified and dialyzed sample free of imidazole is represented by dashed lines ( max,abs 602 nm, max,em 629 nm). E, absorption (heavy lines) and fluorescence spectra (thin lines) of PCB-CpcB biosynthesized in E. coli. The supernatant of the cell lysate ( max,abs 618 nm, max,em 644 nm) is shown by solid lines, and the purified and dialyzed sample free of imidazole is represented by dashed lines ( max,abs 619 nm, max,em 644 nm). cpcb was from M. laminosus, and cpcb from Anabaena PCC 7120 gave very similar results (see Table 1); F, absorption (heavy lines) and fluorescence spectra (thin lines) of PCB-PecB biosynthesized in E. coli. The supernatant of the cell lysate ( max,abs 602 nm, max,em 629 nm) is shown by solid lines, and the purified and dialyzed sample free of imidazole is represented by dashed lines ( max,abs 602 nm, max,em 629 nm). pecb was from M. laminosus, pecb from Anabaena PCC 7120 gave very similar results (see Table 1). G, absorption (heavy lines) and fluorescence spectra (thin lines) of the in vivo (E. coli) reaction product of PCB with CpcB(C84S) (gene from M. laminosus), in the presence of CpeS. The supernatant of the cell lysate ( max,abs 635 nm, max,em 657 nm) is shown by solid lines, and the purified and dialyzed sample free of imidazole is represented by dashed lines (no distinct absorption 550 nm, minor fluorescence with max,em 657 nm), the arrow at 619 nm indicates the absorption maximum of the CpeS-catalyzed addition product of PCB to cysteine-84 of CpcB; H, absorption (heavy lines) and fluorescence spectra (thin lines)ofthein vivo (E. coli) reaction product of PCB with PecB(C84S) (gene from M. laminosus), in the presence of CpeS. The supernatant of the cell lysate ( max,abs 609 nm, max,em 636 nm) is shown by solid lines, and the purified and dialyzed sample free of imidazole is represented by dashed lines ( max,abs 609 nm, max,em 637 nm); the arrow at 601 nm indicates the absorption maximum of the CpeS-catalyzed addition product of PCB to cysteine-84 of PecB. MARCH 31, 2006 VOLUME 281 NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8577

6 TABLE 1 Quantitative absorption and fluorescence data of the reconstituted and biosynthesized PCB-CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), and PCB-PecB The data were averaged from two independent measurements. Phycobiliprotein Absorption a Fluorescence max (Q Vis/uv ) Vis max F nm M 1 cm 1 nm PCB-CpcB(C155I) b,c 337/619 (3.3) 63,500 ( 500) ( 0.01) PCB-CpcB(C155I) c 338/619 (2.5) 122,000 ( 2000) ( 0.02) PCB-CpcB c 338/619 (2.6) 132,000 ( 4000) ( 0.01) PCB-CpcB d 343/618 (2.8) 100,000 ( 2000) ( 0.02) PCB-PecB(C155I) b,c 337/602 (2.2) 69,300 ( 700) ( 0.01) PCB-PecB(C155I) c 338/602 (2.6) 116,000 ( 2000) ( 0.00) PCB-PecB c 337/602 (3.0) 101,000 ( 2000) ( 0.01) PCB-PecB d 338/603 (3.1) 90,400 ( 4500) ( 0.01) a Q Vis/uv denotes the absorbance ratio of the visible and near-uv bands; Vis is the extinction coefficient of the visible band. b Reconstituted in vitro, others were biosynthesized in E. coli. c From M. laminosus. d From Anabaena sp. PCC chromophore from PCB-CpcB(C155I) or PCB-PecB(C155I). In this system, chromophore transfer would generate the photoactive -PEC, which can be quantified by its photochromic signal (51). No such signal was detected (not shown); therefore, binding of PCB to cysteine- 84 therefore seems so strong that it is practically irreversible under the conditions tested. Chromophore Analyses of Reconstitution Products After denaturation in acidic urea solution (8 M, ph 2.0), reconstituted PCB- CpcB(C155I) and PCB-PecB(C155I) gave maximal absorption at 662 nm (Fig. 4A). This is evidence for an intact, cysteine-bound PCB and the absence of mesobiliverdin, which would absorb at longer wavelengths (16, 17). Again, the same results were obtained with PCB-CpcB(C155I) and PecB(C155I) biosynthesized in E. coli (see below), and then purified (not shown). Purified PCB-CpcB(C155I) and PCB-PecB(C155I) were finally digested with pepsin under acidic conditions. Chromopeptides were enriched by chromatography on Bio-Gel under acidic conditions, and they absorbed maximally at 656 nm (dilute HCl), which