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

Vol. 269, No. 12, Issue of March 25, pp. 8686-8694, 1994 Printed in U.S.A.

Oligomeric Structure, Enzyme Kinetics, and Substrate Specificity of the Phycocyanin a Subunit Phycocyanobilin Lyase*

(Received for publication, October 12, 1993, and in revised form, December 28, 1993)

Craig D. Fairchild4 and Alexander N. Glazers From the Department of Molecular and Cell Biology, Uniuersity of California, Berkeley, California 94720

Phycobiliproteins carry linear tetrapyrrole chromo- phores (bilins) thioether-linked to specific cysteine residues. The process of bilin attachment to apoprotein in vivo has been characterized for only one bilin attachment site on one phycobiliprotein, that on the a subunit of phycocyanin (apc). In the cyanobacterium Synecho- coccus sp. PCC 7002, the addition of phycocyanobilin to apo-aPC is catalyzed by the protein products of the cpcE and cpcF genes. We have purified and further characterized the recombinant CpcE and CpcF proteins. CpcE and CpcF form an enzymatically active 1:l complex (Cp- cEF), stable to size exclusion chromatography. CpcEF causes a reduction in apc fluorescence and strongly af- fects its absorption spectrum but has no effect on the p subunit. The CpcEF bilin addition activity exhibits simple Michaelis-Menten kinetics with respect to the apo-aPC and to bilin. CpcEF also catalyzes the addition of phycoerythrobilin to apo-aPc; phycoerythrobilin is thought to be on the biosynthetic pathway of phycocyanobilin. CpcEF shows a preference for phycocyanobilin relative to phycoerythrobilin, both in binding affinity and in the rate of catalysis, sufficient to account for selective attachment of phycocyanobilin to apo-aPC.

In cyanobacteria and red algae, phycobiliproteins are the major components of a thylakoid membrane-associated macro- molecular light-harvesting complex, the phycobilisome (1). The phycobiliproteins carry covalently attached linear tetrapyrrole prosthetic groups (bilins). In different species, the number of different bilins on the various phycobiliproteins ranges from one to three, and the number of distinct attachment sites ranges from eight to more than 20.

The bilins phycocyanobilin (PCB)’ and phycoerythrobilin (PEB) can be cleaved from phycobiliproteins by refluxing in methanol (2, 3). This treatment results in the elimination of a phycobiliprotein cysteine from the 3‘ carbon of the bilin to yield a linear tetrapyrrole with an ethylidene at the C-3 position (the IUPAC numbering scheme for bilins and the structures of PCB and PEB is shown in Ref. 4).

The conformation and interactions of bilins with the poly-

Grant GM 28994 (to A. N. G.). The costs of publication of this article * This work was supported in part by National Institutes of Health

were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

(i To whom correspondence should be addressed: MCB:Stanley/

94720. Tel.: 510-642-3126; Fax: 510-643-9290. Donner ASU, 229 Stanley Hall, University of California, Berkeley, CA

erythrobilin; cypc, C-phycocyanin cy subunit; DTT, dithiothreitol; PAGE, The abbreviations used are: PCB, phycocyanobilin; PEB, phyco-

polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; PCB*, addition-competent form of PCB; SEC, size exclusion chromatography; MBV, mesobiliverdin.

peptide within native phycobiliproteins greatly affect their spectroscopic properties. Generally, a native phycobiliprotein has a strongly enhanced long wavelength visible absorption peak and a much higher fluorescence quantum yield relative to its denatured form or to bilin not associated with protein.

Phycocyanin is a phycobiliprotein that bears adducts of PCB a t residues a-Cys-84 and p-Cys-82 and p-Cys-155. PCB reacts spontaneously with apophycocyanin in vitro at a-Cys-84 and p-Cys-82 (but not a t p-155), to form an unnatural adduct, 3‘- cysteinylmesobiliverdin, which contains an extra double bond between carbons 2 and 3 of ring A of the bilin (5, 6). PEB also reacts at a-Cys-84 and 6-Cys-82 to form a similar adduct with a double bond at C2-C3 of ring A (7).

In the cyanobacterium Synechococcus sp. PCC 7002, the attachment of PCB to the a subunit of phycocyanin (apc) is catalyzed by a specific protein bilin lyase, which is encoded by the genes cpcE and cpcF. The phycobiliproteins of this organism have eight PCB attachment sites on seven polypeptides. Inac- tivation of cpcE or cpcF by interposon insertion leads to loss of correct bilin attachment to apc but not to any of the other PCB attachment sites (8, 9). The protein products of these genes, CpcE and CpcF, have been expressed separately in Escherichia coli and shown to catalyze together the correct addition of PCB to apo-aPc (10). A number of genes with varying degrees of homology to cpcE and cpcF have been characterized in other cyanobacteria (for a review see Ref. ll), although none of these has been shown to encode a protein bilin lyase.

The present study presents further characterization of CpcE and CpcF and of their enzymatic activity. Purification schemes for recombinant CpcE and CpcF are presented. The purified proteins were used to establish that CpcE and CpcF form a 1:1 complex, which is henceforth termed CpcEF. Various assays for catalysis and substrate binding failed to show any activity for the individual CpcE or CpcF polypeptides.

Since PEB is a biosynthetic intermediate in the formation of PCB in a red alga (12), and presumably in cyanobacteria as well, the specificity of CpcEF for its bilin substrate is of particular interest. Kinetic data presented here show that CpcEF, although it can catalyze the addition of PEB to apo-aPC, exhibits specificity for PCB both in binding afflnity and the rate of catalysis.

CpcEF has also been shown to catalyze the rapid transfer of bilin from holo-aPC to apo-aPc (10). This result implies that Cp- cEF can access the thioether linkage between protein and bilin in apc, which should require at least partial unfolding of the holo subunit. In support of this notion, we report here that CpcEF alters the absorption and dramatically reduces the fluorescence emission of the a subunit, but not the p subunit, of phycocyanin. A change in the fluorescence of a holo subunit may be a conve- nient assay for phycobiliprotein bilin lyases yet to be discovered.

EXPERIMENTAL PROCEDURES CpcE and CpcFPurification-Crude CpcE and CpcF inclusion bodies

were prepared and solubilized in 9 M urea at pH 1.9, as described

8686

Phycocyanin a Subunit Phycocyanobilin Lyase 8687

previously (10). The protein solution was brought to 10 mM DTT and pH 7.5-8.0 by the addition of solid DTT and 1.5 M Tris-HC1, pH 8, and incubated at room temperature for 1 h. CpcE and CpcF solutions were then ultracentrifuged (30 min at 100,000 x g) and filtered through a 0.2-pm pore size membrane. Each solution (4-5 ml) was then exchanged into 8 M urea, 20 mM Tris-HCI, pH 7.5 (plus 1 nm DTT for CpcF) by passage through a Sephadex G-25 column (80-ml bed volume) in the same solvent.

Aliquots of each protein (9 ml, 35-40 mg of protein) were loaded onto a Mono Q HR 10/10 column (Pharmacia LKB Biotechnology, Inc.) on a Perkin-Elmer series 410 Bio LC system; the column was preequili- brated in 8 M urea, 20 nm Tris-HC1, pH 7.5, and the flow rate through- out was 4 mumin. The column was washed for 2 min in the starting solvent and then eluted with a linear gradient of NaCl in the same solvent: 35 min, 0-0.35 M NaCl for CpcE; 35 min, 0-0.2 M NaCl for CpcF. Two-ml fractions were collected. The fractions were evaluated by SDS- PAGE, and those containing CpcE or CpcF were pooled, brought to 5 mM DTT, and acidified to pH 2.5 with concentrated HCI.

