the journal of biological chemistry vol. 269, 46, issue of ... · zymatic addition of peb and...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 46, Issue of November 18, pp. 28988-28996, 1994 Printed in U.S.A.

Nonenzymatic Bilin Addition to the CY Subunit of an Apophycoerythrin*

(Received for publication, August 1, 1994, and in revised form, September 23, 1994)

Craig D. Fairchild$ and Alexander N. Glazer§ From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

C-Phycoerythrin is a light-harvesting protein whose a and p subunits carry thioether-linked phycoerythrobilin (PEB) at cysteine residues a-82, a-139, p-48,59 (doubly-linked), p-80, and p-165. The two subunits of Calothrix sp. PCC 7601 C-phycoerythrin, overexpressed together as apopolypeptides in Escherichia coli, formed inclusion bodies. Purified apo-a was soluble in the absence of urea, whereas the apo-P subunit was only soluble at high urea concentrations. Products of nonenzymatic addition of PEB and phycocyanobilin (PCB) to apo-cu were characterized by isolation of bilin peptides and spectroscopy. Reaction of PEB with the apo-a subunit led primarily to 15,16-dihydrobiliverdin (Cys-82) or urobilin (Cys-139) adducts, and small amounts of the natural PEB adducts at both Cys-82 and Cys-139. PCB reacted primarily with Cys-82 to form phycocyanobilin and mesobiliverdin adducts. Both PEB and PCB also formed relatively small amounts of adducts with Cys-59, which is not a bilin attachment residue in natural phycoerythrin. Sodium azide was found to promote the addition of PEB to simple thiols but not to apo-a phycoerythrin.

Phycobiliproteins are light-harvesting proteins that are a part of the photosynthetic apparatus of cyanobacteria, red al- gae, and the cryptomonads (1, 2). Four covalently attached isomeric bilins, phycocyanobilin (PCB),’ phycobiliviolin, phycoerythrobilin (PEB), and phycourobilin (PUB), are responsible for the visible absorption spectra of the phycobiliproteins. Their distinctive spectroscopic properties arise from the differing ar- rangements of their double bonds. PCB and phycobiliviolin have been found only singly-linked to cysteine residues at the 3’ carbon; PEB and PUB have been found both singly- and doubly-linked (at C-3’ and C-18’) to cysteine residues (see Fig. 1). In addition to PCB and PEB, cryptomonad phycobiliproteins contain several more oxidized bilins, not seen in cyanobacterial and red algal phycobiliproteins. Among these are bilins with acryloyl substituents on ring C (at (3-12) in place of the common

* This work was supported in part by National Institutes of General Medical Sciences Grant GM28994 (to A. N. G.). 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 ac- cordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: University of California BerkeleymSDA Plant Gene Expression Center, 800 Buchanan St., Albany, CA 94710.

MCB:Stanley/Donner ASU, 229 Stanley Hall #3206, University of Cali- § To whom correspondence and reprint requests should be addressed:

fornia, Berkeley, CA 94720-3206. The abbreviations used are: PCB, phycocyanobilin; a’‘, C-phycocya-

nin a subunit; aPE, pPE C-phycoerythrin a or p subunit; DBV, 15,16- dihydrobiliverdin; DTT, dithiothreitol; HPLC, high performance liquid chromatography; PEB, phycoerythrobilin; MBV, mesobiliverdin; UB, urobilin; PAGE, polyacrylamide gel electrophoresis; TPCK, L-l-tosyl- amido-2-phenylethyl chloromethyl ketone.

propionyl substituent, mesobiliverdin (MBV), and both singly (C-3’) and doubly-linked (at C-3’ and C-18“) 15,16-dihydrobiliverdin (DBV) (6, 8, 9).

The pathway of PEB and PCB biosynthesis has been eluci- dated in only one organism, the atypical, acidophilic red alga Cyanidium caldarium. Here, biliverdin formed from heme is converted to PEB, and then PEB is isomerized to PCB (10-12). PCB and PEB can be cleaved from phycobiliproteins under reflux in methanol and purified. This treatment, which results in the elimination of a phycobiliprotein cysteine residue from C-3’ of the bilin to yield an ethylidene substituent at C-3, provided the bilins for the original determination of the structures of PEB and PCB (13, 14).

Studies of the next step, the attachment of bilins to apophycobiliproteins, have been limited to extensive analysis of nonenzymatic and enzymatic addition of bilins to the C-phycocyanin of the cyanobacterium Synechococcus sp. PCC 7002. C-Phycocyanin carries PCBs a t a-84, p-82, and p-155. In vitro, PCB and PEB both react with apophycocyanin to form adducts at a-84 and p-82, but not at p-155. The major product of nonenzymatic PCB addition is MBV, and that of nonenzymatic PEB addition is DBV. Thus, in contrast to the adduct formed in vivo, the in vitro reaction in each case leads predominantly to more oxidized adducts with an extra double bond between C-2 and C-3 of ring A (15-17). In vivo, in Synechococcus sp. PCC 7002, interposon mutations in either the cpcE or cpcF gene lead to failure to attach PCB a t a-84 on phycocyanin (18). PCB addition a t seven other sites, including p-82 and p-155 on phycocyanin, is normal in these mutants (19). Subsequent in vitro studies of recombinant CpcE and CpcF showed that these proteins form a 1:l complex that functions as a PCB lyase specific for the a subunit of phycocyanin (20, 21).

From these results, it is tempting to speculate that all bilin addition to apophycobiliproteins might be enzymatic. In support of this notion, a number of genes with varying degrees of homology to cpcE and cpcF have been found in other cyanobacteria (discussed in Ref. 22). However, the question is far from being settled. In plants, addition of the natural bilin (phyto- chromobilin) or of PCB to apophytochrome leads to functional adducts without oxidation of ring A both i n vitro and in vivo, leading to the conclusion that the bilin C-S lyase activity is an intrinsic property of apophytochrome (23, 24).

In this study, we examine the i n vitro addition of bilins to apophycoerythrin. Whereas the only bilin found in the phycobiliproteins of Synechococcus sp. PCC 7002 is PCB, the situa- tion is more complex in cyanobacteria which produce the PEB- bearing phycoerythrins. These organisms invariably produce substantial amounts of PCB-bearing phycobiliproteins as well (25). A study of i n vitro bilin addition to apophycoerythrin is indispensable to the eventual characterization of any phycoerythrin bilin lyases that catalyze in vivo bilin addition to the apoprotein.

Phycoerythrin from Calothriz sp. PCC 7601 was chosen for four reasons: it was of interest to compare the reactions of a

28988

I

I1

Bilin Addition

x x x x x x c v s x x x x x x

i HOOC COOH S / \

A

H H H 10


to Apophycoerythrin 28989


HOOC COOH

IV O0 ;o H H H


V

x x x x x x c y s x x x x x x

HOOC COOH x x x x x x c y s x x x

VI O0 so H H H

PEB (4). III, doubly-linked PEB (5). N , singly-linked DBV (6). V, singly- FIG. 1. Bilin structures. I, singly-linked PCB (3). II , singly-linked

linked PUB (7). VI, doubly-linked PUB (7).

PEB-containing protein to those already characterized for a PCB-bearing protein, C-phycocyanin; the sequence of this phycoerythrin has been determined and its bilin attachment sites identified (26, 27); genes (orfu and or@) proposed to be in- volved in bilin addition to apophycoerythrin have been cloned from this species (28); and the genes for the apophycoerythrin subunits, a and p, have also been cloned (27). This phycoerythrin carries five PEBs, attached at a-82, a-139, p-48,59 (doubly-linked), p-80, and p-165 (26). The only other post-

translational modification is a y-N-methylasparagine residue at position 70 of the p subunit (29).