is characteristic for PCB-chromopeptides (16, 52). HPLC analyses (52) resulted, in both cases, with one major and several minor peaks (Fig. 4), which were subsets of the peaks obtained from -PEC and -CPC, respectively, isolated from PEC of M. laminosus. This, at the same time, identifies the chromopeptides marked with x in Fig. 4 (B and C) as those arising from the -155 chromophores of PEC and CPC, respectively. In summary, therefore, we conclude that CpeS catalyzes correct attachment of PCB to cysteine- 84 of both apoproteins. Biosyntheses of PCB: 84 Chromoproteins in E. coli PCB can be synthesized from heme in E. coli by the action of two enzymes, namely heme oxygenase and PCB:ferredoxin oxidoreductase. The respective genes, ho1 and pcya from Synechococcus PCC 6803, have previously been introduced into E. coli (34) to generate biliproteins (32, 33, 35). This system has been adapted to produce -subunits of CPC and PEC that, by the action of CpeS, had a single chromophore attached to cysteine- 84. The respective genes were taken from Anabaena sp. PCC There is a single gene in this organism, alr3707, that is homologous to pcya from Synechococcus PCC 6803, but there are two genes, all1897 and alr3125, that are both homologous to ho1 and ho2 (22). Of these, alr1897 was used in these experiments: it shows a higher homology to ho1 (72%) than to ho2 (57%) from Synechococcus PCC6803 and, therefore, was also annotated ho1. The plasmids pho1-pcya and pcdf-cpes were co-transformed into E. coli BL21(DE3), together with either one of the plasmids containing genes for the -subunits of CPC (pet-cpcb(c155i) and pet-cpcb) or PEC (pet-pecb(c155i) and pet-pecb). After induction, these cells became fluorescent, with emission maxima identical to the respective PCB proteins produced in vitro FIGURE 4. A, absorption (solid line, max 662 nm) of the purified chromoprotein, PCB-PecB(C155I) (M. laminosus) in acidic urea (8 M, ph 2.0), and of the purified peptide obtained by pepsin digestion, in diluted HCl (ph 2.5, dashed line, max 656 nm). B, HPLC ( detect 650 nm) of the chromopeptides obtained from peptic digestion of the reconstituted PCB-PecB(C155I) (top) and of -PEC isolated from M. laminosus (bottom). Extra peaks in the digest of -PEC (marked with X ) relate to chromopeptides derived from the second binding site, Cys C, HPLC ( detect 650 nm) of the chromopeptides obtained from peptic digestion of reconstituted PCB:CpcB (C155I) (top) and of -CPC isolated from M. laminosus. Marks are as in B. Reconstitutions were done in E. coli with CpeS from Anabaena sp. PCC7120 and apoproteins from M. laminosus JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 13 MARCH 31, 2006

7 TABLE 2 Quantification of chromoproteins produced in E. coli in the presence and absence of CpeS and yield of purified chromoprotein after Ni 2 -affinity chromatography The data were averaged from two independent measurements. Relative yield of chromoprotein in Apoprotein E. coli a E. coli b,c Supernatant d Purified Control e % CpcB 176 ( 21) 40 ( 1) ( 2) 11.8 ( 0.6) CpcB(C155I) 153 ( 12) 75 ( 2) ( 2) 7.6 ( 0.1) PecB 142 ( 15) 15.3 ( 0.3) ( 1) 6.0 ( 0.2) PecB(C155I) 131 ( 10) 21.1 ( 0.1) ( 1) 2.9 ( 0.1) a Relative yield evaluated from Zn 2 -induced fluorescence intensity of the covalently bound chromophore of the respective chromoprotein on SDS-PAGE. b Relative yield evaluated from the fluorescence of E. coli cells and of the respective chromoprotein at different stages of purification. c Low value in E. coli ascribed to particle effects of the respective chromoprotein in E. coli cells. d Set to 100% for each series with a certain apoprotein. e Supernatant of cells lacking cpes and grown under otherwise identical conditions. (Fig. 2). The chromoprotein contents of the E. coli cells were estimated by two methods, namely the fluorescence of the unbroken cells and the Zn 2 -induced fluorescence on SDS-PAGE of extracts obtained by treatment with SDS-lysis buffer. The error limits of both methods are high, and the former is, in particular, prone to underestimate the chromoprotein contents due to packing effects. The results of all four