Characterization of the bilin attachment sites in R-phycoerythrin.

The amino acid sequence around the sites of attachment of all the bilin prosthetic groups of Gastroclonium coulteri R-phycoerythrin, (alpha beta)6 gamma, have been determined. The sequences of tryptic peptides derived from the alpha and beta subunits are (Formula: see text) where the designations alpha and beta refer to the subunits from which the peptides derived. Cysteinyl residues involved in bilin attachment are indicated with an asterisk. Each peptide carries a single bilin, either phycoerythrobilin (PEB) or phycourobilin (PUB). Spectroscopic studies on the gamma subunit indicate the presence of one PEB and three PUB groups. However, five unique tryptic peptides, gamma-A through gamma-E, were characterized, indicating that Gastroclonium R-phycoerythrin is a mixture of at least two species, (alpha beta)6 gamma and (alpha beta)6 gamma', with gamma subunits differing in amino acid sequence. The sequences of the gamma subunit bilin peptides (see below) were not homologous to those from alpha and beta subunits of any biliprotein. (Formula: see text) The bilins in all these peptides are attached through single linkages to a cysteinyl residue, except for the phycourobilin on peptide beta-3 which is attached through two thioether linkages to cysteinyl residues 10 amino acids apart. The availability of small bilin peptides was exploited to obtain more accurate molar extinction coefficients for peptide-linked PEB and PUB groups. Application of these extinction coefficients in the calculation of the bilin content of R-, B-, and C-phycoerythrins shows that there are 5 bilins/alpha beta in each of these three biliprotein types.

where the designations a and 8 refer to the subunits from which the peptides derived. Cysteinyi residues involved in bilin attachment are indicated with an asterisk. Each peptide carries a single bilin, either phycoerythrobilin (PEB) or phycourobilin (PUB).
Spectroscopic studies on the 7 subunit indicate the presence of one PEB and three PUB groups. However, five unique tryptic peptides, 7-A through 7-E, were characterized, indicating that Gastroclonium R-phycoerythrin is a mixture of at least two species, (C$?) S7 and (C$?)67', with 7 subunits differing in amino acid sequence. The sequences of the y subunit bilin peptides ( s e e below) were not homologous to those from a and / 3 subunits of any biliprotein. ?
The availability of small bilin peptides was exploited to obtain more accurate molar extinction coefficients for peptide-linked PEB and PUB groups. Application of these extinction coefficients in the calculation of the bilin content of R-, B-, and C-phycoerythrins shows that there are 5 bilins/C$? in each of these three biliprotein types.
Phycobiliproteins are a family of light-harvesting macromolecules which function as components of the photosynthetic apparatus in cyanobacteria and in two groups of eukaryotic algae, the red algae and the cryptomonads (1,2). Among these proteins, phycoerythrins are characterized by strong absorption bands in the green region of the spectrum. These red-colored proteins fall into three spectroscopically distinct classes: C-phycoerythrins ( X , , , -565 nm), B-phycoerythrins (Xmnx -540-560 nm, shoulder -495 nm), and R-phycoerythrins ( X, = -565,545, and 495 nm) (3). Only a single type of phycoerythrin is present in any organism.
B-and R-phycoerythrins are proteins of -240,000 daltons and have a subunit composition ((~/3)~y. All of the subunits bear bilin chromophores (4). The -550 nm absorbance of phycoerythrins is due to the phycoerythrobilin (PEB)' chromophore whose structure and modes of linkage to the protein have been the subject of recent detailed studies (5)(6)(7)(8). The peak at 495 nm is due to a different bilin, phycourobilin (PUB) (9). The spectroscopic differences between the three classes of phycoerythrins reflect the content and ratio of the PEB and PUB groups (4). A number of attempts were made to determine the structure of PUB, a major prosthetic group in the phycoerythrins of higher red algae, but proof of structure has not yet been achieved nor the mode(s) of linkage to the polypeptide clearly established. Probably the best study on PUB was that of Vaughan (10) who concluded that this bilin was closely related to d-urobilin.
Bilins are linked to the polypeptide chains of the biliproteins through thioether linkages (2). The colored chromophores serve as convenient markers in the purification of bilin peptides. Short chromopeptides provide excellent objects for the determination both of the structure of the bilin and its mode of attachment by nondestructive procedures (5-8,ll).
Only limited information is available on the primary structure of phycoerythrins. Muckle et ~l .
(12) described the amino acid sequences of tryptic PEB peptides from the cy and /3 subunits of C-phycoerythrins. In a comprehensive study, we have recently determined the amino acid sequences and modes of linkage for the five PEB peptides derived from the a and Bilin Peptides from R-Phycoerythrin 4857 dence for the structure of both singly and doubly linked PEB This report is concerned with the structure of the chromopeptides from the R-phycoerythrin of the higher red alga Gastroclonium coulteri. We present here amino acid sequences of the two PEB peptides from the a subunit and of two PEB and one PUB peptide from the /3 subunit. These peptides account for all the bilins on the (Y and p subunits. Spectroscopic analysis of the y subunit showed the presence of one PEB and three PUB groups on this polypeptide. However, we isolated five unique y subunit bilin peptides, one containing PEB and four containing PUB. Consequently, there are at least two y subunits differing in amino acid sequence in Gastroclonium R-phycoerythrin. Further studies, documented in the accompanying paper (13), of small bilin peptides isolated in the course of this work, permitted the unambiguous determination of the structure both singly and doubly linked PUB groups. groups (5, 7,8).