We describe the preparation of apophycoerythrin subunits overexpressed in Escherichia coli. The partially purified apo-P subunit (apo-PPE) required high urea concentrations for solubi- lization and could not be renatured in the presence or absence of apo-aPE or bilin. Thus this report focuses on the nonenzymatic bilin addition to the apo-a subunit. As seen for nonenzymatic bilin addition to apophycocyanin (16, 171, PEB addition to apo-aPE yielded predominantly bilin adducts other than the natural PEB. A major product was an urobilin adduct, which was not a product of PEB reaction with apophycocyanin.

EXPERIMENTAL PROCEDURES Bilins-Biliverdin dihydrochloride was obtained from Sigma. PCB

and PEB were obtained by refluxing phycobiliproteins in methanol and purified as described previously (15, 21). For desalting, bilin solutions were loaded onto C,, cartridges equilibrated with aqueous 10 mM trifluoroacetic acid, washed with the same solvent, and then eluted with 90% acetonitrile, 10% aqueous trifluoroacetic acid.

Expression and Purification of Apophycoerythrin Subunits- Apophycoerythrin was overexpressed in E. coli strain K38 (30) bearing the plasmid pGP1-2 (31). This strain was transformed with plasmid pPM90, which consists of the vector pTZ18R (Pharmacia Biotech Inc.) and the 7.3-kilobase EcoRI fragment from pPM40 (27), kindly provided by Dr. Nicole Tandeau de Marsac of the Institut Pasteur. The 7.3- kilobase fragment contains the cpeB and cpeA genes, encoding the p and a subunits of Calothrix sp. PCC 7601 phycoerythrin; in pPM9O these genes are 3' t o the lac2 and T7 promoters of pTZ18R.

The K38/pGP1-2/pPM90 cells were grown in rich medium (2% tryp- tone, 1% yeast extract, 0.5% NaCl, 0.2% glycerol, 50 mM mi, pH 7.2) containing 50 pg/ml kanamycin and ampicillin, in either 1-liter Fern- bach flasks or a 10-liter fermenter, with vigorous agitation at 30 "C to an absorbance a t 600 nm of 1.5-3.0. The culture was then abruptly shifted to 4 2 4 3 "C by addition of one-fourth volume of medium a t 90-100 "C. After 25 min at this temperature, rifampicin from a 20 mg/ml solution in methanol was added to a final concentration of 100 pg/ml. After 5-10 min more, the culture was cooled to 37 "C, and agi- tated for 2 h. Cells were then harvested by centrifugation and stored a t -20 "C.

In a typical protein preparation, frozen cells (15.7 g wet weight) were suspended using a manual homogenizer in 45 ml of lysis buffer (50 mM Nap,, 2 mM EDTA, pH 7). The cell suspension was passed through a French pressure cell twice at 12,000 psi, and then centrifuged for 15 min at 27,000 x g. The supernatant was removed, the pellet thoroughly suspended in lysis buffer containing 1% (w/v) Triton X-100, and the suspension then centrifuged for 10 min a t 12,000 x g . The supernatant was discarded, and the pellet suspended in 40 ml of 9 M urea-HC1,lO mM DTT, pH 2.5. The suspension was brought to pH 8 with concentrated ammonium hydroxide; after 30 min at room temperature, the suspension was re-acidified with concentrated HC1 and centrifuged for 10 min at 27,000 x g. The supernatant was dialyzed against 500 ml of 3 M

urea-HC1, 10 mM 2-mercaptoethanol, pH 2.5, overnight a t 4 "C. The dialyzed, apophycoerythrin-containing 3 M urea solution was

clarified by ultracentrifugation (30 min, 100,000 x g ) and passage through a 0.22-pm filter. It was then split into two 24-ml aliquots for Bio-Gel PlOO (Bio-Rad) gel filtration chromatography (75 x 5-cm column) in 3 M urea-HC1, 10 mM 2-mercaptoethanol, pH 2.5. Fractions (15 ml) were collected and assayed for apophycoerythrin by SDS-PAGE.

Preparation of Holo-crPE"Calothrix sp. PCC 7601 (32) was grown in continuous green light, in 11-liter carboys of BG-11 medium (33), with continuous bubbling of 5% CO,, 95% N,. Cells were harvested by centrifugation, washed once with 50 mM Napi, 1 mM NaN,, pH 7, and stored at -20 "C.

The preparation of apE was essentially by the protocol of Sidler et al. (26) with minor modifications. In the lysis buffer, 5 mM EDTA was included to inhibit proteolysis of the phycoerythrin, and 0.1% Triton X-100 was used in place of 0.02% benzalkonium chloride. The purified aPE in 7 M acid urea was diluted 1:l with water (aPE concentration 0.12 mg/ml), and renatured by dialysis against 50 mM Nap,, 50 mM ammonium acetate, 2 mM EDTA, 1 mM NaN,, pH 7, at 4 "C. The dialysate was clarified by centrifugation (30 min, 25,000 x g ) , and stored a t 4 "C.

Spectroscopy-Absorption spectra were measured with a Perkin- Elmer A6 spectrophotometer using quartz cuvettes of 1-cm pathlength with the appropriate solution as a reference. The concentration of uro-

28990 Bilin Addition to Apophycoerythrin

bilin (UBI adducts was calculated with the extinction coefficient for phycourobilin peptide in acid 8 M urea of = 94,000 M" cm" (34). For PEB peptides, the t550 = 53,700 M - ~ cm" was used (34), and for DBV peptides, = 35,000 M" cm" (6).

Fluorescence spectra were recorded with a Perkin-Elmer MPF-44B fluorescence spectrophotometer, with 10 x 10-mm cuvettes. Excitation and emission slits were 4 nm; uncorrected emission spectra were taken in the energy mode. The peak absorbance of all protein solutions used was less than 0.08.

Renaturation ofApo-o?E and Apo-pPE-Apo-aPE concentration in pool 111 was calculated using the extinction coefficient calculated from the amino acid sequence (E''' = 17.9 mM-I cm", A::: = 0.96). The concentration in pools I and I1 was estimated by comparison to Coomassie- stained standard proteins in polyacrylamide gels.

Attempts to renature the apophycoerythrin subunits were performed by dialysis of the stock solutions in acid urea against various buffers. Aliquots of pools I, 11, or I11 from gel filtration in 3 M acid urea, 10 mM 2-mercaptoethanol were diluted with the same solvent to 0.1-0.2 mg/ml apophycoerythrin subunit. The dialysis buffers were: 1 mM ammonium acetate, pH 7,50 mM Nap,, 0.1 mM NaN,, pH 7, or, 100 mM Tris-HC1, pH 8.5, containing 0.1 mM D m in each case. The protein solutions in 3 M acid urea were neutralized with ammonium acetate prior to dialysis. In one additional experiment, with the 1 mM ammonium acetate dialysis buffer, the starting protein solution was in neutral 8 M urea. The dialysis was at 4 "C, overnight. The dialysates were centrifuged, and the pellets were washed once with dialysis buffer. The pellets were suspended in a small volume of 8 M acid urea. Equal fractions of each supernatant and pellet were analyzed by SDS-PAGE (14% resolving gel). Protein concentrations were estimated by comparison to Coo- massie-stained standards.

Renatured apo-aPE for bilin addition experiments was prepared by the same protocol, with 50 mM Napi, 1 mM DTT, 0.1 mM NaN,, pH 7, as the dialysis buffer.

BiZin Addition Reactions with Apophycoerythrin-For all addition reactions the protein was pre-reduced with 5 m~ DTT a t pH 7, for at least 30 min at room temperature. DTT was removed from the pre- reduced protein by passage through a Sephadex G-25 (Pharmacia) column in the appropriate solvent. For reactions of bilin with apo-aPE dissolved in buffer, bilin in Me,SO (final Me,SO < 2%) was mixed with protein, the tube was flushed with N,, and sealed with Parafilm.

