Proc. Natd. Acad. Sci. USA Vol. 89, pp. 7017-7021, August 1992 Biochemistry a-subunit Iyase CRAIG D. FAIRCHILD*, JINDONG ZHAOt, JIANHUI ZHOUtI, SUE ELLEN COLSON*, DONALD A. BRYANTt, AND ALEXANDER N. GLAZER*§ *Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and tDepartment of Molecular and Cell Biology, The Pennsylvania State University, University Park, PA 16802 Communicated by Daniel I. Arnon, April 27, 1992

ABSTRACT , unlike other light-harvest- elimination of cysteine at the 3' carbon of the protein-bound hmg proteins involved in photosynthesis, bear covalently attached (3,3'-dihydro-3'-cysteinylphycocyanobilin; referred to . The bilin chromophores are attached through below as PCB adduct). thioether bonds to cysteine residues. The cyanobacterium Syn- The inference that 3(E)-PCB is the immediate precursor of echococcus sp. PCC 7002 has eight distinct blin attachmnt sites phycocyanin-bound bilin in vivo is based on the observations on seven poypeptides, all ofwhich carry the same , that PCB accumulates in C. caldarium cells in presence of phycocyanobilin. When two genes in the phycocyanin operon of added precursor 6-aminolevulinate (6) and that this organisn, cpcE and cpcF, are inactivated by insertion, PCB spontaneously forms a photoactive adduct with apo- together or separately, the srping result is elimination of both in vivo and in vitro (7). correct biln attachment at only one site, that on the a subunit There is evidence suggesting that addition of PCB to of phycocyanin. We have overproduced CpcE and CpcF in apophycocyanin requires enzymatic catalysis. Phycocyanin Escherichia coli. In vitro, these proteins catalyze the attachment carries three PCB groups, at a-Cys-84, 1-Cys-82, and P-Cys- of phycocyanobilin to the a subunit of apophycocyanin at the 155. In vitro mixing of PCB and apophycocyanin results in appropriate site, a-Cys-84, to form the correct adduct. CpcE bilin attachment at residue 84 of the phycocyanin a subunit and CpcF also efficiently catalyze the reverse reaction, in which (a"c-84) and P~c-82, but not at P"c-155, and the principal the bilin from holo-a subunit is transferred either to the apo-a adduct formed is a 3'-cysteinylmesobiliverdin (8, 9). Further- subunit of the same C-phycocyanin or to the apo-a subunit of a more, insertional inactivation of either cpcE, cpcF, or both heterologous C-phycocyanin. The forward and reverse reactions genes in Synechococcus sp. PCC 7002 results in loss of each require both CpcE and CpcF and are specific for the proper bilin addition at aP-84 (10, 11). These mutations have a-Cys-84 position. Phycocyanobilin is the immediate precursor no effect on PCB addition at any of the other seven bilin of the protein-bound bilin. attachment sites, even though four of these show substantial local sequence homology to that on a . Phycobiliproteins are homologous proteins found in cyano- We describe an assay for bilin addition to resin-bound bacteria, , and the (1). In vivo they apo-aPc. With appropriate modifications this assay can be form highly ordered macromolecular light-harvesting assem- applied for studies ofbilin addition to apophycobiliproteins at blies, , in which directional energy transfer is other sites. This and other assays, using proteins overpro- determined by the spectroscopic properties and relative po- duced in Escherichia coli, were used to demonstrate that the sitions of the bilin prosthetic groups. cpcE and cpcF genes encode a protein-bilin lyase specific for The number of bilin attachment sites on all of the phycobil- the aPc-84 attachment site. The immediate bilin precursor for iproteins ofa particular cyanobacterium or red alga ranges from addition to aPC was shown to be PCB. a minimum of 8, all carrying the same bilin, to >20, with up to three different bilins. To study the process of bilin attachment MATERIALS AND METHODS we have chosen a simple case, that in Synechococcus sp. PCC Recombinant Proteins. The Pst I-Xho I fragment contain- 7002, in which there are 8 attachment sites for the same bilin, ing the cpcA gene of Synechococcus sp. PCC 7002 was phycocyanobilin (PCB), on seven polypeptides. transferred from the plasmid pAQPR1 (12) to the Pst I and Sal Posttranslational modification of proteins usually involves I sites of pUC19, and a Pst I-EcoRI fragment from this recognition of simple amino acid sequence determinants. plasmid was subsequently transferred to the corresponding Local sequence homology around modification sites gener- sites ofpTZ19R (Pharmacia LKB). Apo-apc was synthesized ally suggests that there is one enzyme that recognizes all of from this construct in E. coli K38/pGP1-2 (13, 14) with these sites. Surprisingly, this does not appear to be the case kanamycin and ampicillin selection in rich medium [2% for bilin attachment to phycobiliproteins. tryptone/1% yeast extract/0.5% NaCl/0.2% (wt/vol) glyc- Until recently little was known about the processes ofbilin erol/50 mM KP1, pH 7.2] at 300C in 1-liter cultures. High-level synthesis and attachment to the apoproteins. Beale and synthesis of aPc in the absence of inducer was observed; Comejo (2-4) have examined bilin biosynthesis in the red late-exponential phase cells were collected by centrifugation. alga Cyanidium caldarium and have provided evidence for In a typical preparation, cells (11 g) were suspended in 33 the following pathway: -- IXa - 15,16- ml of 50 mM NaPs, pH 7/5 mM EDTA (NaPi/EDTA) and dihydrobiliverdin IXa -. 3(Z)- - 3(Z)- disrupted by two passages through a French pressure cell at PCB -+ 3(E)-PCB. 12,000 psi (1 psi = 6.89 kPa). Cell lysates were centrifuged at PCB can be obtained by cleavage from phycocyanin or 18,000 x g for 20 min; the supernatant, which contained some allophycocyanin by methanolysis (5). PCB has an ethylidene group at the C-3 position (the IUPAC numbering system for Abbreviations: aPc and PPC, a and p subunits of phycocyanin; bilins is shown for PCB in ref. 2) and is the product of CpcE/F, mixture ofCpcE and CpcF proteins; PCB, phycocyanobilin. *Present address: Department of Plant Biology, University of Cali- fornia, Berkeley, CA 94720. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at: MCB: Stanley/ payment. This article must therefore be hereby marked "advertisement" Donner ASU, 229 Stanley Hall, University of California, Berkeley, in accordance with 18 U.S.C. §1734 solely to indicate this fact. CA 94720.

7017 Downloaded by guest on September 27, 2021 7018 Biochemistry: Fairchild et al. Proc. NatL Acad. Sci. USA 89 (1992) apo-a"C, was discarded. Most of the apo-apc was in the quenched by addition of 0.05 volume of 1 M glycylglycine pellet, and was brought into solution by suspension in 450 ml (pH 7). After 1 hr at 4°C, the resin was washed extensively of50mM NaP,, pH 7/200mM NaCl/1 mM dithiothreitol. The with NaPi/EDTA/0.5 M NaCl, followed by NaPi/EDTA. suspension was clarified by centrifugation (18,000 x g, 15 The apo-aIpc resin was stored in NaP,/EDTA/5 mM dithio- min) and nucleic acid contaminants were removed by passage threitol under vacuum at 4°C. through a DEAE-cellulose column (2.5 x 17.5 cm) equili- Assay for Bim Addition to Apo-a"c Resin. Synechococcus brated in 50 mM NaPi, pH 7/200 mM NaCl. Fractions sp. PCC 7002 was grown in 8-liter carboys ofmedium A+ (17) absorbing at 280 nm were pooled and concentrated to 1 at 38°C under constant illumination and an atmosphere of5% mg/ml by ultrafiltration. Aliquots (30 ml) were loaded onto a CO2 in air. Cells were harvested by centrifugation, washed 600-ml (bed volume) Bio-Gel P100 column in NaPi/EDTA; with 2 mM EDTA (pH 8) and 50 mM Tris-HCl (pH 8) and then fractions were analyzed by SDS/PAGE. Apo-a"c (calculated suspended in 4 volumes of the latter buffer and passed A28o mg-ml-lcm-1 = 1.08) from this column was >95% pure. through a French pressure cell twice at 18,000 psi. The lysate The HindIII-Xho I fragment ofplasmid pPC50 (10) encod- was clarified by ultracentrifugation for 90 min at 100,000 x g. ing the cpcE and cpcF genes ofSynechococcus sp. PCC 7002 The apo-aPc resin for each round of assays was packed in was cloned into the HindIII and Sal I sites of pUC9 to a small column, incubated in fresh NaPi/EDTA/5 mM di- generate