Cyanobacterial PhycobilisomesRober t MacColl

Wadsworth Center, N ew York S tate Departm ent of Health , P.O. Box 509, Albany, N ew York 12201-0509Received J uly 9, 1998, and in revised form November 11, 1998

Cyanobacteria l phycobilisomes harve st ligh t andcause energy migration usually tow ard photosys-tem II reaction cen ters . Energy transfe r from phyco-bilisomes directly to photosystem I may occur undercerta in ligh t conditions . The phycobilisomes areh igh ly organ ized complexe s of various biliprote in sand linker polypeptide s . Phycobilisomes are com-posed of rods and a core . The biliprote in s have the irbilin s (chromophore s) arranged to produce rapidand directional energy migration through the phyco-bilisomes and to ch lorophyll a in the thylakoidmembrane . The modu lation of the energy leve ls ofthe four chemically diffe ren t bilin s by a varie ty ofinfluence s produce s more effic ien t ligh t harve stingand energy migration . Acclimation of cyanobacte -ria l phycobilisomes to grow th ligh t by complemen-tary chromatic adaptation is a complex proce ss thatchanges the ratio of phycocyan in to phycoerythrinin rods of certa in phycobilisomes to improve ligh tharve sting in changing habitats . The linkers governthe assembly of the biliprote in s in to phycobili-somes , and, even if co lorle ss , in certa in case s theyhave been shown to improve the energy migrationproce ss . The Lcm polypeptide has several functions ,inc luding the linker function of de te rmin ing theorgan ization of the phycobilisome core s . De ta ils ofhow linkers perform the ir tasks are sti ll topics ofin te re st. The transfe r of exc itation energy from bilinto bilin is cons idered, particu larly for monomersand trimers of C-phycocyan in , phycoerythrocyan in ,and allophycocyan in . Phycobilisomes are one of thew ays cyanobacteria thrive in varying and some-times extreme habitats . Various biliprote in proper-tie s perhaps not re lated to photosyn the s is are con -s idered: the photorevers ibility of phycovio lobilin ,b iophys ica l s tud ie s , and bi l ip ro te in s in e vo lu -tion . r 1998 Academic P re ssKey Word s: bi lipro te in s ; complemen tary ch ro -

matic adaptation ; energy migration in photosyn the -s is ; phycobilisomes .

PHYCOBILISOMES

Phycobilisomes are protein complexes tha t func-t ion in ligh t harvest ing and energy migra t ion , usu-

a lly to photosystem II. When the energy absorbed bythe chromoproteins (biliproteins) of the phycobili-somes reaches the react ion centers of photosystemII, there occurs a t ransduct ion of the excita t ionenergy to chemica l energy. Biliproteins absorb radia -t ion in regions of the visible spect rum where chloro-phyll a has low absorpt ivit ies. Phycobilisomes arepresent in procaryot ic cyanobacter ia and eucaryot icred a lgae. Although th is review fea tures cyanobacte-r ia l phycobilisomes, when per t inent , red a lga l phyco-bilisomes and biliproteins will be ment ioned selec-t ively. The composit ion of phycobilisomes var ies fromorganism to organism, and individua l organismshave phycobilisomes that are changed by the environ-ment in diverse ways.The beginning of study of the phycobilisome came

in 19651966 when Gant t and Cont i, using reda lgae, per formed a ser ies of exper iments tha t re-su lted in the isola t ion of ext r insic granules from theou t er or st r oma l su r face of the ch lorophyll a-conta in ing thylakoid membrane. These water-solubleisola tes were shown to conta in the biliproteins of thea lgae (Gant t and Cont i, 1965, 1966a , 1966b).Following th is lead, other research showed by