For reactions with apophycoerythrin starting in acid urea (1 or 3 M), protein was mixed with bilin and then diluted with neutral buffer; where necessary the pH was adjusted to 7 with ammonium hydroxide. These reaction mixtures were also flushed with N, and sealed. All incubations were a t room temperature in the dark. Addition reactions were terminated by acidification or by passage through a Sephadex G-25 column to remove unreacted bilin.

Isolation of Dyptic Chromopeptides for Amino Acid Analysis-Large- scale preparation of tryptic chromopeptides from aPE, and from the products of addition of PCB or PEB to apo-aPE (from reactions in 1 M urea, 0.1 mM NaN,) was performed for the spectroscopic characterization of the peptides, and for amino acid analysis to identify the cysteine residues derivatized by bilin addition. Each protein sample, in a volume of 75-100 ml, with concentration of about 0.1 mg/ml, was dialyzed against 2 mM Nap,, 1 mM NaN,, pH 7. The pH was adjusted to 2 with 1 N HCl, and the protein solution was concentrated approximately 6-fold by ultrafiltration with a 10-kDa cut-off membrane (PM10, Amicon).

Digestion with TPCK-trypsin (Worthington), and initial purification of the tryptic chromopeptides by Sephadex G-50 and SP-Sephadex C-25 (Pharmacia) chromatography, were performed as described previously (15). Fractions from the SP-Sephadex C-25 columns were collected and pooled as follows: aPE, two 560-nm absorbing peaks, not cleanly sepa- ra ted apo-aPE PEB addition product, three pools based on bilin content, I = PEB (early, minor peak), I1 = PEB, and UB, 111 = DBV; apo-aPE PCB addition product, six overlapping peaks of 640 nm absorbance, I-VI in order of elution.

The peaks from ion-exchange chromatography were further fraction- ated by reverse-phase C,, HPLC, on a Supelco LC-I8 column (250 X 10 mm). HPLC was performed on a Waters 600E system with a Waters 991 photodiode array detector. The aqueous solvent was 0.1 M Napi, pH 2.1; the organic solvent was acetonitrile. The PEB addition product and aPE peptides were eluted with the following gradient: 20-25% acetonitrile in 5 min, 2530% in 25 min, and 30-80% in 5 min. PCB addition products were eluted with a different gradient: 20-35% acetonitrile in 5 min, convex gradient; 3540% in 20 min, linear gradient; 40-80% in 5 min, concave gradient.

HPLC-purified chromopeptides were desalted on C,, cartridges, and acetonitrile was evaporated under a stream of N,. For desalting,

samples were loaded onto C,, cartridges equilibrated with aqueous 10 mM trifluoroacetic acid, washed with the same solvent, and then eluted with 60% acetonitrile, 40% aqueous trifluoroacetic acid. The absorption spectra of the chromopeptides were taken in 10 mM trifluoroacetic acid. Analytical reverse-phase C,, HPLC was performed with a fraction of each peptide to confirm purity. Chromopeptides which were purified in sufficient quantity were submitted to the Protein Structure Laboratory at the University of California, Davis, for amino acid analysis.

Comparative Dyptic Chromopeptide Mapping-The products of 14-h PCB or PEB addition to apo-aPE (0.1 mg/ml, 2 ml) in 50 mM Nap,, pH 7, with 0.15 or 1 M urea, were centrifuged, and protein in the supernatant separated from unreacted bilin by passage through Sephadex G-25 in 50 mM Nap,, 2 mM EDTA, pH 7. After gel filtration, the absorption spectra of the pooled protein fractions were recorded. The protein was then precipitated by addition of 0.25 volume of 50% trichloroacetic acid (w/v), followed by a 2-h incubation on ice, and the trichloroacetic acid pellets were suspended in 100 p1 of 9 M urea-HC1, pH 2.5.

TPCK-trypsin 1125 pg in 25 pl) was added to each of these 100-pl protein solutions (and to a 100-pl aliquot of holo-cyPE in acid urea), and then each was mixed with 800 pl of 35 mM Tris base to give a final pH of 7.5-8. These mixtures were incubated a t 30 "C for 2 h, with addition of another 125 pg of trypsin to each after 1 h. Digestions were quenched by addition of 2.1 ml of concentrated HC1 to each, and the mixtures kept at -20 "C overnight.

Tryptic peptides were desalted on C,, cartridges, and acetonitrile was then removed by centrifugation under vacuum, without heating. These samples were then analyzed by HPLC on a Hi-Pore RP-318 (Bio-Rad, 250 x 4.6 mm) C,, column with a gradient of IO-30% acetonitrile over 30 min, followed by 3045% over 10 min.

Gel Electrophoresis-SDS-PAGE was with the buffer system of Laem- mli (351, an acrylamide monomer:bis ratio of 37.5:1, and 5% acrylamide stacking, 14% resolving gels. Proteins precipitated with trichloroacetic acid were suspended in a pH 8 sample buffer; other samples were mixed with a pH 6.8 sample buffer.

RESULTS

Apophycoerythrin Expression and Purification--Two polypeptides of the appropriate apparent molecular masses for apophycoerythrin subunits appear after heat shock of E. coli K38/pGP1-2/pPM90, and are more abundant 2 h after addition of rifampicin to 100 pg/ml. These polypeptides are not present in cells of the control strain treated similarly. These polypeptides behave as if they are in inclusion bodies: they are insoluble until suspended in acid urea (Fig. 2, lanes 2-7).

The composition of large-scale preparations of apophycoerythrin inclusion bodies dissolved in acid urea was similar to that shown in Fig. 2, lane 7. For purification, the acid urea solution of crude apophycoerythrin was dialyzed to 3 M acid urea and applied to a Bio-Gel PlOO column in the same solvent. This resulted not only in separation of most of the contaminating proteins from the apophycoerythrin subunits, but also in some resolution of the subunits. Some apophycoerythrin, mainly apo-P subunit, and most of the contaminating polypeptides eluted early, near the void volume. In later-eluting fractions, there were partly overlapping peaks of apo-PPE and apo- aPE. These later-eluting fractions were combined into three pools, in order of elution: I, apo-PPE; 11, approximately equimo- lar apo-PPE and apo-aPE; 111, apo-aPE (Fig. 2B).

Renaturation ofApophycoerythrin Subunits-Attempts were made to renature the proteins in these three pools by neutral- ization and removal of urea by dialysis. The results of dialysis against low and moderate ionic strength pH 7 buffers, or against 100 mM Tris-HC1, pH 8, were the same: apo-PPE in pool I or pool 11 precipitated almost completely (although the higher molecular mass contaminant remained in solution); some apo- aPE, whether from pool I1 or pool 111, remained in solution. The highest concentration of apo-aPE obtained on renaturation was about 0.1 mg/ml. Apo-aPE precipitated when concentrated by ultrafiltration.