experiments, summarized in Table 2, indicate that the cellular contents are similar to or even higher than those of the supernatants obtained after breaking the respective cells. This shows that the chromoproteins are formed in vivo and no significant reconstitution took place after cell disruption. The spontaneous in vivo addition of PCB to the apo-biliproteins was furthermore excluded by an identical series of control experiments, where pcdf-cpes was omitted in the transformation of BL21(DE3) and, hence, the lyase was absent in the cells after induction. As a result, only trace amounts were formed of the chromoproteins (PCB- CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), or PCB-PecB), namely 3 10% (by fluorescence assay) relative to the production in the presence of CpeS (Fig. 2 and Table 2). The biosynthesized PCB-CpcB(C155I), PCB-CpcB, PCB-PecB(C155I), or PCB-PecB in E. coli were designed to have a His tag, whereas HO1, PcyA, and CpeS were untagged; hence, the former could be easily purified via the Ni 2 -affinity column. The purified products from these in vivo reconstitutions had identical absorption and fluorescence spectra as the respective chromoproteins produced in vitro. In particular, PCB-CpcB(C155I) and PCB-PecB(C155I), produced in vitro from the respective apoproteins and PCB under the action of CpeS, had the same spectra as biosynthesized PCB-CpcB and PCB-PecB in E. coli: this also shows that only a single PCB had been covalently bound in vivo to Cys-84 of CpcB and PecB, respectively (Fig. 1, E and F). The mutants CpcB(C84S) and PecB(C84S) gave very much reduced amounts of chromoproteins (Fig. 1, G and H), in which the chromophore furthermore absorbs at longer wavelengths than the CpeScatalyzed addition products to cysteine-84 (the absorption positions of the latter are indicated by arrows in Fig. 1, G and H). Such red-shifts are typical for oxidation products of the chromophore like mesobiliverdin, which can be formed during spontaneous addition reactions (17). Isolated CpcB(C84S) from this test has no distinct absorption peak in the 600 nm range, indicating non-covalent binding of the chromophore, which is lost on the Ni 2 column. Small amounts of chromoprotein were isolated from the test with PecB(C84S) after Ni 2 chromatography, indicating either a tighter non-covalent binding, or the covalent addition of a modified chromophore to cysteine Enzyme Kinetics of CpeS Enzyme kinetics of CpeS, carried out under the optimal conditions (see above), showed Michaelis-Menten behavior, and were analyzed from the Lineweaver-Burk plots shown in Fig. 5. With respect to PCB, the following values were obtained with FIGURE 5. Kinetic analyses of PCB-binding to CpcB(C155I) and PecB(C155I) (both derived from M. laminosus, expression in E. coli) under catalysis of CpeS from Anabaena sp. PCC7120. A, CpcB(C155I) (25 M), CpeS (25 M), KPP (500 mm), NaCl (150 mm), and EDTA (1 mm). B, PecB(C155I) (25 M), CpeS (25 M), KPP (500 mm), NaCl (150 mm), and EDTA (1 mm). CpcB(C155I): K m (PCB) M, k cat s 1, and with PecB(C155I): K m (PCB) M, k cat s 1. These values fall well into the range of those obtained for the -84 lyases CpcE/F (21) (K m (PCB) M and k cat s 1 ), and PecE/F (28) (K m (PCB) M and k cat s 1 ). DISCUSSION In this work, the genes coding for CpcB and PecB and their mutants were cloned from M. laminosus (Fischerella sp. PCC7603) and Anabaena sp. PCC7120, and that coding for CpeS was cloned from Anabaena sp. PCC7120. Not only are the genes and proteins of these two organisms highly homologous (22), 5 but they also have the same type of pigmentation and phycobilisome structure (57, 58). The success of the in vivo reconstitutions described, in which the genes for PCB-CpcB(C155I) and PCB- PecB(C155I) were from M. laminosus, and all other genes from Anabaena sp. PCC7120, further supported this close relationship. Compared with enzymatic chromophore attachment to the -subunits, relatively little has been known, until recently, about the catalytic chromophore attachment to the -subunits of phycobiliproteins. The field has been opened by Shen et al. (36) who identified lyase activities MARCH 31, 2006 VOLUME 281 NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8579