pUC9EF. For overproduction of CpcE, the Dra thiothreitol for 30 min, and washed with several volumes of I-Hpa I fragment of pUC9EF was cloned into the Sma I site 50 mM Tris HCI (pH 8). Each assay mixture contained 300 ,l ofpUC19. The Xba I-Nde I fiagment ofthe resultant plasmid of apo-a"pc resin (settled bed volume); variable amounts of was cloned into the Xba I and Nde I sites of expression cyanobacterial extract, purified , or bilin plasmid pET-3a (15) to generate pET-3a-CpcE. For overpro- (from stock solutions in dimethyl sulfoxide); and 50 mM duction of CpcF, plasmid pUC9EF was digested with Sal I Tris HCI (pH 8) with or without additions (described below) and the ends were filled in by incubation with dNTPs and the in a total volume of 1.1 ml. The reaction was performed in Klenow fragment of DNA polymerase I. The blunt ends of 1.5-ml Eppendorf tubes, protected from light, with agitation the plasmid were ligated with Nco I linkers (hexamers; at 37°C. Assays were quenched by cooling on ice. Pharmacia LKB). The Nco I-BamHI fragment of the result- Assay resins were transferred to minicolumns and washed ant plasmid, denoted pUC9F-Nco, was cloned into expres- successively with NaPi/EDTA; 9 M urea/HCI, pH 2.5; sion plasmid pET-3d (15) to generate pET-3d-CpcFl. Plas- water; 2:1 (vol/vol) acetonitrile/2-propanol with 0.1% tri- mids pET-3d-CpcFl and pET-3a-CpcE were transformed fluoroacetic acid; and NaPi/EDTA. The fluorescence emis- into E. coli B121(DE3) (15). sion spectra of settled assay resins in a 4-mm-pathlength CpcE and CpcF proteins were produced separately in square cuvette were taken with 600-nm excitation and 4-nm 1-liter cultures grown in rich medium under ampicillin selec- slit widths on a Perkin-Elmer MPF-44B fluorescence spec- tion. At an OD600 of 1.2-1.5, solid isopropyl f3-D- trophotometer. thiogalactopyranoside was added (1 mM); cells were col- Tryptic digestion of assay resin suspension and a soluble lected by centrifugation 3 hr later. Large quantities of CpcE phycocyanin standard, with separation ofchromopeptides by and CpcF were found predominantly as inclusion bodies and reverse-phase HPLC, was performed as described (8). were purified by a conventional method (16). Briefly, this Other Methods. [35S]phycocyanin and [35S]allophycocya- entailed cell lysis in a French pressure cell, centrifugation of nin were purified from Synechococcus sp. PCC 7002. A 50-ml the lysate, and washes of the CpcE- or CpcF-containing culture in medium A+, with 6 mM MgSO4 and 14 mM MgCl2 pellet first with buffer containing EDTA and 1% Triton X-100 rather than 20 mM MgSO4, and with 0.8 ;LCi (29.6 kBq) of detergent and then with buffer alone. The inclusion bodies [35S]sulfate (Amersham), was grown to near-stationary were solubilized with acidic 9 M urea containing 1 mM phase. Cells were harvested by centrifugation, washed with dithiothreitol. Concentration and purity of CpcE and CpcF 0.5 M sucrose/50 mM Tris-HCI, pH 8, and frozen. Thawed were estimated by SDS/PAGE with comparison of cells were suspended in 2 ml of20 mM Tris-HCI, pH 8/10 mM Coomassie-stained bands to those of protein standards. EDTA/0.1% hen eggwhite lysozyme (Sigma) and incubated The urea-solubilized proteins were clarified by centrifuga- 1 hr at 25°C. The cell lysate was centrifuged and the super- tion, and CpcE and CpcF were renatured, separately or natant was fractionated as follows: (NH42SO4 precipitation together, either by dilution ofprotein (1.5 mg/ml) in acid urea (20-55% of saturation) of all phycobiliprotein; hydroxylapa- 1:10 into 50 mM Tris'HCI, pH 8/75 mM NaCl or by dialysis tite column chromatography with a 10-200 mM Pi gradient to of protein (0.3 mg/ml), in urea neutralized with Tris base, separate phycocyanin and allophycocyanin; Mono Q FPLC against 50 mM Tris HCl, pH 8/75 mM NaCI/1 mM thiogly- (Pharmacia) chromatography in 20 mM Tris-HCl (pH 8) with colate/1 mM MgCl2/1 mM NaPPj. Precipitate was removed aO-0.35 M NaCI gradient. The specific activity ofthe purified by centrifugation. proteins was 860 cpm/,ug of phycocyanin and 1050 cpm/,ug CpcE expressed in E. coli