Bilin Addition to Renatured Apo-dE-1ncubation of the renatured apo-aPE with biliverdin, PCB, or PEB resulted in the formation of bilin-protein complexes which were stable to Seph-

Bilin Addition to Apophycoerythrin 28991

*

c P + a-

FIG. 2. Overexpression of apophycoerythrin in E. coli and partial purification of subunits. Coomassie-stained polyacrylamide gels. Panel A, samples from overexpression of apo-PE in E. coli K38/ pGP1-2/pPM9O, compared to similarly treated K38/pGP1-2 lacking pPM9O. Lane 1, apophycocyanin size standard: upper band, apo-P subunit, 18.3 kDa; lower band, apo-cy subunit, 17.6 kDa. Lanes 2-7, trichloroacetic acid-precipitated samples from small-scale inclusion body preparations; the first lane of each pair is from cells lacking pPM90, the second from cells containing pPM9O: 2 and 3, supernatants from lysis; 4 and 5, 1% Triton X-100 wash of the pellets from lysis; 6 and 7, supernatants from acid urea suspension of the pellets after the deter- gent wash. The calculated molecular masses of apo-PE subunits are: apo-cy, 17.2 kDa; apo-P, 19.2 kDa (26). Panel B, samples of the three apo-PE pools from Bio-Gel PlOO chromatography in 3 M acid urea, pools I, 11, and 111, numbered in order of elution.

adex G-25 chromatography. The spectrum of the biliverdin complex is similar to that of the corresponding complex formed with the apo-a subunit of phycocyanin (not shown), with the long wavelength absorbance peak lower relative to that in the near UV and a shoulder a t 450 nm (Fig. 3B). The spectra of the products of reaction of PCB with apo-aPE are very different from those of the reaction of PCB with apo-a". Whereas the spectra of C-phycocyanin, the MBV adduct of apophycocyanin, and the MBV adduct of apo-a phycocyanin (15, 20) all have a red:near UV peak absorbance ratio greater than 2.4, the products of PCB addition to apo-aPE have a red:near UV peak absorbance ratio of ~ 0 . 7 .

In vitro reaction of PEB with apo-cup" produces a complex mixture of adducts. The spectrum of the apo-aPE-bilin adducts (after removal of free bilin) has a major peak at 490 nm diagnostic of the presence of a significant amount of a urobilin (or urobilins) (Fig. 3A). The two peaks of similar height centered around 550 nm, similar to those seen with holo-a'", indicate the presence of PEB adducts (Fig. 3A). The peaks at 606 and 330 nm correspond to those seen in a DBV adduct formed in the i n vitro addition of PEB to apo-aPc (16).

The following observations provide additional support for the assignment of the 606 and 330 nm peaks to a DBV adduct. In both native and denatured phycobiliproteins, the 330-nm peak is diagnostic of polypeptide-bound DBV (6) and that at 308 nm is diagnostic of polypeptide-bound PEB (see Fig. 4 in Ref. 36). The ratio of peak heights, 330:308 nm, in the acid urea spectra (Fig. 4B) correlates with the ratio of the 606-nm peak to the 550-nm peaks in the native spectra (Fig. 4A).

Experiments were performed to assess the effect of the formation of bilin adducts on the renaturation of the phycoerythrin subunits. Aliquots of Bio-Gel PlOO pools I, 11, and I11 were dialyzed against 1 M acid urea, then mixed with bilin (biliverdin, PCB, or PEB), and diluted 1:7 with 100 mM ammonium acetate. The pH of each mixture was then adjusted to 7 with 1 M ammonium hydroxide. The reaction mixtures were incubated a t room temperature for 24 h. They were then cen-

0.04

0.03

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a

e B o 8 0.06

0.05

0.04

0.03

0.02

0.01

0 250 350 450 550 650 750

a. (nm) 0

apo-aPE. Products of incubation of 5 PM bilin with 0.09 mgml renatured FIG. 3. Absorbance spectra of bilin complexes with renatured

apo-cy"" in 50 mM Nap,, 1 mM NaN,, pH 7, for 24 h. The mixtures were clarified by centrifugation and bilin-protein complexes were separated from free bilin by Sephadex G-25 chromatography in the same buffer. The relative absorbance spectra of holo-cy"" and biliverdin in this buffer are provided for comparison. Panel A, solid line, product of apo-crl'E + PEB; broken line, holo-cyPE. Panel B, thick solid line, product of apo-a'" + PCB; thin solid line, product of apo-a'" + biliverdin; broken line, biliverdin.

trifuged, and the pellets and supernatants analyzed by SDS- PAGE. With respect to the recovery of soluble protein, the results were similar to the outcomes of renaturation trials on these pools in the absence of bilin.

The covalent addition products were examined by SDS- PAGE. The mobility of holophycobiliproteins on SDS-PAGE is less than that of the corresponding apoproteins, and the difference in apparent molecular mass roughly reflects the difference in the calculated mass due to the bilin content (observed for apo- versus holophycoerythrin subunits (data not shown); see Ref. 37 for apo-PC versus PC).

The mobility of the apo-aPE band from the supernatants of renaturation mixtures with PCB or PEB was slightly less than that of untreated apo-a'", or that from renaturation in the presence of biliverdin. There was no detectable ap0-P'" in the supernatants of pool I or pool I1 renaturations.

The shift in mobility for apo-aPE incubated with PCB or PEB was examined further using SDS-PAGE with zinc staining, a sensitive fluorescence method for visualizing polypeptides bearing tetrapyrroles (38). PEB/apo-a'" mixture yielded a poorly resolved doublet of fluorescent bands, one with a mobility similar to that of holo-apE, and one with a mobility inter- mediate between that of apo-aPE and holo-a'". The PCB/apo-aPE mixture yielded a similar pattern. The two bands were not well resolved, but they do provide clear evidence for covalent addition of PCB and PEB to apo-a'" and the formation of adducts


0.016

0.012

0.008

0.004

0 250 350 450 550 650 1 I

FIG. 4. Effect of urea on PEB addition to apo-gE. Apo-aPE (0.06 mg/ml) and 12 VM PEB were incubated in a volume of 4 ml at the concentration of urea indicated, in 50 mM Nap,, 50 mM ammonium acetate, 0.1 mM NaN,, pH 7. To start the reaction, pre-reduced apo-aPE in 1 M acid urea was diluted 1 : l O into the appropriate solution containing PEB. The spectra in each panel have been adjusted to the same 280-nm absorbance. Panel A, native absorbance spectra. After a 19-h reaction, 0.8-ml samples were centrifuged and the supernatants passed through Sephadex G-25 columns in 50 mM Nap,, 0.1 mM NaN,, pH 7. Panel B, absorbance spectra in acid urea. Samples (1.1 ml) were centrifuged, and the supernatant acidified with concentrated HCI and passed through Sephadex G-25 columns in 3 M urea-HC1, pH 2.5. Thick solid line, 0.1 M urea, 18-h reaction; thin solid line, 0.1 M urea, I-h reaction; broken line, I M urea, 18-h reaction.

bearing either one or two bilin per aPE. Biliverdin.apo-aPE complex did not yield fluorescent bands

diagnostic of bilin-zinc complexes on SDS-PAGE. Similarly, the complexes of apophycocyanin with biliverdin were unstable to electrophoresis,’ or tryptic digestion with subsequent acidification (16).

Effect of Urea on PEB Addition to Apo-dE-The effect of urea on PEB addition to apo-aPE was examined by carrying out reactions in 0.1, 1, 5, or 8 M urea. Substantial bilin adduct formation took place in 0.1 or 1 M urea, but little or no bilin was found attached to protein after 19 h of reaction in 5 or 8 M urea (Fig. 4A).

More extensive addition of bilin to apo-aPE took place in 0.1 than in 1 M urea. This was evident from the observation that addition in 0.1 M urea led to products with a higher ratio of visible to 280-nm absorbance than did addition in 1 M urea (Fig. 4A). However, the 280-nm total absorbance recovered in the supernatant fraction after centrifugation of the 0.1 M urea reaction mixture was about 40% less than that in the supernatant from the reaction mixture in l M urea. These results show that more protein is lost to precipitation during reaction at the low urea concentration.

As noted above, the absorption spectrum of the products of the reaction of PEB with renatured apo-aPE has peaks characteristic of three types of polypeptide-bound bilins: DBV, PEB, and UB.