8 specific for the 84 site for the products encoded by a family of genes, cpcs, -U, and -V, and by the more distantly related cpct from Synechococcus sp. PCC7002. The gene, cpes, which shows high homology to cpcs, had originally been identified in Calothrix PCC7601 on the phycoerythrin linkerpolypeptide operon (cpec,-d,-e,-s,-t, and -R), which in addition to the linker genes, cpec, -D, and -E and to cpes, contained cper, which was described as an activator required for expression of the phycoerythrin operon.(37). Shen et al. (36) assigned lyase activities, specific for cysteine- 84, to CpcS and to CpcT from Synechococcus sp. PCC7002, which increased in mixtures of them, and in the presence of CpcU and CpcV. The genome of Anabaena sp. PCC7120 contains a single gene homologous to cpcs, namely alr0617, which was expressed in E. coli in this work, and the activity of the product, CpeS, 6 studied in detail. Under catalysis of CpeS, PCB is attached regioselectively to only one of its two binding sites, cysteine- 84, thus confirming the results of Shen et al. (36). For CPC and PEC, this leaves the second binding site, cysteine- 155, as the only one to which no lyase can yet be assigned. Non-enzymatic attachment in mutants of CpcB and PecB from M. laminosus indicated that an autocatalytic binding of PCB to Cys-155 is favored under the influence of the detergent, TX100 (41). Experiments reported here, however, with multiply transformed E. coli indicate that a spontaneous addition to either site is unlikely in vivo, at least in this organism, and that yet a third lyase is required for chromophore attachment to cysteine There is only very little residual binding to mutants lacking cysteine-84, and the absorption of the resulting products are red-shifted, indicating an oxidation of the chromophore during the spontaneous addition reaction. The need for yet another enzyme catalyzing the attachment to cysteine- 155 is further emphasized by the different stereochemistry of PCB bound to this position, as compared with those bound to cysteines 84 or 84 (59). In vitro, binding of PCB with CpcB and PecB at both binding sites, Cys-84 and Cys-155, is conceivable, simultaneously or sequentially, by a clever combination of enzymatic (CpeS) and detergent (TX100) treatment, but, hitherto, attempts have failed to form correctly bi-chromophorylated -CPC or -PEC by attaching PCB first to Cys- 84 by the action of CpeS, and then to Cys- 155 under control of TX100, or vice versa. Irrespective of whether a complete reconstitution may eventually become possible, we conclude that one or more other unknown factors responsible for the binding of PCB at Cys- 155 probably exist in cyanobacterial cells, which are absent in E. coli. These findings complement the results of Shen et al. (36), who found that knockouts of cpcs (the homologue of cpes) and the homologous cpcu in the cyanobacterium, Synechococcus sp. PCC7002, resulted in CPC containing -subunits carrying only a single chromophore at cyteine-155 (consensus numbering) and devoid of a covalently bound 84 chromophore. Although the PCB binding sites at Cys- 84 and Cys- 84 are phylogenetically related and similar not only in sequence but also spatially, and the chromophores have the same stereochemistry and very similar conformations (59), there exist considerable differences between the lyases responsible for chromophore attachment at these two sites. The 84-lyases act as heterodimers (CpcE/F or PecE/F) and are often encoded by genes contained in the same operon as the apoproteins, like, for example, in M. laminosus and Anabaena sp. PCC7120 (21, 28, 60). 