DISCUSSION
The results presented here (Figs. 5 and 7) demonstrate that the (Y and /3 subunits of G. coulteri R-PE yield five unique tryptic bilin peptides. The CY subunit carries two PEB groups, each linked to the polypeptide through a single thioether bond. The /3 subunit carries three bilins: two PEB groups, each linked through a single thioether bond, and a PUB linked through two such bonds. Our earlier study on B-PE produced the unexpected finding that both singly and doubly linked PEB groups were present within a single protein (6). The present work demonstrates that this situation holds for phycourobilins as well.
All five of the a and /3 subunit bilin peptides show great similarity to the corresponding chromopeptides derived from Porphyridium cruentum B-PE. In fact, three of the peptides from R-and B-PE have identical amino acid sequences (Fig.  9); the two remaining peptides differ in only a few residues (Figs. 9 and 10). These results are remarkable in two respects. The unicellular alga P. cruentum belongs to the subclass Bangiophycidae and is a member of the order Porphyridiales which includes the simplest red algae of this subclass, whereas Gastroclonium is a seaweed belonging to the much more complex subclass Florideophycidae, order Rhodymeniales.
The close correspondence of the sequences of the Band Rphycoerythrins, which are chloroplast components, suggests that the chloroplasts of red algae are much more closely related to each other than are the algae within which they are contained. The second noteworthy observation is that in peptides p-3 from B-and R-PE, 37 of the 40 residues are identical (Fig. 10). Yet, the Gastroclonium peptide carries a doubly linked PUB whereas the P. cruentum peptide possesses a doubly linked PEB. Further, this sequence bears a strong resemblance to residues 38-77 in a region of C-PC which does not contain bilin (Fig. 10). The finding of high sequence conservation over a 40-residue stretch for the p-3 peptides Portions of this paper (including "Experimental Procedures," "Results," Figs. 1-8, and Tables I-XI) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3364, cite the authors, and include a check or money order for $11.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. reinforces the view that the amino acid sequence at the bilin attachment site is not the sole determinant of the bilin identity at that site. Conformational parameters must also play an important role (29).
Spectroscopic analysis of the R-PE y subunit shows that this polypeptide contains one PEB and three PUB groups. However, five tryptic bilin peptides (Fig. 6), one containing PEB and four containing PUB, assigned to this subunit, were isolated in comparable yield. Yu et al. (30) have shown that Callithmnion R-PE contains two y subunits differing in molecular weight. From studies on the composition of P. cruentum phycobilisomes, it appears that B-PE may contain three different y subunits (31). The present study of the amino acid sequences of bilin peptides derived from the y subunit indicates that Gastrocloniurn R-PE contains at least two y subunits, but that these subunits must be very similar in amino acid sequence. It would be more appropriate to describe R-PE as a set of at least two biliproteins differing only in the nature of the y subunit, i.e. (

(Y/3)6y and ( ( Y / 3 ) 6 7 ' .
The y subunit sequences reported here are the first determined for such a subunit. All of the bilins on the y subunit(s) are attached through single thioether bonds. These peptides comprise -25% of the total sequence for this polypeptide. In view of the fact that the CY and /3 subunits of all biliproteins show considerable homology (2, 6 , 25), it is striking that the y subunit bilin peptides show no homology to any known (Y and /3 subunit sequences. The sole exception is the PEBcontaining peptide y-A which possesses the sequence Cys(bi1in)-X-Arg, common to several a and /3 subunit bilin peptides (Fig. 9). The structures of both singly and doubly linked PUB chromophores were established by 'H NMR and mass spectrometry analyses of peptides p-3, y-BV8, and y-DP (Tables VI  FIG. 10. Homology between polypeptide regions bearing doubly linked bilins. The homologous peptides from Gastmlonium R-PE and P. c a ntum B-PE, which carry doubly linked PUB and PEB groups, respectively, are compared to each other and to a homologous sequence, not containing bilins, encompassing residues 38-77 of the Synechococcus 6301 C-phycocyanin j3 subunit (28). The asterisks indicate the positions of the two bilin-linked cysteine residues and the dots represent identical amino acid residues. #38-77 and XI), as detailed in the accompanying report (13).

R-PE
Previously the molar extinction coefficients for PEB and PUB were determined from absorption spectra in acid urea and amino acid analyses of entire biliproteins as well as of subunits (4,32). The value reported for PEB was 43.3 m"' cm-' at 555 nm (32) and for PUB, 104 mM" cm-' at 495 nm (4). The availability of several pure small PUB-and PEBcontaining peptides permitted a much more accurate determination of the tnolar extinction coefficients applicable to these protein-bound bilins in 8 M urea at pH 3. An e (550 nm) value of 53.7 m"' cm-' was obtained for PEB and a value for e (495 nm) of 94 m"' cm" for PUB. The molar extinction coefficient for PUB is identical to that reported for d-urobilin hydrochloride in chloroform (33). Application of the newly determined extinction coefficients to the absorption spectra of B-PE (4) and R-PE in 8 M urea at pH 3 gives chromophore contents in accord with the numbers determined from sequence studies. The new values also lead to a reassessment of the bilin content of C-phycoerythrin. This biliprotein contains only PEB groups. The extinction coefficient of C-phycoerythrin at 555 nm in acid urea is 260 m"' cm"/a@ (32). The molar extinction coefficient, determined by Glazer and Hixson (32) for protein-bound PEB, of 43.3 m"' cm", yielded a value of 6 PEB/a@. The extinction coefficient of 53.7 m"' cm-' determined in the present study leads to an estimate of 4.84 PEB/a@ in C-phycoerythrin. It is evident that R-, B-, and C-phycoerythrins all have 5 bilins/a@ monomer. 14.