C. D. Fairchild, unpublished observation. . ~~~ ~ ~

The dependence of the adduct absorption spectrum on the reaction time shows that the appearance of urobilin adducts trails that of the PEB and DBV adducts both in 0.1 and 1 M urea. This is best seen in the acid urea spectra of 1- and 18-h samples from a reaction in 0.1 M urea (Fig. 4B). The intensity of the 495-nm peak (UBI is greater, relative to the 563-nm peak (DBV and PEB), in the 18-h sample than in the 1-h sample. UB adducts represent a higher fraction of the total bilin adducts for the reaction performed in 0.1 M urea than for that performed in 1 M urea (Figs. 4, A and B ) . Parenthetically, UB adducts represent a similar fraction of total adducts for reactions carried out in the absence of urea (Fig. 3 A ) and in the presence of 0.1 M urea (Fig. 4A).

The ratio of DBV to PEB adducts also depends on the urea concentration at which the addition of PEB to apo-aPE is carried out. This is revealed by the ratio of absorbances in acid urea at 330 and 308 nm, diagnostic of DBV and PEB adducts, respec- tively. Relatively, less DBV adduct is formed in 0.1 M urea than in 1 M urea.

Fluorescence Emission of dE and Apo-dE Bilin Adducts- The holo-aPE subunit carries two PEBs. As shown previously for aPE from Pseudanabaena and Nostoc species (39, 401, the fluorescence emission spectrum of aPE has a single peak, despite a double-peaked excitation spectrum (similar to the absorbance spectrum). This result implies that one of the two PEB chromophores on aPE transfers energy efficiently to the other, which fluoresces. Since the aPE from Pseudanabaena was monomeric by gel filtration and sedimentation velocity, the differences in the absorption spectra of the two PEB chromophores must be attributed to their different protein environments (39).

The excitation and emission peaks of aPE and the apo-aPE addition products are listed in Table I. The fluorescence emission peaks of the products of PEB addition reflect the complex- ity of the absorbance spectrum, except that the 540-550-nm excitation (and absorbance) peak, as in apE, has no corresponding emission peak (Table I). There is some energy transfer from UB to the other chromophores, as evidenced by the 490-nm excitation peak for 620-nm emission, but there is also a clear 501-nm emission maximum, indicating that this transfer is not complete. This observation suggests that a fraction of the aPE adducts carries PUB as the sole bilin chromophore.

Effect of Urea on the Fluorescence Emission of dE and Apo-dE Bilin Adducts-The relative stability of apE and of the various bilin adducts formed with the apo-a subunit in uitro was further characterized by exploiting their fluorescence emission properties. With increasing urea concentrations, the fluorescence emission of cypE, apo-aPE, and apo-aPE bilin addition products is quenched. A two-state urea denaturation function was fit to these data in order to obtain a C,, the concentration of urea at the midpoint of the transition, for each set, and these are listed in Table 11.

The intrinsic fluorescence of apo-aPE appears to be already in transition in the absence of urea, and this transition is complete at 0.4 M urea (42). This suggests that the apo-aPE is partially unfolded even under “native” conditions. The two natural PEBs on native aPE stabilize the subunit and shiR the C, to greater than 5 M urea. The various bilins present in the products of PEB or PCB addition reactions with apo-aPE also stabilize the protein, to various degrees. The order of the midpoints (with the bilin adduct presumed to be responsible for the emission), is: Ci78 (PEB) > Ckl’ (DBV), C r l (UBI >> Cg03690 (PCB, MBV).

TFyptic Chromopeptides of the Apo-dE PCB and PEB Addi- tion Products--Trypsin digestion of the bilin addition products of apo-aPE yielded a number of chromopeptides; the major chromopeptides were purified and their amino acid content deter-


TABLE I Absorbance, fluorescence emission, and excitation maxima of aPE and the products of PEB or PCB addition to apo-aPE

Bilin addition reactions were performed in 1 M urea, 50 mM Nap,, 0.1 mM NaN,, pH 7. Spectra were taken in 50 mM Nap,, 1 mM NaN,, pH 7.

mPE Products of PEB Products of PCB addition addition

Absorbance maxima

Emission (visible light)

Excitation A Emission maximum

Emission A Excitation maximum

Excitation

544,566"

530 573

590 542, 562

492, 550, 572, 606

470 501, 578, 610

620 490, 540sh, 570, 602

560sh, 604, 670

580 597,623, 688

700 590, 673

Wavelengths are in nanometers. Suffix "sh, shoulder.

TABLE I1 Urea denaturation ofapo-aPE, holo-aPE, and the products ofPEB or

PCB addition to apo-aPE, monitored by fluorescence emission Protein solutions (2 ml) were dispensed into tared 10 x 10-mm

cuvettes by weighing. The starting protein concentrations were between 0.05 and 0.12 mg/ml. Each solution contained 50 mM Nap,, 50 mM ammonium acetate, 0.1-0.5 mM NaN,, pH 7; the holo-aPE solution also contained 0.2 mM EDTA. The absorbance of each solution at its visible absorption maximum was less than 0.08. Urea concentration was in- creased in steps by addition of 8 M urea-HCI, pH 7, with mixing of capped tubes by inversion. After 10 min, the emission value (or, for the PEB and PCB addition products, values) was recorded for each step. Each value was then corrected for the change in volume. Except for apo-aPE (see text), the data for each protein were fit to a two-state

(Robelko Software, Michael L. Johnson, Absoft Corp.; described in Ref. denaturation function using the program "NonLin for Macintosh

41). Six variables, the linear constants for the upper (folded) and lower (unfolded) base lines, and the relation between free energy and [urea] in the transition zone, are fit simultaneously. The value for AGO, the apparent free energy of unfolding in absence of urea, and m, the slope of the line describing the transition zone, were used to calculate C,, the concentration of urea at the transition midpoint.

Protein Excitation A Emission A c m

nm M urea Apo-aPE 280 340 Holo-aPE

- O? 530 573

PEB addition product 470 5.4

50 1 PEB addition product

4.4 530 573

PEB addition product 4.7

530 612 4.5 PCB addition product 580 630 1.9 PCB addition product 580 690 1.8

mined. The amino acid composition of each chromopeptide, together with the amino acid sequence, allowed assignment of the position of the bilin-bearing cysteine residues. The results of this analysis are presented in Table 111.

The absorbance spectra of the chromopeptides in 10 mM trifluoroacetic acid were used to identify the bilin adduct; the criteria used for chromopeptides formed in the reaction of apo- aPE with PEB were: UB, single visible peak at about 490 nm (Ref. 32; Fig. 5 ) ; DBV, peaks at 562 and 330 nm, a shoulder at about 690 nm, and a 562:330 ratio of less than 1.5 (to distin- guish it from free PEB, which also has a peak at 330 nm, but has a higher red :W peak ratio); and PEB, peaks at 308 and 550-560 nm, without a 590-nm shoulder (6). The criteria for PCB-derived chromopeptides were: MBV, 355-365 and 680- 690 nm peaks, near W r e d ratio > 2; PCB, 345-352 and 650- 660 nm peaks, near W.red ratio < 1.5 (15).

The yields roughly reflect the relative amounts of the chromopeptides prior to fractionation. One additional chromopeptide from the PEB addition product was purified but not analyzed. It eluted at a lower percent organic solvent than the major DBV peptide on preparative HPLC, and had an absorbance peak in the elution solvent at 520 nm. With time, 90% of this chromopeptide converted to a species of HPLC elution time and absorbance spectrum identical to the major DBV peptide. A similar phenomenon was observed by Wedemayer et al. (6) for

TABLE I11 Amino acid analysis of major chromopeptides formed on PCB or PEB

addition to apo-aPE Tryptic chromopeptides were isolated as described under "Experi-

mental Procedures." The elution times are from a Supelco LC-18 (250 x 4.6 mm) column with different gradients for chromopeptides from aPE and apo-aPE + PEB (20-30% acetonitrile, 20 min) and for those from apo-aPE + PCB (20-40% acetonitrile, 20 min). For each chromopeptide, the nature of the bilin and yield were determined from the absorbance spectrum, and the attachment site was inferred from the amino acid composition with reference to the published sequence of aPE (26, 27).