Reverse-phase HPLC was performed with a semipreparative scale C, column (Hi-Pore RP-304, Bio-Rad) in the solvent system used for the separation of phycobiliprotein subunits (13): aqueous, 0.1% trifluoroacetic acid; organic, 2:l acetonitri1e:isopropyl alcohol, 0.1% trifluoroacetic acid. Aliquots of CpcE (1.4 ml, 2 mg of protein) and CpcF (1.5 ml, 1.5 mg of protein) Mono Q pools were loaded without further treatment onto the C, column in 35% organic solvent, 3 mumin flow rate. The elution gradients, after 2 min at 35% organic solvent, were: CpcE, 3 5 4 5 % organic in 2 min, 5.571% in 32 min; CpcF, 3 5 4 5 % organic in 5 min, 5540% in 5 min, and 60-70% in 30 min.

CpcE and CpcF pools were reduced to 0.20 volume by rotary evaporation. Some precipitate formed during this step; this precipitate dissolved on dilution of the concentrated pools 1:2 (v/v) with 9 M urea-HCI, 10 m Tris-HC1, pH 2.5. The pools were dialyzed against the urea diluent containing 1 nm DTT. Dialyzed CpcE and CpcF were stored at 4 "C (DTT was added to the CpcF solution to a final concentration of 5 mM).

Renaturation of CpcE and CpcF-For assay of fractions from chromatography, CpcE or CpcF solutions in urea (HPLC fractions were reduced to 0.20 volume by evaporation with a stream of N, and diluted 1:5 with 9 M acid-urea) were diluted 1 : l O (v/v) into 50 mM Tris-HC1, pH 8, 75 nm NaCl, and clarified by centrifugation; small aliquots of these solutions were then used in the bilin addition assays.

For all other studies, purified CpcE, CpcF, or CpcE and CpcF stock solutions in 9 M acid-urea were diluted to final concentrations: 45 pg/ml CpcE; 30 pg/ml CpcF in 3 M urea, 150 mM Tris-HC1, pH 8,O. 1 mM MgCl,, 1 nm thioglycolate. These solutions were then dialyzed a t 4 "C against 75 nm NaCl, 50 mM Tris-HC1, pH 8, 1.1 m MgCl,, 1 nm disodium pyrophosphate, 1 mM thioglycolate. For some experiments the renatured proteins were concentrated by ultrafiltration with a 10.000 molecular weight cutoff membrane (YM-10, funicon). Protein solutions were then ultracentrifuged (30 min, 100,000 x g ) and filter-sterilized through a 0.2-pm filter prior to storage a t 4 "C.

Determination of CpcE and CpcF Concentrations-The concentrations of impure CpcE and CpcF, and of CpcE renatured together with the various pools of purified CpcF, were determined by SDS-PAGE and comparison of Coomassie-stained bands with protein standards run on the same gel.

Concentrations of solutions of purified CpcE and CpcF were determined spectroscopically, with czSo calculated from the Trp and Tyr content ascertained from the amino acid sequences: under denaturing conditions, 35,640 cm" (A;:; = 1.22/cm) for CpcE and 20,220 M - I cm-' (A::: = 0.91/cm) for CpcF; under native conditions, P o of 38,440 M-' cm" (A::: = 1.32/cm) for CpcE and 21,060 M-' cm-' (A:$ = 0.95/cm) for CpcF (14). The concentration of native CpcEF was calculated assuming a 1:l ratio of CpcE to CpcF.

Assays for the Addition of Bilin to apo-aPc-All addition assays were performed at 37 "C on a Perkin-Elmer MPF-44B fluorescence spectro- photometer with slit widths of 5 nm for excitation and 8 nm for emission, in a square cuvette of 4-mm path length.

For relative CpcEF activity with PCB as the bilin substrate, the increase in uncorrected 640 nm emission with 600 nm excitation was monitored continuously over time. For each concentration of apo-aPc and PCB, the nonenzymatic rate of fluorescence gain was determined with an aliquot of the appropriate buffer in place of CpcEF. Unless otherwise noted, each reaction mixture contained 75 mM NaCl, 50 mM Tris-HC1, pH 8.0, 1 nm MgCl,, 1 nm disodium pyrophosphate, 1 mM thioglycolate. The latter three ingredients were added to apo-aPc and Tris-NaCI with mixing, after which CpcEF was added with mixing, and then the assay was initiated by the addition of bilin in dimethyl sulf-

oxide with mixing (the final concentration of dimethyl sulfoxide did not exceed 1.2% by volume). CpcE activity was assayed in the presence of a 2-fold molar excess of CpcF and vice versa.

CpcEF Kinetics-The rate of gain in uncorrected fluorescence emission at 640 nm was converted to the rate of formation of holo-aPc by comparison with the 640 nm emission of a known concentration of holo-aPc under assay conditions.

The proportion of PCB* (addition-competent PCB; see Ref. 10) in the stock PCB was estimated as follows. An assay with a large amount of CpcEF (200-300 nM) was performed with 20 &m PCB and 10 p~ apo-aPc. Under these conditions a clear, biphasic rate of fluorescence gain is observed (10). The slower, second rate was extrapolated back to the time of mixing, and this level of fluorescence was taken as being a result of the addition of all of the PCB* to apo-aPc. This fluorescence was converted into a concentration by comparison with a solution of holo-aPc of known concentration.

The higher concentrations of PCB used in some assays were sufficient to reduce the observed fluorescence by attenuation of the excitation and emission energies (the so-called "trivial reabsorption" phenom- enon). For experiments where [PCB] varied, the function relating apc fluorescence reduction and [PCB] was determined empirically. This function was then used to correct the rates of fluorescence gain in each assay. When [PCB] was held constant, this correction was not necessary; the conversion from fluorescence gain to holo-aPc formed was made using a solution of holo-aPc in 20 1" PCB.

Kinetic parameters were determined by iterative curve fit to the Michaelis-Menten equation, V, = [SIV,,,,,/([Sl + K,,,), where S is the substrate whose concentration was varied and V, is the initial rate, using the program KaleidaGraphTM (Abelbeck Software), with weight- ing proportional to V, (15). The KI for PEB was calculated from the observed K , (K)) for PCB in the presence of PEB using the formulas K: = dm and a = 1 + [Il/KI.

For comparison of the rates of the CpcEF-mediated addition of PEB or PCB to apo-aPc in near saturating concentration of bilin, larger volume reactions were used (3 ml), and assays were allowed to proceed for 5 min. Bilin concentrations were 20 p ~ , apo-aPc was 10 p ~ , and CpcEF was 2.0 nM for the PCB reactions and 30 nM for PEB reactions. Reactions were quenched by the addition of 0.25 volume of cold 50% (w/v) trichloroacetic acid. The quenched assay mixtures were kept at -20 "C overnight. Protein precipitates were collected by centrifugation a t 4 "C, dissolved in 0.3 m18 M urea-HC1,lO mM 2-mercaptoethanol, pH 2.5, and protein separated from unreacted bilin by Sephadex G-25 chromatography in the same solvent (20-ml column volume). The first 280 nm-absorbing peak was collected in each case, in a volume of 1 ml, and its absorption spectrum was taken with the urea solvent as a reference.