elect ron microscopy tha t cyanobacter ia and cy-anelles likewise conta in phycobilisomes (Wildmanand Bowen, 1974; Edwards and Gant t , 1971; Gant tand Cont i, 1969; Bourdu and Lefor t , 1967; Lefor t ,1965; Edwards et al., 1968). There are differen tst ructura l types of phycobilisomes, but in th is reviewthe hemidiscoida l phycobilisomes having a t r icylin-dr ica l core and six rods, which are found extensivelyin the cyanobacter ia , will be discussed in most deta il.The hemidiscoida l phycobilisomes are divided in to

three core types: bicylindr ica l; t r icylindr ica l (F ig. 1);and pentacylindr ica l (Ducret et al., 1996, 1998; Sidler,1994; Yamanaka et al., 1980; Williams et al., 1980;Glauser et al., 1992a; Isono and Katoh , 1983, 1987).The pentacylindr ica l phycobilisomes have eight rodsassocia ted with a five-cylinder core. In addit ion tothe hemidiscoida l types, there are other st ructura llydifferen t phycobilisomes: hemiellipsoida l, bundle-shaped, block-shaped, and hemiellidiscoida l (Ducretet al., 1998; Wehrmeyer et al., 1988; Wehrmeyer,

JOURNAL OF STRUCTURAL BIOLOGY 124, 311334 (1998)ARTICLE NO. SB984062

311 1047-8477/98 $25.00Copyr ight r 1998 by Academic PressAll r igh ts of reproduct ion in any form reserved.

1983). A cyanobacter ium with biliproteins not orga-n ized in to phycobilisomes has a lso been repor ted(Reuter et al., 1994).The work of Gant t and co-workers (e.g., Gant t and

Cont i, 1965, 1966a , 1966b; Gant t and Lipschultz,1973, 1977; Gant t et al., 1976, 1988; Mimuro et al.,1986b; Redlinger and Gant t , 1981, 1982) was semi-na l. In the more than three decades since the or igi-na l breakthrough, there has been excit ing progress.The techniques of biochemist ry, molecula r biology,spect roscopy, x-ray diffract ion , elect ron microscopy,and biophysics have served to add to the understand-ing of the way these granules per form their funct ionsin photosynthesis. There are previous reviews oncer ta in of these topics (e.g., Sidler, 1994; Gant t ,1975, 1990; Scheer, 1987; MacColl and Guard-Fr ia r,1987; Thomas, 1989; Zuber, 1987; Bryant , 1991;Stadnichuk, 1995).Biliproteins are ca tegor ized in to three types by

bilin energy: those of h igh energy (phycoerythr ins orphycoerythrocyanin), in termedia te energy (phycocya-n ins), and low energy (a llophycocyanins). Energywill flow from highest - to lowest -energy pigmentsand th is is how the phycobilisomes are organized(Fig. 1). There are six rods in hemidiscoida l phycobili-somes having three cylinders in their core. The coreis situa ted in proximity to the thylakoid membraneand photosystem II, where chlorophyll a is loca ted(Figs. 1 and 2). The rods have the phycoerythr in orphycoerythrocyanin , if either is presen t , fur thestfrom the core. The core has the a llophycocyanins, ofwhich there are two funct iona l types: a llophycocya-n in having a 650-nm maximum and two lower-energy a llophycocyanins, the Lcm polypept ide and

the aB polypept ide, which t ransfer energy to chloro-phyll (Gant t , 1975; MacColl and Guard-Fr ia r, 1987;Bryant et al., 1979; Sidler, 1994; Bald et al., 1996).The rods are composed of stacks of disks, and thedisk adjacent to the core is invar iably phycocyanin .Dividing the bilins in to rods will make it simpler tohave ar rangements of the bilins for efficien t energyt ransfer. There are typica lly two to six disks in a roddepending on the organism and the impact of theenvironment .Tandeau de Marsac and Cohen-Bazire (1977) pro-

pelled the understanding of phycobilisomes to ahigher level with the discovery of the linker polypep-t ides. Their work was a ll the more outstandingbecause, pr ior to their 1977 publica t ion , there was nohin t of these linkers. They found tha t a new group ofpolypept ides existed with in the phycobilisomes, andit is now known tha t most of these are color less whileothers have chromophores, like the biliproteins. Tan-deau de Marsac and Cohen-Bazire (1977) poin tedout tha t the linkers would funct ion in a t tach ing the