Protein Elution time Attachment Bilin Yield site

min Holo-aPE 4.9 Cys-82 Holo-aPE 5.7 cys-139

Apo-aPE + PEB 3.6 cys-139 Apo-aPE + PEB 4.3 Apo-aPE + PEB 4.7 Apo-aPE + PEB 5.1 Apo-aPE + PEB 6.0 Apo-aPE + PEB Late" cys-59

Apo-aPE + PCB 5.6 Apo-aPE + PCB 6.6 Apo-aPE + PCB 8.5 Apo-aPE + PCB 16.5 Apo-aPE + PCB

cys-59 18.1 cys-59

cys-139 CYS-82 CYS-82 cys-139

CYS-82 CYS-82 CYS-82

" Eluted at greater than 30% acetonitrile.

PEB PEB

UB UB DBV PEB PEB PEB

PCB MBV MBV PCB MBV

nmol 6.5

14.5

7.1 1.1

1.6 1.6 0.8 1.7

1.1 6.0 1.5

26

45

3

FIG. 5. Comparison of urobilin absorbance spectra. Spectra of urobilin-bearing peptides, purified by HPLC, taken on-line with a mul- tiple diode array detector and adjusted to the same peak absorbance. Solid line, major UB peptide from the product of PEB addition reaction with apo-a" (Table 111,3.6-min peak). The solvent is -80% 0.1 M Nap,, pH 2.1 (aq), 20% acetonitrile. Broken line, the a subunit of the major phycoerythrin of Synechocystis sp. WH8501 in 0.1% trifluoroacetic acid with -55% 2:l (v/v) acetonitri1e:isopropyl alcohol (from Ref. 43). This subunit bears two singly-linked PUB chromophores.

a DBV peptide from a cryptomonad phycobiliprotein, and the conversion from the 520-nm form to the 560-nm form was shown to be driven by light.


0.25 I I

' 0.10 - p :: ::

e 5 0.05 j, ~ <

:: ' .:;- A . -

i i i : I

o ' ~ 5 " " " " " " " " " " 20 25 30 35 Time (min)

n

20 25 30 35 Time (min)

0 " " " " ~ " " " " " "

FIG. 6. Products of PCB and PEB addition to apo-gE: tryptic

are described under "Experimental Procedures." The identities of the chromopeptide maps. Addition reactions and preparation of products

bilins indicated on the chromatograms were inferred from on-line absorbance spectra. Panel A, products of PEB addition. Tryptic chromopeptides from: top chromatogram, reaction in 1 M urea; middle chromatogram, reaction in 0.15 M urea; bottom chromatogram, holo-cyPE. Panel B, products of PCB addition. Tryptic chromopeptides from: top chromatogram, reaction in 1 M urea; bottom chromatogram, reaction in 0.15 M urea. Insets, absorbance spectra of native reaction products in 1 M (solid lines) and 0.15 M (broken lines) urea.

HPLC separations of tryptic digests of the products of PEB and PCB reactions with apo-a", in 0.15 or 1 M urea, along with that of holo-aPE for comparison, are shown in Fig. 6. The absorbance spectra of the products prior to trypsin digestion are also shown (Fig. 6, insets).

In the chromatograms from apo-a"/PEB reaction products there is an unresolved shoulder of PEB adduct (18.9 min) eluting at the front edge of the main DBV adduct peak (19.07 min); this shoulder has the same elution position as the early-eluting holo-aPE chromopeptide, that corresponding to Cys-82 (Fig. 6A). There is another PEB adduct peak (20.35 min) in the chromatogram of the PEB addition product which elutes in the same position as the other holo-aPE chromopeptide. The nature of the other PEB peptide peaks is not known; they might differ in the geometric isomer of the bilin, although their on-line absorbance spectra differ only slightly (42). A similar multiplicity of PCB and MBV peptide HPLC peaks for a bilin peptide with a unique amino acid sequence has been observed previously for peptides obtained from in vitro apophycocyanin-bilin adducts (15).

The late-eluting chromopeptides from the products of PEB (32-36 min) and PCB (34-38 min) addition represent bilin adducts at Cys-59. Spectroscopy showed that both DBV and PEB Cys-59 bilin peptides resulted from PEB addition (Fig.

6-41, although only the PEB peptide was purified and analyzed (Table 111).

The peak labeled "DBV?" (Fig. 6 A ) is the 520-nm species discussed above. The peak labeled "PCB?" (Fig. 6B) appears to be PCB adduct, but has absorbance peaks that are blue-shifted by 10 nm and a higher ratio of red:UV absorbance.

The primary difference between the chromatograms of products from reactions performed in 0.15 and 1 M urea is the total yield of chromopeptides. From the same starting amount of apo-cuPE, reaction in 0.15 M urea yields a lower amount of chromopeptides than reaction in 1 M urea, mainly due to greater precipitation of protein in the low urea reaction mixture. As noted above, the PEB addition products from reaction at the lower urea concentration have higher relative amounts of UB. The difference is due mainly to an increase in amount of the earlier-eluting UB peptide (Fig. 6-4). There is no obvious difference in the relative amounts of bilin peptides resulting from PCB addition in 0.15 or 1 M urea.

Addition of Small Thiol Compounds to PEB in the Presence of Sodium Azide-In bilin addition reactions with apo-aPE, the presence of a thiol such as DTT resulted in spectroscopic changes in the visible region which are additional to those attributable to the formation of polypeptide-bilin adducts. This phenomenon was explored further: 2 mM DTT or 5 mM 2-mercaptoethanol were incubated for 8.5 h under the conditions of the addition reaction, and the products analyzed by reverse- phase HPLC. The predominant products had a urobilin spectrum, with very minor components with spectra resembling those of DBV and PEB adducts. Little or no unreacted PEB remained after incubation with either thiol. The products of PEB reaction with each thiol gave a distinct elution pattern, and these patterns were dissimilar to that of PEB similarly incubated without thiol (see Ref. 42 for a full description).

These results differ from those of Arciero et al. (151, who reported no reaction of simple thiols with PEB in neutral Napi. This difference was found to arise from the presence of 0.1 mM sodium azide (used as an antimicrobial agent) in our addition reactions. Thus, sodium azide, or a contaminant thereof, is the agent promoting bilin adduct formation with simple thiols. It is important to note that azide had no qualitative effect on bilin addition to apo-aPE. Reaction mixtures identical to those used for the tryptic chromopeptide maps in Fig. 6 (which did not contain azide) were performed with the addition of 0.1 mM sodium azide. A small reduction in the total amount of protein- bilin adducts formed was observed in the presence of azide, but this decrease was not accompanied by changes in chromopeptide maps or adduct spectra (42).

DISCUSSION

The number of distinct bilin attachment sites in the phycobiliproteins in a cyanobacterium depends on the particular set of phycobiliproteins present. For organisms studied in detail to date the number of such sites ranges from a minimum of 8 to a maximum of 22 (see Ref. 44, for a review); in Calothrix sp. PCC 7601, whose phycoerythrin we study here, there are at a minimum 13 bilin attachment sites (2). Only two bilin lyases have been identified to date. Synechococcus sp. PCC 7002 phycocyanin a subunit lyase has been extensively characterized, and shown to be responsible for the specific addition of PCB to Cys-84 in the a subunit of C-phycocyanin both in vivo and in vitro (19-21). We have recently demonstrated that the genes pecE and pecF in Anabaena sp. PCC 7120 encode a specific phycoerythrocyanin a subunit lyase responsible for the attachment of phy~obiliviolin.~ A number of genes suspected to encode other bilin lyases have been found in the genomes of various

L. J. Jung and A. N. Glazer, unpublished results.


cyanobacteria (reviewed in Ref. 22), but the roles of the gene products remain to be established. As discussed in the Intro- duction, the possibility that addition at some sites does not require enzyme catalysis has not been ruled out.