The spectra from the +CpcEF and -CpcEF reaction mixtures were adjusted to the same A,,, with an allowance for the absorbance caused by CpcEF. This was done to correct for unequal recovery of the protein from the reactions and does have a significant effect on the final values determined (the factors were 1.039 for +CpcEF PEB, and 1.095 for -CpcEF PCB). Since PEB and PCB adducts have some absorbance at 280 nm, the adjustment leads to a small underestimation of the CpcEF- mediated addition rates, which should to some extent cancel out in the ratio of these rates.

The amount of CpcEF-mediated adduct formation was determined from the difference spectra, +CpcEF minus control, using the following extinction coefficients for bilin peptides in 8 M acid-urea: PEB adduct,

= 53,700 M-' cm-' (16); PCB adduct, P 3 = 33,800 M" cm-' (17). Other Methods-PCB and apo-aPc, holo-aPC, and holo-P phycocyanin

were prepared, and their concentrations were determined a s described previously (10). Denatured apo-aPc was prepared from protein exposed to 10 nm D m , dialyzed extensively against 5 m ammonium acetate, and lyophilized. The lyophilized protein was suspended a t 16 mg/ml in 8 M urea, 75 nm NaCl, 50 nm Tris-HC1, pH 8, and diluted 1:lO in assay buffer.

PEB was prepared as described previously (7). The main PEB peak from reverse-phase HPLC purification was used for the experiments presented here, and its concentration was determined using the c594 of PEB dimethyl ester (25,200 M-I cm"; Ref. 18) with PEB diluted into 5% (v/v) HCl in methanol.

RESULTS

Recombinant CpcE and CpcF proteins were subjected to two purification steps under denaturing conditions: ion exchange chromatography in 8 M urea on Mono Q, and reverse-phase C4 HPLC. Fractions were analyzed by SDS-PAGE and pooled fractions by a bilin addition assay.

8688 Phycocyanin (Y Subunit

" 29 -1

c l 2 3 4 5

66 - 45 -

12.4 - """

" - -

FIG. 1. CpcE and CpcF proteins overexpressed in E. coli. Coo- massie-stained SDS-polyacrylamide gels (Laemmli system (19), mono- mer:bis ratio of 37.5, 10% polyacrylamide stacking gel). Size standards (in kDa) were: bovine serum albumin, 65; ovalbumin, 45; carbonic an- hydrase, 29; avidin, 18; horse heart myoglobin, 17; hen egg white ly- sozyme, 14.4; cytochrome c, 12.4. The predicted molecular mass for CpcE is 29.2 kDa and for CpcF is 22.2 kDa. For the gels in panels A and B, the amount loaded in each lane is a similar percentage of the preparation at each step. Panel A, CpcE expression; 14% polyacrylamide. Lanes 1 and 2, whole cells solubilized and samples trichloroacetic acid- precipitated, uninduced (lane 1 ), and induced (lane 2 ) with isopropyl-

of cell lysate; lane 4, 1% Triton X-100 wash of the pellet from cell lysis; 1-thio-P-n-galactopyranoside; lane 3 , supernatant from centrifugation

lane 5, supernatant of acid-urea solubilization of washed inclusion bodies. Panel B, CpcF expression; 12% polyacrylamide. Lane 1, supernatant from centrifugation of cell lysate; lane 2, 1% Triton X-100 wash of the pellet from cell lysis; lane 3,50 mM Tris-HC1, pH 8, wash of the pellet from the detergent wash; lane 4, supernatant of acid-urea solubilization of washed inclusion bodies. Panel C, purified CpcE and CpcF, 146 polyacrylamide. Lanes 1 (CpcE) and 5 (CpcF), purified proteins in 9 M acid-urea. Lanes 2 (CpcE), 3 (CpcEF), and 4 (CpcF) are proteins renatured by removal of urea by dialysis. CpcE and CpcEF had been concentrated by ultrafiltration.

Purification of CpcE-The result of an inclusion body preparation of recombinant CpcE is shown in Fig. LA. As described previously (lo), when the cpcE gene is induced with isopropyl- 1-thio-P-D-galactopyranoside, three new bands become apparent on SDS-PAGE of solubilized E. coli cells (Fig. LA, lanes 1 and 2): a band with apparent molecular mass 31 kDa, and two closely spaced bands of 29 kDa (labeled CpcE) and 28.5 kDa. Most of the 28.5-kDa band is lost in the soluble fraction of cell lysate (lane 3), and what remains after washing and is solubilized in acid-urea solution is predominantly the 29- and 31-kDa bands (lane 5 ) . The amount of the 29- and 31-kDa bands relative to that of the 28.5-kDa band varies from preparation to preparation of CpcE-producing cells, and some preparations contained almost exclusively the 28.5-kDa product.

The 31-kDa polypeptide eluted from the Mono Q column in two peaks early in the NaCl gradient; these fractions did not exhibit any CpcE activity in bilin addition assays and were not purified further. The 28.5-kDa polypeptide coeluted with the earliest eluting 31-kDa peak. In a separate preparation en- riched for the 28.5-kDa polypeptide, this peak was collected for further purification. The 29-kDa polypeptide eluted later in the NaCl gradient than the 31-kDa peaks and was collected for further purification.

Portions of the 28.5- and 29-kDa Mono Q fractions were loaded onto a C4 column and eluted with a gradient of increas-

Phycocyanobilin Lyase

ing organic solvent. The 29-kDa polypeptide eluted in 67.5% organic solvent; CpcE activity in the bilin addition assays cor- related with this peak. The final punty of the 29-kDa CpcE fraction is illustrated in Fig. lC, lane 1. The 28.5-kDa polypeptide eluted a t a much lower organic solvent content, around 54%; it lacked CpcE activity in the bilin addition assays.

Purification of CpcF-CpcF protein overexpressed in E. coli is to some extent lost in the soluble fraction of cell lysate (Fig. 1 B , lane 1 ), but the bulk of CpcF ultimately is solubilized only in acid-urea (lane 4 ).

Polypeptides of the apparent molecular mass of CpcF (22 kDa) eluted in three peaks from the Mono Q column. These were pooled separately and are referred to below as early, main, and late. The three Mono Q pools of CpcF were purified further by reverse-phase HPLC. The 22-kDa polypeptide of the main Mono Q fraction eluted in 64% organic solvent. That of the early Mono Q fraction eluted in two peaks, in 63% (early-1) and 65% (early-2) organic solvent. That of the late fraction eluted in 65% organic solvent.

To assess the relative activity of these four CpcF fractions, each was renatured in the presence of active CpcE. By SDS- PAGE, the concentration of CpcE and CpcF in each renatured protein mixture was roughly equivalent. The results of the bilin addition assays for each, expressed as the percent of main CpcF activity, were: main, 100%; early-1, 29%; early-2, 64%; late, 19%. Although assay error and disparity in enzyme concentration may account for some of the variation observed, it is clear that all of the CpcF fractions, with the possible exception of early-2, have lower activity when renatured in the presence of CpcE than does main CpcF. The main fraction of CpcF was used in all experiments described below. The final purity of this fraction of CpcF is illustrated in Fig. lC, lane 5 .