exhibited three main polypep- of allophycocyanin. tides on SDS/PAGE (data not shown): a presumably full- Unlabeled from Anabaena sp. PCC 7120 and length product of29-30 kDa and two polypeptides of slightly Synechococcus sp. PCC 7002 were isolated (18) and their a higher mobility, presumed to be proteolysis products. The and (3 subunits were separated (19). Phycocyanin subunits polypeptide ofhighest mobility represented the most soluble were renatured by removal of organic solvents by rotary material, which was discarded with the supernatant from evaporation followed by 2:9 dilution of the aqueous subunit centrifugation. The other two products remained in solution with 9 M urea/HCI, pH 2.5/10 mM 2-mercaptoethanol and to a similar extent on removal of urea from the solubilized dialysis against NaPi/EDTA. inclusion bodies. The concentrations of CpcE specified be- PCB was isolated from partially purified phycocyanin and low are those of the larger product. stored at -20°C in dimethyl sulfoxide (8). The yield of soluble CpcE and CpcF on renaturation was 50-60%. Renatured CpcE and CpcF proteins were used RESULTS without further purification. Incubation of a crude cyanobacterial extract in pH 8 buffer Preparation of Apo-aPc Resin. Purified apo-aPC (0.4-1.0 with resin-bound apo-a"c results in a protein-bilin adduct mg/ml) was dialyzed extensively against NaP,/EDTA and with an emission spectrum very similar to that of authentic mixed (1 mg of protein per ml of resin) with Affi-Gel 15 aPC holoprotein. The spectrum is readily distinguishable from (Bio-Rad) in NaPi/EDTA. The reaction was allowed to that of mesobiliverdin adduct, the product of nonenzymic proceed with gentle agitation for 30 min at 4°C and was reaction of PCB with apo-apc resin (Fig. 1). After tryptic Downloaded by guest on September 27, 2021 Biochemistry: Fairchild et al. Proc. NatL. Acad. Sci. USA 89 (1992) 7019 120

8 -. 15

0 %2 _- C4 E U r0_

8 10 VQ D0i 0 S. .:D 610 690 ii X (n) FIG. 1. Fluorescence emission spectra ofbilin adducts to apo-apc resin. Solid line, PCB adduct, product of incubation with crude cyanobacterial extract; dashed line, mesobiliverdin adduct, product of nonenzymic addition. Excitation wavelength was 600 nm. The spectra are scaled to approximate the difference in quantum yield between the two adducts. Fraction number digestion of the adduct that was formed in the presence of FIG. 2. Hydroxylapatite column fractionation of Synechococcus cyanobacterial extract, reverse-phase HPLC separation of sp. PCC 7002 crude extract. The extract (29 ml) was loaded onto a the products yields a single chromopeptide that is eluted in hydroxylapatite column (2.5 x 15 cm) in 75 mM NaCl/50 mM the position of the a-1 bilin-tripeptide from phycocyanin and Tris'HCl/10 mM NaPi, 1 mM thioglycolic acid, pH 8; 6-ml fractions has an identical absorbance spectrum (data not shown). were collected. After washing with the same buffer, a 600-ml 10-150 Formation of the correct PCB adduct was largely abolished mM NaPi gradient was applied; the initial elution of the phosphate gradient corresponds to the initial rise in A622 of the eluate. In this by pretreatment of the cyanobacterial extract with trypsin portion ofthe column profile A622 reflects phycocyanin content. Bilin [8% (wt/wt) vs. extract protein for 30 min at 300C; an equal addition to apo-aPc resin was assayed with and without CpcE/F weight of soybean trypsin inhibitor was added before assay]. proteins (4 jg of each), with 50 tul of each fraction, in 50 mM To determine whether the crude extract contained un- Tris HCl/75 mM NaCl/1 mM MgPPi/1 mM thioglycolate, pH 8, with bound precursor bilin, extract (9.7 ml) was passed through a a 1-hr incubation at 37°C; the amount of addition is expressed as Sephadex G-25 column (1.5 x 72 cm) in 50 mM Tris HCl (pH relative fluorescence emission at the peak emission wavelength of 8) at 40C. PCB and other bilins bind strongly to Sephadex holo-aPc. Excitation wavelength was 600 nm. resins under these conditions. No observable chromophore bound to the top ofthe column, but little ofthe correct adduct