We address the following questions in this study. What are the properties of apo-aPE and apo-PPE? Does uncatalyzed addition of bilins to these subunits i n vitro proceed and does it show attachment site selectivity? Is there discrimination between PEB and PCB in such addition? What are the products of the in vitro addition?

Calothrix sp. PCC 7002 apo-aPE and apo-pPE in E. coli were expressed in E. coli. The recombinant subunits were recovered in an insoluble form suggesting that they had not folded cor- rectly in the E. coli cytoplasm and had formed inclusion bodies. Purification of the subunits under denaturing conditions allowed isolation of pure apo-aPE and apo-pPE. At low protein concentration, apo-aPE was recovered in a soluble form in the absence of denaturants, after dialysis against appropriate buffers. However, no conditions allowing “renaturation” of apo-PPE were found. Consequently, further studies were restricted to apo-aPE.

The limited solubility of apo-aPE is attributable to its mar- ginal stability under native conditions, as determined from its urea denaturation behavior. Intrinsic tryptophan fluorescence at 340 nm was used to assess the extent of folding in renatured apo-aPE. By this criterion, the tertiary structure of this subunit was totally disrupted at 0.4 M and higher urea concentrations, suggesting that it was partially unfolded even in the absence of denaturant. The apparent mass of renatured apo-cuPE relative to globular proteins, determined by HPLC size exclusion chromatography, is 18.2 kDa, similar to its calculated mass of 17.2 kDa (42); thus, under native conditions, there is no evidence for the increase in hydrodynamic radius of apo-aPE associated with complete unfolding of globular proteins. In summary, these results suggest that apo-aPE in the absence of urea or at low urea concentrations forms something like a molten globule (45).

In the renaturation experiments in the presence of bilin, more protein remained in solution on dilution to 1 M urea than on dilution to lower concentrations of urea. The 1 M concentration of urea is below that required for denaturation of apo-aPE bilin adducts, and may serve to delay aggregation of apo-aPE until it can form a complex with bilin. The simplest explanation for this observation is that a small fraction of the apo-aPE exists in a conformation(s) that allows bilin addition and that the formation of the more stable bilin adduct (see Table 11) allows repopulation of the addition-competent conformation(s).

Reaction of PEB with apo-aPE led to the formation of bilin adducts at Cys-82 and Cys-139, the cysteine residues that function as sites of PEB attachment i n vivo. At Cys-82, the major adduct was identified spectroscopically as DBV, an oxidized product differing from PEB by the presence of a double bond between the C-2 and C-3 of ring A. DBV is also formed in high yield in the i n vitro addition of PEB to apophycocyanin (17). A small amount of a Cys-59 PEB adduct is also formed (Table 111). The major adduct at Cys-139 is a urobilinoid (UB) adduct with an absorption spectrum indistinguishable from that of authen- tic cysteine-linked phycourobilin (Fig. 5; Ref. 43).

The source of UB adduct(s) is an interesting puzzle. A clue to its origin is that the rate of UB addition appears to lag behind that of PEB and DBV. This lag can be explained in three ways: ( a ) addition of PEB at Cys-139 with concomitant isomerization to UB is favored when the Cys-82 site is already occupied by a bilin. If Cys-82 is free, a PEB adduct forms at Cys-139. ( b ) A PEB or DBV adduct at Cys-139 site isomerizes or is oxidized to a UB over time. This explanation appears unlikely because no increase in UB relative to the DBV and PEB adducts is seen

after free bilins are removed from the reaction mixture. ( c ) A UB precursor increases in concentration over time in the reaction buffer. The third possibility is attractive, given that a pigment with a UB spectrum is always present in stock soh- tions of PEB (for example, see Ref. ll), and that this component increases in quantity over time under the conditions of the addition reaction. Strong acids kg. 12 N HC1) promote the isomerization of both free and phycoerythrin-bound PEB to urobilin pigments of undetermined structure (46,471. The rate and extent of such isomerization under other conditions has not been explored.

The on-line absorbance spectrum of the main PEB peak (Fig. 6 A , 17.8 min; spectrum not shown) lacks the 495-nm absorbing (UB) component, but all efforts to obtain a concentrated stock solution of PEB lacking a UB component were unsuccessful. In phycourobilin, only rings B and C are in conjugation. Conse- quently, the identity in the absorption spectra does not provide information on the state of oxidation of rings A and D. It is not clear whether this urobilin is simply isomerized PEB, or a solvent adduct or oxidation product derived from PEB. It is also unclear whether any of the UB adducts described in this report correspond to the phycourobilin found linked to many phycobiliproteins.

The major product of PCB addition to apo-aPE is a Cys-82 MBV adduct. This is also the major adduct seen in the in vitro addition of PCB to apo-aPc (15, 20). However, the absorption spectra are very different in the two cases. The apo-aPC MBV adduct has the high ratio of long wavelength to near-UV absorbance characteristic of the extended conformation of the PCB chromophore, whereas the spectrum of the apo-aPE MBV adduct indicates that it is in the cyclohelical conformation (48, 49). This is an unexpected finding because the crystal structure of B-phycoerythrin shows that the PEB conformation a t Cys-82 is similar to that of PCB in the analogous site in C-phycocyanin (50). A small amount of a PCB adduct is also formed at Cys-82.

In addition to the adducts at Cys-82 and Cys-139, adducts formed at Cys-59 to a limited extent with PEB, and to a greater extent with PCB (Table 111). Cys-59 is not a binding site for PEB in natural phycoerythrins. Although DTT and 2-mercaptoethanol form adducts with PEB under these reaction conditions in the presence of sodium azide (see “Results”), the extent of reaction at this cysteine is not affected by the presence of azide. It is possible that Cys-59 resides in a domain of the apoprotein with a weak bilin binding site. In the crystal structure of B-phycoerythrin, Cys-59 is very near one of the attachment sites for the doubly-linked PEB of the p subunit (50); the p subunit is structurally similar to the 01 subunit, and thus the (Y

subunit may have a partial bilin binding site in this region of the protein.

The mechanism of nonenzymatic bilin addition to apophycobiliproteins is unclear. Addition of thiol to the ethylidene of PCB dimethyl ester in chloroform has been shown to be catalyzed by methyl-2-mercaptoacetate (51). The proposed mechanism involves reversible addition of the nucleophilic catalyst to the C-10 methyne bridge. Two major PCB-thiol adducts were produced by addition to the ethylidene, one of which is unstable and easily oxidized to MBV-thiol. By analogy, it is possible that a nucleophilic amino acid plays the role of the catalyst in addition of bilin to apophycobiliprotein, and that ring A-oxidized bilin adducts (MBV and DBV) result from formation of an unstable stereoisomer of PCB or PEB adduct. Similarly, azide may catalyze simple thiol addition to PEB by reversible addition at the C-10 bridge.