Evidence for Physical Interaction between CpcE and CpcF- The SDS-polyacrylamide gel of the renaturation products of CpcE, CpcF, or both together (Fig. 1C) illustrates three interesting points. First, the molar ratio of CpcE to CpcF left soluble after renaturation is approximately 1:l (Fig. lC, lane 3; the ratio of Coomassie stain densities is 3:2, the approximate ratio of calculated molecular masses CpcE:F). This ratio is main- tained even with renaturation of higher concentrations of CpcE and F (greater than 0.2 mg/ml each), when most of the protein is lost to aggregation. Second, on storage of renatured CpcE at 4 "C slow proteolysis takes place (Fig. lC, lane 2); this proteolysis is retarded in the presence of CpcF. Third, CpcEF together can be concentrated by ultrafiltration without substantial loss of activity (to a t least 0.8 mg/ml), whereas CpcF, although it can be concentrated in soluble form, loses nearly all of its activity in the process. CpcE can be concentrated to a limit of about 0.3 mg/ml without loss of activity.

CpcE and CpcF proteins, separately renatured, can be combined to yield active enzyme, although the activity is less than that for co-renatured CpcEF (Table I, line 3, compared with line 1). Extra CpcE, with the amount of CpcF held constant, led to an increase in addition activity with separately renatured proteins (Table I, lines 4 and 51, but additional CpcE added to co-renatured CpcEF did not increase addition activity (line 7; compare with line 1). Extra CpcF, whether with separately renatured CpcE (Table I, line 6) or co-renatured CpcEF (line 8), not only does not increase addition activity, but it may inhibit i t slightly.

That both CpcE and CpcF are required for bilin addition activity, along with the additional evidence discussed above, together suggested that the two proteins might form a complex of 1:l stoichiometry. To test this hypothesis, SEC was performed with renatured, CpcE, CpcF, and CpcEF concentrated by ultrafiltration; 280 nm absorbance profiles are shown in Fig. 2, and the apparent molecular masses of these and other pro-


TABLE I Bilin addition activity of CpcE and CpcF renatured separately and

together CpcE and CpcF where separated by a slash were renatured together;

where separated by a plus sign they were renatured separately. Bilin addition activity is expressed as either the gain in relative fluorescence (with the rate of nonenzymatic gain in fluorescence, 5.05 min", subtracted from each) or as the percent activity relative to the first entry.

Enzyme Gain in relative Relative

fluorescence (per min)

addition activity

1. 1 p~ CpcE/CpcF 2. 0.5 p~ CpcE/CpcF 3. 1 VM CpcE + 1 PM CpcF 4. 2 VM CpcE + 1 PM CpcF 5. 3 p~ CpcE + 1 PM CpcF 6. 1 p~ CpcE + 2 CpcF 7. 1 p~ CpcE/CpcF + 1 p~ CpcE 8. 1 p~ CpcE/CpcF + 1 p~ CpcF

4.53 * 0.10" 1.93 1.45 3.00 3.52 1.19 4.71 3.99

%

(100) 42 32 66 78 26

104 88

1 p~ CpcE/CpcF the results from two assays were averaged and the observed range is indicated.

0.05 670 158 45 17 + + + +

c

o , , , , , , , , , , F?

4 6 8 10 12 14 Time (min)

FIG. 2. Size exclusion chromatography of CpcE, CpcF, and CpcEF. HPLC SEC was performed with a 300 x 7.5 mm Bio-Si1 TSK- 250 column (Bio-Rad). The solvent was 100 mM Napi, pH 6.8, at a flow rate of 1 mumin; 100 pl of protein solution was injected. Upper truce,

tained 47 pg of CpcEF; 4.8 pg of CpcE; and 11 pg of CpcF. The elution CpcEF. Middle truce, CpcE. Lower trace, CpcF. 100-pl injections con-

times of A,,, peaks corresponding to globular protein standards are marked with urrows: thyroglobulin (670 kDa), 6.25 min; y-globulin t 158 kDa), 9.50 min; ovalbumin (45 kDa), 11.00 min; and myoglobin (17 kDa), 13.00 min. The rise in around 14 min is caused by buffer

buffer (not shown) from that of CpcF protein did not reveal any late components; subtraction of the chromatogram of CpcF renaturation

eluting Azso peak.

teins of interest relative to globular protein standards are listed in Table 11. The elution time of CpcE is consistent with its monomeric molecular mass. CpcF, however, elutes as a broad region of 280 nm absorbance. As noted above, concentrated CpcF lacks addition activity in the presence of CpcE; active (not concentrated by ultrafiltration) CpcF was also loaded onto the size exclusion column, but no substantial 280 nm absorbance eluted in the size range of 15-670 kDa, nor did any fractions collected contain CpcF activity (not shown).

The elution time of the main CpcEF 280 nm peak is consistent with the calculated molecular mass of a 1:l complex of CpcE and CpcF (Table 11). Furthermore, the presence of both CpcE and CpcF proteins and CpcEF activity correlate well with the 280 nm peak (not shown). When separately renatured CpcE and CpcF are mixed prior to SEC, the height of the 11.8-min CpcE peak is reduced, and there is a new (shoulder) peak eluting a t around 10.9 min (not shown).

Apo-ape, apc MBV adduct, holo-aPC, and PCB were individu- ally mixed with CpcEF and the SEC elution profiles of the mixtures compared with those of the individual components. The elution profiles of the mixtures in each case closely re-

TABLE I1 Apparent molecular mass of proteins by HPLC SEC relative to

globular protein standards

Protein sample Apparent mass Calculated mass

kDa kDa CpcEF renatured together 48 51.4" "29 kDa" CpcEh 30 29.2 "28.5 kDa" CpcEb 21 ? Holo-a PC 19 18.2 Apo-a PC 17 17.6

Calculated mass for a CpcEF heterodimer. The apparent molecular masses determined by SDS-PAGE are

given in quotation marks.

sembled the sum of the profiles of the two components. SDS- PAGE analysis of fractions from SEC of CpcEF, apo-ape, and the two mixed together reinforce this analysis (not shown).

The 28.5-kDa product of the CpcE preparation was renatured alone and in the presence of CpcF and subjected to SEC. The 28.5-kDa protein eluted in a sharp peak with an apparent molecular mass of 21 kDa, and its elution was unaltered in the sample renatured with CpcF. Whether the mobility of the 28.5- kDa polypeptide is anomalous on SDS-PAGE or SEC or both is not clear.

Quenching of Holo-aPc Fluorescence Emission by CpcEF-It has been demonstrated that CpcEF can transfer the bilin of holo-cyPC to apo-cyPc (10). To do this, CpcEF presumably must access the thioether bond that links the bilin to the polypeptide chain of holo-aPC, a bond that is buried in the folded subunit. If this notion is correct, interaction of CpcEF with holo-cyPC must require, at the least, disruption of the local environment of the chromophore. Any such disruption should result in changes in the absorbance and emission spectra of the cy subunit. Further- more, these spectroscopic changes should only occur when CpcEF is mixed with the a subunit, not with the p subunit of phycocyanin.

Spectroscopic changes are in fact observable upon mixing of CpcEF with ape, or the MBV adduct of apo-aPC, but not with p phycocyanin. As shown in Table 111, the fluorescence of holo-aPC is reduced in the presence of CpcEF. The fluorescence of p phycocyanin is unaltered by CpcEF. The extent of fluorescence loss of holo-aPC depends on the ratio of CpcEF to ape as well as the concentration of cypc. Compare the first line of Table I11 with the first line of Table IV; both are 1:l ratios of CpcEF to ape, but the reduction of fluorescence at 642 nm in the mixture with 1.5 p~ apc is 49%, whereas that of the mixture with 0.77 PM apc is only 35%.