6 By contrast, CpeS alone is capable of correctly binding PCB to Cys- 84, with kinetic constants comparable to those of the 84 lyases, and this protein is encoded by a gene, alr0617, that is remote from the cpc and pec operons. Also, whereas the 84-lyases bind to other proteins and aggregate among each other, in the present study we have been unable to observe stable complexes with any of the other lyases or apo-biliproteins tested using Ni 2 -affinity chromatography. It should be noted that Shen et al. (36) described more complex functions for the new type of lyase: they observed synergistic effects with combinations of four gene products, three of them deriving from cpes-like genes (cpcs, cpcu, and cpcv), and a fourth, more distantly related gene (cpct). They furthermore reported specificities of the different -subunits, which depend on the particular mixture of proteins used. Chromophore attachment by CpeS to Cys- 84 of both CpcB and PecB supports this broader specificity, as does the high degree of homology of alr0617 (from Anabaena sp. PCC7120, which, like Synechococcus sp. PCC7002, lacks phycoerythrin), to genes involved in C-phycoerythrin (CPE) synthesis. CpeS has first been identified in Calothrix PCC 7601, as part of an operon, cpecdestr, which has also been found in several other phycoerythrincontaining cyanobacteria (37). Anabaena sp. PCC7120 does not produce CPE and does not contain the cpecdestr operon. Of the other genes, two cpet homologues (all5339 and alr0647) and one cper homologue (alr3496) are present in Anabaena sp. PCC7120 but are not contiguous with cpes (alr0617) (22). Also distant are the linker genes of Anabaena sp. PCC 7120 that are homologous to cpec, -D, or-e. The cpes gene is in the center of a cluster of three genes: interestingly, the 5 -flanking one (alr0616) is homologous to nblb, coding for a protein involved in chromophore removal from biliproteins, whereas the 3 -flanking gene (alr0618) has no obvious homology with genes related to biliprotein metabolism or to phycobilisomes. It should be noted, however, that the genes coding for -84 lyases are also often not on an operon containing the respective substrate apoprotein (9). A third difference is that, unlike CpcE/F, CpeS did not catalyze cleavage or transfer of PCB covalently bound to Cys- 84. Among other phycobilin:biliprotein lyases studied for cleavage activity, cyanobacterial phytochrome has only a weak reverse (cleavage) enzymatic activity (15), while binding-only activity has been observed with the core-membrane linker, ApcE (11), and with the isomerizing lyase, PecE/F (25, 26). Currently, this leaves only CpcE/F as a lyase that readily catalyzes both the forward and backward reactions (20). Under certain physiological conditions, like nitrogen-starvation, phycobiliproteins are degraded in cyanobacteria (42), and chromophore cleavage has been implicated as an early event of this process. Currently, there is only one protein known, namely NblB, that is directly involved in the cleavage, but the reversible action of CpcE/F may indicate that it also has some related functions and is involved in regulation and/or degradation of biliproteins (42, 23). It is curious in this context, that in Anabaena sp. PCC7120 the gene immediately 5 of cpes (alr0616) has a high homology to nblb from several cyanobacteria, thus placing genes coding for a biosynthetic enzyme and a chromophore-cleaving protein next to each other; possibly, on the same operon. Restructuring of the phycobilisome might be a process requiring such a combination of functions. Judged from available sequence data, there is no common motif among the lyase domains of proteins binding the bilin chromophores autocatalytically (phytochromes and ApcE) and the 84 lyases. Neither CpcS, -T, -U, or -V from Synechococcus sp (36), nor CpeS from Anabaena sp. PCC7120 studied here, are obviously related to either subunit of the 84 lyases of these organisms or of M. laminosus. Although these results confirm chromophore attachment to the 84 sites of cyanobacterial biliproteins, binding to the 155 site still remains to be demonstrated. Acknowledgments H. S. and K. H. Z. are grateful to Volkswagen Stiftung for the Partnership. H. S. is grateful to Deutsche Forschungsgemeinschaft for support. M. Z. is grateful to the National Natural Science Foundation of China for support. We thank Dr. R. Porra, Canberra for help in preparing the manuscript JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 NUMBER 13 MARCH 31, 2006

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