CpcE/F was much greater (40-fold more than without CpcE/F; formed on incubation ofthe protein-containing fraction ofthe a slightly higher ratio than that shown in Fig. 3). The amount of eluate with the apo-a'c resin. radioactivity on the assay resin was not enough to account for Various compounds were added to assays ofthe Sephadex the transfer of even one sulfur atom per transferred bilin. G-25 eluate in an effort to recover lyase activity. The best Consequently, only bilin is transferred to the resin. There was enhancement of activity (to levels approaching those seen no significant amount of bilin transfer from [35S]allophycocya- before gel filtration) was seen in 75 mM NaCl/1 mM MgPP1/1 nin with or without CpcE/F. mM thioglycolate at pH 8. Recombinant, partially purified The amounts of CpcE/F-dependent transfer of bilin from CpcE and CpcF enhanced the addition activity of crude phycocyanin and from a similar molar amount ofpurified a"~c extract severalfold when both were added to assays. CpcF were roughly equal (Fig. 3). There was a smaller but signif- alone did not enhance activity; CpcE alone enhanced activity by about 50%. 40 An attempt was made to purify the source of bilin in the C4 E~~~~~~~~~+pE crude extract, along with any endogenous enzymatic activ- S ~~~~~~~~~ -CpcEF ities involved in the addition process. The results of hydrox- C 30- ylapatite fractionation of the cyanobacterial extract are shown in Fig. 2. Most intrinsic addition activity copurified with a subfraction of phycocyanin that is eluted at a lower C 20 phosphate concentration than bulk phycocyanin (20). When both CpcE and CpcF (CpcE/F) were added to each assay, the main peak of intrinsic activity was enhanced severalfold, but a new, larger peak of activity appeared that coincided with the main phycocyanin peak. 0- This result suggested that phycocyanin itself might be a bilin donor in the resin assay in the presence of CpcE/F. In accord with this supposition, an assay with pure phycocyanin in place 07002 7002 7002 7120 7120 7120 of cyanobacterial extract showed strong, CpcE/F-dependent (a13 a 13 a13 a 5 transfer ofbilin to apo-aPc resin (Fig. 3). CpcE alone allowed a 25 jig 14.5 jg 15.5 jg 75 jig 43.5 jg 46.5 jig small amount ofbilin transfer; with CpcF alone there was none FIG. 3. Phycocyanins and their subunits as bilin donors to apo-aPC (data not shown). When 35S]phycocyanin was used as a bilin resin. The bilin donor in each assay was phycocyanin or its purified donor, only a trace ofradioactivity remained bound to the assay a or , subunit in the amount indicated; "7120" is Anabaena sp. PCC resin. This amount was no greater in assays with CpcE/F than 7120, "7002" is Synechococcus sp. PCC 7002. Assay conditions were in those without, although the amount of bilin transfer with as in Fig. 2, with or without CpcE/F proteins (8 ,ug of each). Downloaded by guest on September 27, 2021 7020 Biochemistry: Fairchild et al. Proc. Natl. Acad Sci. USA 89 (1992)

icant amount of addition with purified .8Pc as donor; this addition was not CpcE/F-dependent, and the product had a 2 i}81- peak emission (628 nm) distinct from that ofPCB adduct (642 I-, nm) or mesobiliverdin adduct (668 nm). The product of I CpcE/F-independent transfer ofbilin from phycocyanin also 1 had a blue-shifted emission spectrum, whereas CpcE/F- Ca 10 i~~~~~~~~~~~~~~~~~~~~~~~~~~ 3 dependent transfer from phycocyanin or aI'C resulted in the §I emission spectrum characteristic of the PCB adduct. 5 - -EF Phycocyanin from another cyanobacterium, Anabaena sp. go PCC 7120, also served as a bilin donor for CpcE/F-mediated lvlin En'- -I tb1% 1 A r- 0 1.is 0 U 30 60 YU 1lU 2 4 6 1U bilin transfer (Fig. 3). This reaction was much slower than TIME (m that with Synechococcus sp. PCC 7002 phycocyanin, even at a 3-fold higher concentration. This most likely reflects FiG. 5. (A) CpcE/F-mediated transfer of bin between identical CpcE/F specificity for the polypeptide but may also be a polypeptides: from soluble Synechococcus sp. PCC 7002 holo-apC to result of greater stability of the quaternary structure of apo-aPc resin. A plot of identical assays, stopped at various times Anabaena phycocyanin. The latter possibility is supported under conditions as in Fig. 2 and read as in Fig. 2, is shown. The soluble holo-aPC was 1.25 ,uM (25 CpcE/F proteins, 0.1 ,uM each aPC was a better bilin .g), by the fact that purified Anabaena (3.2 ,ug of CpcE, 2.8 .g of CpcF). (B) Addition of PCB to soluble donor than the Anabaena phycocyanin, whereas this was not apo-aPc with or without CpcE/F. Relative fluorescence emission at the case for Synechococcus aPc and phycocyanin. 