The absorbance spectrum of holo-cyPC in the presence of sufficient CpcEF to cause a 71% loss of fluorescence emission at 642 nm has two notable features (Fig. 3A). First, the ratio of the absorbance peaks red:near UV is shifted from 4.2 without CpcEF to 0.8 with CpcEF. This change is the result of increase in the near UV peak and decrease in the red peak. Second, the near UV peak is sharpened, with a A,,, of 364 nm, in the presence of CpcEF. The reason for splitting of the near UV peak in apc in the absence of CpcEF is not known; although this preparation of apc had this feature, others prepared by the same method have a single, broad peak at 357 nm and a red: near UV ratio of 3.3 (see Ref. 20).

The fluorescence emission spectrum of ape with an amount of CpcEF sufficient to cause a 52% loss of emission at 642 nm (Fig. 3 A ) is not significantly shifted relative to the apc emission spectrum in the absence of CpcEF. For these samples, the absorbance a t the excitation wavelength, 580 nm, was reduced 20% in the presence of CpcEF. Thus, there is a real reduction in the quantum yield of apc in the presence of CpcEF, here a 40% loss relative to that of cypc alone, and not just a change in absorbance.

8690 Phycocyanin a Subunit Phycocyanobilin Lyase

TABLE 111 Change in fluorescence emission of apt, P phycocyanin, and gC

MBV adduct in the presence of CpcEF The conditions for assay of addition of PCB to apo-aPc were used, but

without either of the substrates. Phycobiliprotein was mixed with assay buffer, and the fluorescence emission was monitored until it stabilized. CpcE, CpcF, or CpcEF was then added, and the emission was again allowed to stabilize (5-10 min). The X,,c&,, (nm) were: a, P phycocyanin, 600/642; a-MBV adduct, 620/668. The change in fluorescence emission at the wavelength monitored is expressed as a percent relative to a control mixture of phycobiliprotein plus the final dialysis buffer from CpcEF renaturation; a different control mixture was used for each line.

[CpcEFI Phycocyanin Ratio subunit [CpcEFl:

[biliproteinl Change in emission

~

w IC

0.77 0.77 VM a 1: 1 7.70 0.77 PM a 10: 1 -71.0

-35.0

2.25 7.50

0.56 PM a-MBV 4: 1 -37.4

0.77 0.75 PM a-MBV 1O:l 0.77 PM 1:l

-50.6 +0.3

TABLE IV Change in fluorescence emission of phycocyanin subunits in the

presence of CpcE or CpcF alone The change in fluorescence emission at the wavelength monitored is

expressed as a percent, relative to a control mixture of phycobiliprotein plus the final dialysis buEer(s) from CpcE or CpcF renaturation; three different control mixtures were used. The control mixture for lines 1-4 contained CpcE and CpcF dialysis buffers, that for 5 and 6, and that for 7 contained CpcE dialysis buffer. The A,,Jh,, (nm) were: a subunit, 600/642; P subunit, 590/630.

CpcE, CpcF Phycocyanin subunit Change in emission (1.5 PM) k 1.6")

1. 1.5 PM CpcEFb a 2. 1.5 PM CpcE + CpcF a 3. 1.5 PM CpcE only a 4. 1.5 VM CpcF only a 5. 4.8 PM CpcE only a 6. 2.2 PM CpcF only a 7. 4.8 PM CpcE only P

%

-49

+2.7 -25

-2.2 +9.4 -0.8 +10

0.04 0.05i

P 9

0.04 ~

0.03 ~

l ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ n / , ~ ~ , ~ ~ ~ ~ ~ I ~ ~ ~ ~ ~ ~ ~ ~ ~ I ~ ~ ~ ~ , ~ ~ l ~ )

X (nm) 300 400 500 600 700

FIG. 3. Absorbance and emission spectra of aPc CpcEF. Emis- sion spectra were obtained as described in Table 111. Absorbance spectra were then taken of the mixtures, after cooling to room temperature, in a 1-cm path length quartz cuvette on a Perkin-Elmer A6 spectropho- tometer with assay mixture lacking protein as the reference. Thin lines, -CpcEF; thick lines, +CpcEF; solid lines, absorbance; broken lines, uncorrected fluorescence emission. Panel A, spectra of apc. Absorbance

0.63 VM apc k 1.9 VM CpcEF. Panel B , spectra of (xpc MBV adduct (0.75 spectra of 0.77 p apc 2 7.7 VM CpcEF, fluorescence emission spectra of

PM) * 7.5 PM CpcEF. The MBV adduct of apo-aPc was prepared by nonenzymatic reaction of PCB with prereduced apo-aPc in 50 nm Napi, 5 mM EDTA, pH 7, as described previously for apo-ap phycocyanin (5).

Some measurements were taken in duplicate, and the mean per-

CpcE and CpcF renatured together; entry 2 is separately renatured cent error is shown.

CpcE and CpcF.

The change in the absorbance spectrum of aPc MBV adduct in the presence of CpcEF (Fig. 3B) is similar to that seen for holo-aPc (with its natural PCB chromophore). There is an increase and sharpening of the near UV peak, again with a slight shift in the A,,,, from 369 to 367 nm. Also, there is a decrease in the red peak but, as with apt, without a shift in the A,,,, which is 650 nm with or without CpcEF. The red:near UV ratio is changed from 3.0 to 1.3 in the presence of CpcEF, and the red peak is broadened somewhat.

There is a substantial shift in the fluorescence emission spectrum of aPc MBV adduct in the presence of CpcEF (Fig. 3B). The peak is blue shifted, from 668 to 646 nm. Thus, although the reduction in emission a t 668 nm is 50%, there is little change in quantum yield: a loss of only 6% relative to that of aPC MBV adduct without CpcEF. With the reduction in absorbance at the excitation wavelength factored in (13% a t 580 nm), the change in quantum yield of the aPc MBV adduct in the presence of CpcEF, relative to that of apc MBV adduct alone, becomes positive, a 10% gain. Since the spectra are of uncorrected emission, and shifted, these quantum yields are only relative; nevertheless, it is reasonable to conclude that there is no dramatic loss in the quantum yield of the apc MBV adduct in the presence of CpcEF.

CpcE and CpcF were tested separately to assign functions to each subunit of the lyase. Separately renatured CpcE and

CpcF, when added together, reduce aPc fluorescence emission (Table IV, line 2), albeit not as effkiently as when renatured together to form CpcEF (line 1); this difference parallels that observed for the bilin addition assay. Similar concentrations of CpcE or CpcF alone do not have a large effect on aPc emission (Table IV, lines 3 and 4), although CpcE may cause a slight gain in fluorescence and CpcF a slight decrease. A larger amount of CpcF does not cause a decrease (Table IV, line 6), but 3-fold more CpcE causes approximately a %fold greater gain in emission (line 5). However, this CpcE effect is not phycocyanin subunit-specific, since the higher concentration of CpcE yields a similar gain in emission for p phycocyanin (Table IV, line 7).

Since CpcEF causes large spectroscopic changes in (Y subunits bearing bilin, the spectrum of the bilin substrate, PCB, in the presence of CpcEF was examined. After a 20-min incubation at room temperature, the PCB absorption spectrum in the presence of CpcEF showed a very slight decrease in the red: near UV ratio (6%) and a small red shift in the red peak A,,, (5 nm) relative to a control mixture (data not shown). There was no substantial, further change after a 7-h incubation. CpcE or CpcF alone had no significant effect on the absorption spectrum of PCB. There was no significant fluorescence for any of the mixtures at zero time or after 30 min.