The results described above provide partial answers to the questions posed at the beginning of “Discussion.” Apo-aPE is lacking in tertiary structure and is markedly stabilized against


urea denaturation by bilin addition, Although holo-pPE is water- 16. Arciero, D. M., Dallas, J. L., and Glazer, A. N. (1988) J. Bid. Chem. 263,

apo-PPE is in the absence Of denaturants' This 17. Arciero, D. M., Dallas, J. L., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18350-18357

raises the strong possibility that in cyanobacteria this subunit 18358-18363 is either kept in solution by interaction with a chaperone or 18. Zhou, J., Gasparich, G. E., Stirewalt, V. L., de Lorimier, R., and Bryant, D. A.

that bilin addition is co-translational. In vitro bilin addition to 19, Swanson, R, V., Zhou, J,, Leary, J, A,, Williams, T,, de R,, Bryant, D, apo-cuPE does take place, but with incomplete site-selectivity and lack of discrimination between PEB and PCB. A mixture of 20. Fairchild, C. D., Zhao, J., Zhou, J., Colson, S. E., Bryant, D. A., and Glazer, A. bilin adducts is Obtained with PEB adducts as minor

21. Fairchild, C. D., and Glazer, A. N. (1994) J. Biol. Chem. 269, 8686-8694 nents. The types and proportions Of the bilin adducts are dif- 22. Wilbanks, S. M., and Glazer, A. N. (1993) J. Biol. Chem. 268, 1226-1235 ferent at the different addition sites. Consequently, although 23. Cornejo, J., Beale, S. I., Terry, M. J., and Lagarias, J. C. (1992) J. Biol. Chem. the protein environment of particular cysteine residues does 267,14790-14798

of the bilin that reacts most rapidly) in vitro, the results of this 69-94, Elsevier Science Publishers B. V., Amsterdam, The Netherlands

cobiliproteins in vivo is enzyme-catalyzed.

(1992) J. Bid. Chem. 267, 16138-16145

A,, and Glazer, A. N. (1992) J. Biol. Chem. 267, 1614616154

N. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7017-7021

influence the fate Of the attached bilin (Or the nature 25. Glazer,A. N. (1987) in The Cyanobacteria (Fay, P., and Van Baalen, C. eds) pp.

study strongly support the view that all bilin addition to phy- 26. Sidler, W., Kumpf, B., Riidiger, W., Zuber, H. (1986) Biol. Chem. Hoppe-Seyler

27. Mazel, D., Guglielmi, G., Houmard, J., Sidler, W., Bryant, D. A,, and Tandeau

24. Li, L., and Lagarias, J. C. (1992) J. Biol. Chem. 267, 19204-19210

367, 627442

The apophycoerythrin subunit preparation described is a de Marsac, N. (1986) Nucleic Acids Res. 14,82794290 source of substrates for putative phycoerythrin PEB lyases from Culothrix sp. PCC 7601. Since the nonenzymatic reaction leads mainly to DBV and UB adducts, it should be possible to measure the activity of a lyase producing only PEB adducts. The insolubility of the apo-PPE could be overcome by linking it t o a solid matrix, as has been done previously for assay of phycocyanin a subunit PCB lyase (20). It will be of particular interest to determine the mode of formation of the double link- age to the PEB at Cys-48,59 on the P subunit.

REFERENCES 1. Glazer, A. N. (1989) J. Bid. Chem. 264, 1 4 2. Grossman, A. R., Schaefer, M. R., Chiang, G. G., and Collier, J. L. (1993)

Microbiol. Rev. 57. 725-749 ~~ ~

3. Lagarias, J. C., Klotz, A. V., Dallas, J. L., Glazer, A. N., Bishop, J. E., OConnell,

4. Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. J. F., and Rapoport, H. (1988) J. Biol. Chem. 263, 12977-12985

5. Schoenleber, R. W., Lundell, D. J . , Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259,5485-5489

6. Wedemayer, G. J. , Kidd, D. G., Wemmer, D. E., and Glazer, A. N. (1992) J. Biol.

7. Nagy, J. O., Bishop, J. E., Klotz, A. V., Glazer, A. N., and Rapoport, H. (1985)

8. Wedemayer, G. J. , Wemmer, D. E., and Glazer, A. N. (1991) J. Biol. Chem. 266,

9. Wemmer, D. E., Wedemayer, G. J., and Glazer, A. N. (1993) J. Biol. Chem. 268,

Biol. Chem. 259,5481-5484

Chem. 267, 7315-7331

J. Biol. Chem. 260, 4864-4868

47314741

1658-1669

11. Beale, S. I., and Cornejo, J. (1991) J. Bid. Chem. 266, 22333-22340 10. Beale, S. I., and Cornejo, J. (1991) J. Biol. Chem. 266, 22328-22332

12. Beale, S. I,, and Cornejo, J. (1991) J. Biol. Chem. 266, 22341-22345 13. Cole, W. J., Chapman, D. J., and Siegelman, H. W. (1967)J. Am. Chem. SOC. 89,

14. Chapman, D. J., Cole, W. J., and Siegelman, H. W. (1967)J. Am. Chem. Soc. 89, 3643-3645

15. Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 5976-5977

18343-18349

28. Tandeau de Marsac, N., Mazel, D., Capuano, V., Damerval, T., and Houmard,

phic Bacteria (Drews, G., and Dawes, E. A,, eds) pp. 143-153, Plenum Press, J. (1990) in Molecular Biology of Membrane-bound Complexes in Phototro-

New York

". .

29. Klotz, A. V., and Glazer, A. N. (1987) J. Biol. Chem. 262, 17350-17355 30. Russel, M., and Model, P. (1984) J. Bacteriol. 159, 1034-1039 31. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S.A. 82,

32. Tandeau de Marsac, N. (1983) Bull. Inst. Pasteur 81, 201-254 33. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., and Stanier, R. Y.

(1979) J. Gen. Microbiol. 111, 1-61 34. Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260,4856-4863 35. Laemmli, U. K. (1970) Nature 227, 680485 36. Glazer, A. N., and Hixson, C. S. (1975) J. Biol. Chem. 250, 5487-5495 37. Bryant, D. A,, Duhbs, J. M., Fields, P. I., Porter, R. D., and de Lorimier, R.

38. Berkelman, T. R., and Lagarias, J. C. (1986JAnaL Biochem. 156, 194-201 39. Zickendraht-Wendelstadt, B., Friedrich, J., and Riidiger, W. (1980) Photochern.

Photobiol. 31, 367-376 40. Zilinskas, B. A,, Grabowski, J., and Campbell, S. (1984) in Advances in

Photosynthesis Research (Sybesma, C., ed) Vol. 11, pp. 687-690, Kluwer Boston, Hingham, MA

1074-1078

(1985) FEMS Microbiol. Lett. 29, 343-349

41. Johnson, M. L., and Frasier, S. G. (1985) Methods Enzymol. 117,301-342 42. Fairchild, C. D. (1993) Ph.D. thesis, Enzym.atic and Non-enzymatic Addition of

43. Swanson, R. V., Ong, L. J., Wilbanks, S. M., and Glazer, A. N. (1991) J. Biol.

44. Ong, L. J., and Glazer, A. N. (1991) J. B i d . Chem. 266, 9515-9527 45. Ptitsyn, 0. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., and Razgulyaev, 0.

46. 0 Carra. P., 0 hEocha, C., and Carroll, D. M. (1964)Biochemistry 3,1343-1348 47. Cole, W. J. , 0 hEocha, C., Moscowitz, A., and Krueger, W. R. (1967) Euz J.

48. Duening, M., Schmidt, G. B., and Huber, R. (1991) J. Mol. Biol. 217,577-592 49. Scheer, H., and Kufer, W. (1977) Z. Naturforsch. 32c, 513-519 50. Ficner, R., Lobeck, K., Schmidt, G., and Huher, R. (1992) J. Mol. Biol. 228,

51. Stumpe, H., Miiller, N., and Grubmayr, K. (1993) Tetrahedron Lett. 34,4165-

Bilins to Apophycobiliproteins, University of California, Berkeley, CA

Chem. 266,9528-9534

I. (1990) FEBS Lett. 262, 20-24

Biochem. 3,202-207

935-950

4168

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