640 nm (excitation wavelength, 600 nm) was read continually over Anabaena aPc and Synechococcus aPC are separable by the time range shown; the dashed portions are extrapolations to the reverse-phase HPLC. Transfer of bilin from the Anabaena start ofmixing. Each assay mixture contained 10 jM PCB and 10 ,LM holo-aPc to Synechococcus apo-aPC in solution, rather than apo-aPc in 0.5 ml 50 mM Tris HCl/75 mM NaCl/1 mM MgPP,/1 mM attached to a resin, can thus be monitored. Without CpcE or thioglycolic acid, pH 8, at 3rc. +EF, 2 ,uM CpcE and 2 ,uM CpcF. Assays were started by addition of PCB. CpcF, or with either alone, the bilin (Fig. 4A; 350-nm trace) was eluted entirely with the Anabaena aPC. With CpcE/F there was a great deal of bilin transfer to the Synechococcus Transfer from Synechococcus holo-aPc to Synechococcus aPC (Fig. 4B). Under the chromatographic conditions used, apo-caPC resin is much more rapid (Fig. 5A). With concen- an apo-aPc is eluted just ahead of its cognate holoprotein. In trations of holo-aPc and CpcE/F similar to those used for Fig. 4B, the 280-nm trace shows a clear shoulder at the transfer from Anabaena aPC to Synechococcus aPC, this position of the Anabaena ac. This shoulder indicates that reaction is half-complete in 10 min. bilin was removed from this subunit, without degradation of Since free bilins in solution exhibit insignificant fluores- the polypeptide, to yield the corresponding apo-apC. cence compared with those found on native phycobilipro- As noted above, transfer of bilin from Anabaena holo-apc teins, it should be possible, given the correct bilin precursor, to the apo-aPC of Synechococcus is slow. The reaction to monitor enzymatic addition of bilin to apoprotein by an mixtures analyzed in Fig. 4 were incubated 18 hr at 37°C; bilin increase in fluorescence. The result of a representative assay transfer under these conditions is barely detectable after 1 hr. with PCB as the source of bilin, in the presence or absence of CpcE/F, is shown in Fig. 5B. CpcE/F-mediated addition of PCB was biphasic, with a rapid initial rise in emission - 280 nm 0.15 A followed by a slower, linear rate. The extent of the initial increase in emission was propor- .0 tional to the amount of PCB used in the assay. With a higher concentration ofPCB (10-20 ,M) and less CpcE/F (0.01-0.2 ,uM), the initial rate could be accurately measured and was proportional to the concentration of CpcE/F (data not shown). The slower, second rate varies with the concentra- 0.104 tion of enzyme, but not in direct proportion. The amount of fluorescence roughly correlates with the amount of covalent addition of bilin, as measured by reverse-phase chromatog- raphy ofaliquots taken near the end ofthe initial, faster phase and at timepoints thereafter (data not shown). 0.020-0 B The products ofPCB addition to soluble apo-aPc under the conditions described were a mixture of PCB and mesobili- 0.02 verdin adducts, as determined from the emission spectrum. 