Kinetics of CpcEF-mediated Addition of PCB to Apo-gC-In the course of these investigations, a fresh preparation of PCB was found to contain a higher fraction of PCB* than previous preparations (2.0% compared with < 0.5%). This PCB had been prepared in the standard way but with rotary evaporation to a small volume, rather than to dryness, in the final step. This

Phycocyanin a Subunit

FIG. 4. Rate of CpcEF mediated PCB addition to apo-aP" as a function of substrate concentration. Initial rates of holo-aPr formation (Vi) per 0.5-ml assay were determined as described under "Ex- perimental Procedures." Panel A, initial rate versus [PCB], fit to the Michaelis-Menten equation. The data shown are from two separate dilution series of PCB; in each dilution, the final concentration of trifluoroacetic acid was 7.5% (prior to addition to the assay mixture). These assays were performed in 150 mM Tris-HC1, pH 8, rather than 75 mM NaCl, 50 mM Tris-HCI, pH 8, to buffer the acid PCB solutions. The concentration of CpcEF was 15 n ~ . The K,, for PCB from this fit is 1.2 (e 0.2) p, the V,,, 3.97 (k 0.31) pmol min". Panel B, initial rate versus [apo-aPc], fit to the Michaelis-Menten equation. The concentration of CpcEF was 10 nM. The K,,, for apo-aPC is 10.8 (e 2.8) p ~ , the V,,, 7.3 (k

1.4) pmol rnin". Insets, Lineweaver-Burk plots of each data set.

change resulted in a stock solution of PCB in approximately 1:l (v/v) trifluoroacetic acid:dimethyl sulfoxide rather than neat dimethyl sulfoxide. Dilution of this stock solution with dimethyl sulfoxide resulted in slow loss of PCB*. Some attempts were made to separate PCB* from bulk PCB by reverse-phase HPLC, but these were unsuccessful. The PCB stock solution containing 2% PCB* was used for all assays described below.

A plot of the rate of holo-aPc formation uersus [PCB] and the linear fit to a Lineweaver-Burk plot of the same data demon- strate Michaelis-Menten kinetics with a K, for PCB of 1.2 PM (Fig. 4). If the real substrate for CpcEF is PCB*, a K,* of 24 nM ( 2 4 nM) can be calculated, equal to 2% of the K, for bulk PCB.

Plots of the rate of holo-ape formation uersus [apo-aPC] simi- larly exhibit Michaelis-Menten kinetics, with a K,,, for apo-aPC of 10.8 PM (Fig. 4B). This result indicates that the PCB kinetic studies were performed in a subsaturating concentration of apo-aPc (10 p j , so the kcat of CpcEF was calculated from the apo-aPC data only and determined to be 1.5 ( 2 0.3) rnin".

Since some rearrangement of apo-aPC may be required for CpcEF to access a-Cys-84, bilin addition assays were performed with 3 PM denatured apo-aPC as a substrate. The rates of 640 nm emission gain were similar whether denatured apo- aPc was added 5 min before starting the reaction by the addition of bilin (2.49 arbitrary unitdmin), or the reaction was initiated by the addition of denatured apo-ape (2.82 unitdmin). The rate obtained with 3 p~ native apo-ape was slightly higher (3.24 unitdminj.

CpcEF Specificity for Bilin: PCB Versus PEB-PEB serves as a bilin substrate for CpcEF-mediated addition to apo-cwPC. PEB by itself in the assay buffer has significant fluorescence (Fig. 51, which does not change over time. As demonstrated for apo-aP phycocyanin (7), PEB forms a fluorescent adduct with apo-aPC nonenzymatically at a measurable rate. The predominant adduct formed is a 15,16-dihydrobiliviolin rather than PEB, as evidenced by a red shifted fluorescence emission maximum relative to PEB-containing proteins (Fig. 5; Ref. 7). In the presence of CpcEF, the rate of gain in fluorescence is increased, and the product has a blue shifted emission maximum (Fig. 5).

By subtraction of the PEB-only emission spectrum, the spectra of the nonenzymatic and CpcEF-mediated addition products can be derived. The 571 nm emission maximum for the

Phycocyanobilin Lyase 8691

+ CpcEF

PEB only ....,..............

8

550 570 590 610 630 650 670 I ' I ' I - I ' I . I * I

( 4

FIG. 5. Fluorescence emission spectra of PEB and products of PEB addition to apo-aPc z CpcEF. In standard bilin addition assay buffer, emission spectra of 20 p~ PEB only, or PEB and 10 PM apo-aPC e 0.3 PM CpcEF, after a 5-min incubation a t 37 "C. With the emission spectrum of PEB only subtracted from each, the emission maxima are 571 nm for +CpcEF, 587 nm for -CpcEF. A,,, = 540 nm.

CpcEF-mediated addition product is similar to those of natural PEB-bearing proteins, which have emission maxima in the range 570-581 nm (21,221. The a subunit of Calothrix sp. PCC 7601 C-phycoerythrin has an emission maximum of 574 nm. Interestingly, the gain in 571 nm fluorescence in the presence of CpcEF does not have the biphasic character seen for PCB addition. This suggests that all or nearly all of the PEB is competent for CpcEF-mediated addition to apo-aPC.

Given that CpcEF can use PEB as a substrate, it was of interest to determine whether CpcEF might have the PEBPCB isomerase function described in the red alga Cyanidium caldarium (12). Bilin addition assay mixtures containing very high concentrations of CpcEF (0.85 VM) and 1.5 PM PEB were preincubated at 37 "C in the presence of 1 mM thioglycolate or 1 mM reduced glutathione for 45 min. Apo-aPC was added to start the reactions, and after 25 min the emission spectra of the products were compared: the spectra were essentially identical, and similar to that shown in Fig. 5. There was no indication of significant PCB adduct fluorescence in either case with excitation a t 600 nm.

Two methods were used to assess the relative catalytic efficiency of CpcEF with PEB and PCB as its bilin substrate. For relative binding affinity for each bilin, the kinetics of PCB addition in the presence of PEB were used to determine the KI

of PEB as a competitive inhibitor of PCB addition. For relative turnover rate with each bilin, the amount of adduct formed in a period of time with high [bilin] was measured.

For the KI of PEB, the kinetic data from one of the two PCB dilution series was compared with data obtained using the same dilutions of PCB along with 5 VM PEB. The addition of 5 PM PEB did not greatly affect the nonenzymatic rate of fluorescence gain at 640 nm, but PEB addition in the presence of CpcEF did produce a small rate of 640 nm fluorescence gain. This rate was not assessed in the presence of PCB; presumably, PCB should act as a competitive inhibitor for PEB addition, and any correction for the gain in fluorescence caused by the PEB addition should be less in higher [PCB].

Therefore, the CpcEF-mediated fluorescence gain at 640 nm because of the PEB addition in the absence of PCB was first simply subtracted from each observed rate in the presence of PCB. The KI for PEB was calculated, theoretical PCB-inhibited rates of fluorescence gain caused by the PEB addition were calculated for each [PCB], and these rates were subtracted from the observed rates. These rates were used to calculate a new KI, and the procedure was reiterated once. The resultant rates of the PCB addition to apo-aPC in the presence of PEB,

8692 Phycocyanin a Subunit Phycocyanobilin Lyase

1

0 5 10 15 20 25 30 [PCB1 (WM)

FIG. 6. PEB inhibition of CpcEF-mediated PCB addition to apo-aPc. Initial rates of holo-aPc formation (V,) per 0.5-ml assay in the presence of PCB alone were determined as described under “Experi- mental Procedures.” Initial rates of holo-aPc formation in the presence of 5 PM PEB were corrected for the contribution of PEB addition to apo-aPc as described under “Results.” The concentration of CpcEF in each assay was 15 nM. Curve fits are to the Michaelis-Menten equation. The kinetic parameters derived from these fits are: K,,, for PCB only, 1.85 (2 0.83) p; observed K,,, for PCB in the presence of 5 PM PEB, 12.58 (2 0.46) PM; V,, for PCB only, 3.67 (2 0.43) pmol min”; VmaX in the presence of 5 p PEB, 3.33 (20.07) pmol min”. Inset, Lineweaver-Burk plots of the same data.

with a fit of the data to the Michaelis-Menten equation, are shown in Fig. 6.

The kinetic parameters derived from these plots, which show an increase in the observed K, for PCB in the presence of PEB but a similar V,,,, verify that PEB is a competitive inhibitor of PCB addition with a KI of 0.86 (2 0.44) PM. This is illustrated graphically in the Lineweaver-Burk plot of the data (Fig. 6, inset). The ratios of KI to K, and KI to K,*, using the K, for PCB and the K,* for PCB* from the full data set in Fig, 4A, can be considered a measure of the relative affinity of CpcEF for the bilins. These are: K I X m , 0.72; KI:K,,,*, 30 (with a range of 15-65, using error limits for each value in the ratio).

For estimation of the relative efficiency of CpcEF with each bilin substrate, the rate of addition of each to apo-aPC was determined at a near saturating concentration of bilin. Bilin at 20 was used; this is near saturating for PCB containing 2% PCB* and should also be so for PEB, if the KI for PEB inhibition of PCB addition is similar to the K, for CpcEF-mediated PEB addition.

Since the extinction coefficient of native apc PEB adduct is not known, it was necessary to use denatured assay products for quantitation; established extinction coefficients for thioether-linked PCB and PEB in acid-urea could then be used. The difference spectra of the reaction products, +CpcEF minus control, for PCB and PEB addition reactions are shown in Fig. 7. These difference spectra can be considered the spectra of bilin adducts formed by CpcEF.

The difference spectrum for PCB addition product mixture has the correct long wavelength A,,, 663 nm, but a lower red:near UV ratio than do known PCB peptides (0.6 versus l.O), presumably because of an MBV component; MBV peptides have a lower red:near UV ratio (5) . The difference spectrum for PEB addition product mixture has two peaks in addition to that of PEB adduct (560 nm). One, at 665 nm, may be a PCB adduct. It is possible that there is a small amount of PCB in the stock PEB, formed by isomerization during purification, or on storage. As noted above, preincubation of CpcEF with PEB does not change the emission spectrum of the reaction products, so CpcEF does not appear to have PEBPCB isomerase activity. The identity of the other peak, at 784 nm, is unknown.

The amounts of PEB and PCB adducts formed by CpcEF in 5 min, calculated from the difference spectra, were similar, 74.4

t I I I I xx) 400 500 600 700 800

1 (nm) FIG. 7. CpcEF-mediated addition of PEB or PCB to apo-aPc

with a near saturating bilin concentration: difference spectra in acid-urea of the products of addition z CpcEF. The reaction conditions and treatment of the products are described under “Experimen- tal Procedures.” The difference spectra shown are of the +CpcEF and -CpcEF reaction products in 1 m18 of M acid-urea, 10 mM 2-mercaptoethanol. Broken l i n e , difference spectrum of PEB addition products. Solid line, difference spectrum of PCB addition products.

and 96.3 pmol, although 15 times more CpcEF was present in the PEB reaction (91 versus 6.1 pmol). Based on these values, the turnover rate for PEB is 0.16 rnin”, and for PCB, 3.2 min”. Thus, the ratio of kcat PCB to PEB is 19. The error for this method of rate determination is probably large but was not determined; a realistic estimate is an error similar to that for the kinetic constants observed above, about 25%, which trans- lates into a range of 12-32 for the ratio of kcat PCB:PEB.

The turnover rate for PCB from this experiment is substan- tially higher than that calculated from the data for variation of [apo-aPcl in Fig. 4B, 1.5 min”. A better comparison is with the turnover rate of 0.53 min” from the variation of [PCB] in the presence of 10 p~ apo-aPC (Fig. 4A), the concentration used for this experiment. Different preparations of CpcEF were used for these experiments, and the different turnover rates are likely to reflect variation in specific activity of CpcEF. Each renatured preparation of CpcEF differs somewhat in specific activity, and the activity decays over time at 4 “C. The ratio of turnover numbers for PCB and PEB should not be affected by this variation.

DISCUSSION

Overexpression in E. coli yielded what appeared to be mul- tiple forms of both CpcE and CpcF. The four CpcF fractions differed in the specific activity for bilin addition, but all were active in the presence of CpcE. All four forms have similar mobility on SDS-PAGE, but this does not preclude partial proteolysis (near the termini) as the source of variation. CpcF contains two cysteine residues, so it is possible that oxidation might affect the chromatographic and enzymatic properties of the protein.

It is not certain that each of the three polypeptides produced in E. coli on overexpression of CpcE is derived from the cpcE gene. Only one, that with an apparent molecular mass of 29 kDa on SDS-PAGE, has the mass predicted from the coding sequence of cpcE as well as bilin addition activity in the presence of CpcF. One interpretation of the results is that the polypeptide of apparent molecular mass 31 kDa is an E. coli protein which co-aggregates with overexpressed full-length CpcE in vivo. This sort of co-aggregation in inclusion bodies has been observed previously (23). The polypeptide of 28.5-kDa apparent molecular mass may be a proteolysis product of CpcE; if so, this proteolysis must result in loss of a portion of CpcE required for association with CpcF and bilin addition activity.

The results of HPLC SEC, along with the increased solubility and stability of each protein in the presence of the other, indi-


cate that CpcE and CpcF interact to form at least a heterodimer. It is possible that CpcEF may form higher order oligomers that are not stable to SEC under the conditions used. The physical association of CpcE and F helps to explain the lack of function of either protein alone, whether measured as bilin addition activity, cypc fluorescence quenching, or, more tentatively, a n effect on the absorption spectrum of PCB.

The quenching of fluorescence and reduction in red:near UV absorbance ratio of holo-ape by CpcEF verifies that the presence of the lyase can shift up(' into an unfolded or rearranged conformation, at least in the region that surrounds the bilin. It i s an open question whether this shift is actually induced by CpcEF binding or is the result of a change in a preexisting equilibrium between different ape conformations which is driven by the binding of one to CpcEF. That the loss of fluorescence is not caused by proteolysis is demonstrated by the lack of effect of either CpcE or CpcF alone, as well as the lack of a major change in size of a"<' after incubation, as judged by SDS-PAGE (not shown).

Some or all of the loss of cypc fluorescence in the presence of CpcEF may be a result of bilin removal from the protein. It is interesting that the effect of CpcEF on the a''" MBV adduct is to shift the emission maximum without a large effect on the quantum yield. The bilin structure near the thioether linkage is different for the MBV adduct; the chemistry of removal would have to be different than for PCB and, without reduction of the MBV, would presumably result in a 3-vinyl group rather than the 3-ethylidene of PCB. Therefore, it is possible that a"" MBV adduct is essentially a noneliminating product analog and that its +CpcEF absorption and emission spectra are those of thioether-linked bilin in the active site of CpcEF. If this is true, it should be possible to find a nonfluorescent MBV peptide that will become fluorescent on binding CpcEF.