0.0027 A slightly higher fraction of PCB adduct in this mixture was seen at high concentrations of CpcE/F. However, the prod- TIME (min.) uct could be shifted to exclusively PCB adduct by preincu- bation of equimolar PCB and CpcE/F proteins for 30 min at FIG. 4. Transfer of bilin from Anabaena sp. PCC 7120 holo-aPC 37°C with subsequent addition of apo-aPc. to Synechococcus sp. PCC 7002 apo-a"c with CpcE only (A) or with CpcE and CpcF (B). Each assay mixture contained 75 J~g of each a DISCUSSION subunit in 1.3 ml of 50 mM Tris.HCl/75 mM NaCI/1 mM MgPP1/1 Our of the results of CpcE/F-mediated addi- mM thioglycolic acid, pH 8, and was incubated 18 hr at 3rC. The a interpretation subunits were separated by C4 reverse-phase chromatography es- tion of PCB to soluble apo-aPc is summarized in the scheme sentially as described (19), with loading of samples in acid 6 M urea below. at 35% organic solvent, a 1-min linear gradient to 53%, and a 20-min lineargradient to 63% organic solvent. The 350-nm traces reflect bilin Other isomer s content; the 280-nm traces reflect protein content. Holo-aPC of of PCB PCB* +CpcEjF CpcE/F.PCB* CpcE/F Anabaena is eluted in 15.3 min, and that of Synechococcus in 18.9 + + + = + min. apo-a apo-a apo-a a-PCB Downloaded by guest on September 27, 2021 Biochemistry: Fairchild et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7021 The fast initial rate of fluorescence gain would correspond to mined, the fluorescence quantum yield of phycocyanin is addition of a fraction of the PCB (PCB*) immediately avail- similar to that of aPC at pH 8, room temperature or 37TC able for binding to CpcE/F and subsequent transfer to (C.D.F., unpublished results), suggesting that the environ- apo-aPc. The second, slower rate would depend on the rate ment of the bilin on the isolated subunit is similar to that of of isomerization of the bulk of the PCB to PCB*. PCB can the bilins on phycocyanin (ac3). react nonenzymically with apo-a'C, to form mostly the Cleavage of the buried thioether bond of aPc in the pres- mesobiliverdin adduct, at a rate comparable to this isomer- ence of CpcE/F without input from any high-energy com- ization. Thus, the product of most reactions is a mixture of pounds implies either that aPc undergoes a rapid, transient mesobiliverdin and PCB adducts. PCB adduct can be ob- unfolding in solution or that CpcE/F proteins catalyze such tained as the exclusive product by preincubation ofequimolar an unfolding by using only the energy of binding. The rapid PCB and CpcE/F before addition of apo-aPc. This outcome rate of CpcE/F-mediated bilin removal also hints that is most likely due to strong binding ofPCB* by CpcE/F, with CpcE/F may have a role in the degradation of phycocyanin, a resultant shift ofthe equilibrium ofisomerization, but actual which occurs under certain conditions ofnutrient deprivation catalysis of PCB isomerization by CpcE/F cannot be ruled (29). out. The demonstration that CpcE and CpcF together specifi- The nature of PCB* is not known. PCB can undergo cally catalyze the addition of PCB to the correct cysteine rotational isomerization about its three methine bridges and residue of apo-apc represents a definitive identification of the 3-ethylidene double bond; the predominant isomer in catalytic proteins required for the attachment of a bilin solution is all-Z, syn at the bridges, but a minor fraction of a prosthetic group to an apophycobiliprotein. Cytochrome c "stretched" form with distinct absorbance and fluorescence heme Iyase (30) catalyzes a similar reaction but bears no spectra has been detected and attributed to E isomers at the significant sequence homology to CpcE or CpcF. central, C-10 bridge (21). E,Z,Z and Z,Z,E isomers (at the C-5, C-10, C-15 methine bridges) of related bilatrienes can be This research was supported in part by National Science Foun- formed that are stable at room temperature (22-24). The dation Grant DMB 8816727 (A.N.G.), National Institute of General isomer of PCB adduct found on holo-a'c has been deter- Medical Sciences Grants GM28994 (A.N.G.) and GM31625 mined to be Z-anti, Z-syn, Z-anti (25). Another candidate for (D.A.B.), and the Lucille P. Markey Charitable Trust (A.N.G.). a low abundance isomer of PCB is the 3(Z)-ethylidene form (26). 1. Glazer, A. N. (1989) J. Biol. Chem. 264, 1-4. The transfer of bilin (PCB) from the holo-apc of one 2. Beale, S. I. & Cornejo, J. (1991) J. Biol. Chem. 266, 22328-22332. organism to the apo-a'C of another is summarized below 3. Beale, S. I. & Cornejo, J. (1991) J. Biol. Chem. 266, 22333-22340. 4. Beale, S. I. & Cornejo, J. (1991) J. Biol. Chem. 266, 22341-22345. (heterologous transfer). 