The fluorescence quenching assay also confirms the specificity of CpcEF for apt, at least relative to p phycocyanin. This assay is potentially useful for assay of other, putative lyases. It does not require isolation of the apophycobiliprotein, only the more easily obtained holoprotein. It also does not require the bilin precursor, which is not known for every type of bilin found in phycobiliproteins.

That the rates of CpcEF-mediated bilin addition for denatured apo-aP" allowed to fold for 5 min or for the time of mixing (15 s ) are similar can be explained in two ways. Either the portion of apo-a"" which CpcEF recognizes is mostly unstructured even in native apo-a, or this portion of apo-a"c folds from its urea-denatured form in the time of mixing. The latter is quite possible; in pH shift experiments the apo-a chain of hemoglobin regains its native helical content with a half-time of 5 s (24).

The observation that a stock solution of PCB in 50% trifluoroacetic acid has a higher fraction of PCB"' than a solution in dimethyl sulfoxide is a tantalizing but not necessarily a revealing result. Braslavsky et al. (25) suggest that PCB dimethyl ester in solution is in equilibrium between the predominant, cyclohelical form (all 2, syn) and other, stretched forms. One or more of these stretched isomers has a higher quantum yield of fluorescence than does the cyclohelical form. For the analogous compound, biliverdin dimethyl ester, fluorescence increases to a constant quantum yield upon acidification below [HCll = M. Intramolecular proton exchange on the bilin is a n energy wasting process, and this exchange is prevented in the monoprotonated biliverdin dimethyl ester; it is the latter effect, and not a shift of the equilibrium to more stretched forms of the bilin, which is thought to explain the greater fluorescence at acid pH (26). The concentration of acid required for formation of substantial PCB" is far in excess of M and thus is likely to exert its effect through something other than monoprotonation of the bilatriene ring system.

Since the conformation of the bilin in cup'' is a stretched form (all 2, but anti, syn, anti; Ref. 27), it is reasonable to suggest tha t PCB'" may be the particular stretched isomer equivalent to that found in the holoprotein product of addition. Some support for this notion is provided in CpcEF-mediated addition of PEB to apo-ap", which lacks the previously observed clear biphasic character of the CpcEF-mediated addition of PCB (10). PEB lacks the conformational constraint of conjugation across the C-15 bridge between rings C and D and thus does not require synlanti isomerization to convert from a cyclohelical conformation to one closer to that found in the holo-a subunit. This fact may increase the effective concentration of "PEB'"" to the point that the slower, secondary, linear rate seen with PCB is not obvious for PEB.

Although PEB can serve as the bilin substrate for CpcEF- mediated addition to apo-a"", CpcEF shows some specificity for PCB"' both in binding and in turnover rate. The Michaelis- Menten kinetics of CpcEF with respect to PCB allowed meas- urement of the K, for PEB inhibition of CpcEF-mediated PCB addition to apo-cup". The quantity K,:K,,, can be considered a measure of the relative affinity of CpcEF for the two bilins. This ratio is 0.72 for PEB inhibition with respect to bulk PCB, which implies that CpcEF actually binds PEB better than PCB. How- ever, the ratio K1:K,,,'i', where K,,,'!' refers to PCB'", is 30, which implies that CpcEF has a 30-fold higher affinity for PCB:': than for PEB. These ratios are further evidence for at least partial PCB:': character in PEB.

The ratio of turnover rates, PCB:PEB, at near saturating concentrations of bilin, is 19, reflecting discrimination of CpcEF for bilin at the level of catalysis as well as binding. The product of the ratios PCB"':PEB for binding and catalysis is the ratio of the catalytic efficiencies, kCat:Kn,, of the enzyme for each bilin; thus, the ratio (kCat:K,, PCB" to k,,t:K,,, PEB) is 570. Using the error ranges for the binding and catalysis ratios, the range for the ratio of catalytic efficiencies PCB'":PEB is 180- 2,080.

If PEB is on the biosynthetic pathway for PCB in cyanobacteria as well as the red alga C. caldarium, there should be PEB present in the cell as well as PCB, but there is no evidence for a detectable amount of PEB adduct in natural phycocyanin. However, the limit of detection for PEB adduct in phycocyanin i s an open question. Essentially no 570-580 nm fluorescence emission is observed with natural phycocyanin, but energy transfer within the ap protomer might mask steady-state emission from any PEB adduct.

The emission spectrum obtained on 530 nm excitation of purified a''' shows a slight bulge at 570 nm which may be caused by a PEB-like bilin adduct (not shown). The purification of ape requires chromatography under denaturing conditions, and thus any PEB-like bilin may be artifactual, produced by isomerization or oxidation of the natural bilin. Nevertheless, the observed 570 nm emission can be used to estimate an upper limit for the amount of PEB adduct that might be present in natural ape. Using a per bilin of 63,400 M" cm" for PEB, and 9,900 M" cm" for PCB (calculated from the extinction coefficients of native C-phycoerythrin and phycocyanin) and assuming a 1:l ratio of quantum yields for the two bilins in apc (the quantum yield of cypc PEB adduct relative to PCB adduct is not known but can be estimated from CpcEF assays in saturating concentrations of bilin; the ratio is near l:l), the upper limit for the fraction of PEB adduct in ape can be calculated to be 0.026%. For comparison with the ratio of catalytic efficiencies of CpcEF PCB*:PEB, the inverse of this gives the estimated lower limit for the ratio of PCB adduct to PEB adduct in apc, 3,850.

Assuming a ratio of PCB":PEB in vivo of 1:1, the ratio of catalytic efficiencies PCB":PEB alone is not quite sufficient to

8694 Phycocyanin cr Subunit Phycocyanobilin Lyase

explain the low fraction of PEB adduct found in natural a''. Given that the nature of PCB* is not known, it is difficult to say what the ratio of PCB*:PEB is in uiuo. Extract of C. caldarium was shown to catalyze the isomerization of 3(Z)-PEB to 3(2)- and 3(E)-PCB, and the ratio of PCB:PEB after a 20-min incubation appears to be at least 50:1(12). If this ratio reflects that in uiuo, and assuming that most or all of the PCB in vivo is in the form PCB*, when combined with the ratio of catalytic efficiencies of CpcEF it is sufficient to rationalize the fraction of PEB adduct in a''.

The ability of CpcEF to catalyze the addition of a bilin other than PCB to apo-aPc may be a useful property, particularly if it proves general for other phycobiliprotein bilin lyases. With the appropriate 3-ethylidene bilin precursor it would then be possible to design phycobiliproteins with spectroscopic properties not found in natural phycobiliproteins, for use as fluorescent reagents in antibody conjugates or for other purposes.

REFERENCES

2. Cole, W. J., Chapman, D. J., andsiegelman, H. W. (1967) J. Am. Chem. SOC. 89, 1. Glazer, A. N. (1989) J. Biol. Chem. 264, 1 4

3. Chapman, D. J., Cole, W. J., and Siegelman, H. W. (1967) J . Am. Chem. Soc. 89,

4. Beale, S. I., and Cornejo, J. (1991) J. Biol. Chem. 266, 22341-22345

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