5. Beuhler, R. J., Pierce, R. C., Friedman, L. & Siegelman, H. W. (1976) J. Biol. Chem. 251, 2405-2411. apo-7120a 6. Troxler, R. F. & Bogorad, L. (1966) Plant Physiol. 41, 491-499. 7120a-PCB apo-7120a 7. Elich, T. D. & Lagarias, J. C. (1989) J. Biol. Chem. 264, 12902- +CpcE/F + -CpcE/F 12908. + CpcE/F.PCB _ ~ + -CpcE/F + pcE/F 8. Arciero, D. M., Bryant, D. A. & Glazer, A. N. (1988) J. Biol. apo-7002at + 7002a-PCB Chem. 263, 18343-18349. apo-7002a 9. Arciero, D. M., Dallas, J. L. & Glazer, A. N. (1988) J. Biol. Chem. 11 263, 18350-18357. CpcE/F + PCB + apo-a 10. Zhou, J., Gasparich, G. E., Stirewalt, V. L., de Lorimier, R. & Bryant, D. A. (1992) J. Biol. Chem., in press. The product of such a reaction is exclusively PCB adduct, 11. Swanson, R. V., Zhou, J., Leary, J. A., Williams, T., de Lorimier, presumably because CpcE/F proteins bind PCB tightly, so R., Bryant, D. A. & Glazer, A. N. (1992) J. Biol. Chem., in press. that free PCB is never present in sufficient quantity for a 12. de Lorimier, R., Bryant, D. A., Porter, R. D., Liu, W.-Y., Jay, E. & Stevens, S. E., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, significant nonenzymic reaction with apoprotein. The same 7946-7950. scheme applies to transfer of bilin from Synechococcus sp. 13. Russel, M. & Model, P. (1984) J. Bacteriol. 159, 1034-1039. PCC 7002 holo-apc to apo-a': of the same organism (homol- 14. Tabor, S. & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. USA ogous transfer). The rate of heterologous transfer must be 82, 1074-1078. limited by the slow removal of bilin from Anabaena sp. PCC 15. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. 7120 holo-aPC by CpcE/F; the rate of (1990) Methods Enzymol. 185, 60-89. CpcE/F-mediated 16. Marston, F. A. 0. (1986) Biochem. J. 240, 1-12. addition of PCB to 7120 apo-a'C must also be relatively slow, 17. Stevens, S. E., Jr. & Porter, R. D. (1980) Proc. Natl. Acad. Sci. though this rate was not directly measured. USA 77, 6052-6056. That the CpcE/F proteins are capable of catalyzing not 18. Glazer, A. N. (1988) Methods Enzymol. 167, 291-303. only the addition of PCB to apo-aPc but also the back 19. Swanson, R. V. & Glazer, A. N. (1990) Anal. Biochem. 188, 295- reaction, and at a comparable rate, is a surprising result. The 299. 20. Gray, B. H., Cosner, J. & Gantt, E. (1976) Photochem. Photobiol. PCB-protein adduct is stable at room temperature when the 24, 299-302. protein is denatured by urea or methanol, and stable adducts 21. Braslavsky, S. E., Schneider, D., Heihoff, K., Nonell, S., Aramen- at the 3' carbon of PCB form readily in an excess of various dia, P. F. & Schaffner, K. (1991) J. Am. Chem. Soc. 113,7322-7334. nucleophiles, including thiols (5, 27). The bilins of phycocy- 22. Falk, H. & Grubmayr, K. (1977) Angew. Chem. Int. Ed. Engl. 16, anin are held firmly in place in the native protein; this rigidity 470-471. is demonstrated by the high quantum yield of fluorescence of 23. Falk, H., Grubmayr, K., Haslinger, E., Schlederer, T. & Thirring, K. (1978) Monatsh. Chem. 109, 1451-1473. these proteins compared with the very low yields of fluores- 24. Kufer, W., Cmiel, E., Thummler, F., Rudiger, W., Schneider, S. & cence from free bilins or bilin-oligopeptides. The thioether Scheer, H. (1982) Photochem. Photobiol. 36, 603-607. linkage and rings A and B of the PCB prosthetic groups of 25. Schirmer, T., Bode, W. & Huber, R. (1987) J. Mol. Biol. 196, phycocyanin are buried in a hydrophobic pocket, though the 677-695. bilin on aPc is partly solvent-exposed in the af3 phycocyanin 26. Beale, S. I. & Cornejo, J. (1984) Plant Physiol. 76, 7-15. 27. Klein, G. & Rudiger, W. (1979) Z. Naturforsch. 34c, 192-195. monomer (25). 28. Swanson, R. V. & Glazer, A. N. (1990) J. Mol. Biol. 214, 787-7%. The melting temperature of phycocyanin from Synecho- 29. Grossman, A. R. (1990) Plant Cell Env. 13, 651-666. coccus sp. PCC 7002 has been reported to be 63.5°C (28). 30. Dumont, M. E., Ernst, J. F., Hampsey, D. M. & Sherman, F. Though the melting temperature of aPC has not been deter- (1987) EMBO J. 6, 235-241. Downloaded by guest on September 27, 2021