FIG. 1. Model of a t r icylindr ica l hemidiscoida l phycobilisome.In the upper drawing, the phycobilisome is shown at tached tophotosystem II. Two photosystem I par t icles are shown adjacentto the photosystem II par t icle. A t r icylindr ica l core is shown, andthe two bot tom cylinders a t tach to the thylakoid membrane. Thetwo basa l cylinders tha t a re a t tached to the membrane will eachbe ca lled A, and the cylinder loca ted above them will be ca lled B ortop cylinder.

FIG. 2. Phycobilisome deta ils. The core conta ins the a llophy-cocyanins and a t taches to the thylakoid membrane. The bot tomtwo cylinders, the basa l or A cylinders, a re a t tached to thethylakoid membrane. The rod conta ins phycocyanin and, whenpresent , phycoerythr in or phycoerythrocyanin . The core com-plexes are t r imer or t r imer-like complexes: (1) a3b3Lc8.9; (2) a3b3;(3) a2b2b16Lcm; and (4) aBa2b3Lc8.9. Where a and b are the subunit sof a llophycocyanin , Lc8.9 is a core linker, b16 is a bilin -conta in ingpolypept ide, and Lcm and aB are the two lower-energy allophycocya-n ins. The two cylinders adjacent to the thylakoid membrane arecomposed of one copy each of complexes 14. The th ird cylinder,the B or top cylinder, is composed of two copies each of complexes 1and 2. The side view of the core shows two of the cylinders and theth ird is h idden . Since there are four t r imers per cylinder, the tota lnumber of t r imers is 12 in it s core.

312 ROBERT MACCOLL

phycobilisomes to the thylakoid membrane and inassembly of the biliproteins. Exper iments using pur i-fied linkers and biliprotein provided direct evidencetha t linkers funct ion in assembly of biliproteins(Lundell et al., 1981a). As cer ta in of the linkers causeassembly of the biliproteins, they produce changes inthe spect ra of biliproteins, and th is may serve todirect energy migra t ion more efficien t ly through thephycobilisomes (Yu et al., 1981; Wendler et al., 1986).Linkers are found in both cyanobacter ia l and reda lgae phycobilisomes and may be 1015% of the tota lmass (Tandeau de Marsac and Cohen-Bazire, 1977;Yamanaka et al., 1978; Koller et al., 1978).

BILIPROTEINS

Biliproteins of the cyanobacter ia are obta ined asdissocia t ion products of the phycobilisomes. Whenthe procaryot ic cells a re broken and the cellu la rconten ts escape in to a low-ionic-st rength aqueousmedium, the phycobilisomes dissocia te in to the var i-ous components, and the biliproteins, either with orwithout a t tached linkers, a re obta ined for ana lysis.The rela t ive stability of biliproteinlinker complexesvar ies among the biliproteins and differen t sources.Allophycocyanin , with a 650-nm maximum, is foundnear neut ra l pH as a t r imer having three a and threeb polypept ides; each of these polypept ides has onechromophore (bilin ). Tr imers (a3b3) a re r inglike as-semblies of three monomers (ab) having threefoldsymmetry. C-Phycocyanin is found as a complexsolu t ion of a3b3, a6b6, and other oligomers. Thehexamers (a6b6) a re disk shaped, formed by face-to-face assembly of t r imers. Rods are formed by face-to-face assembly of these disks. The a polypept ide hasone chromophore and b has two. C-Phycoerythr in ,which a lso assembles to disks, has an a polypept idewith two chromophores and a b with three. These aand b polypept ides vary in molecula r mass from15 000 to 20 000 Da.Monomers (ab) of a llophycocyanin and C-phycocya-

n in are of in terest in the study of biliproteins be-cause they represent minimum unit s having a ll thebilins. Homogeneous and stable monomers of thesebiliproteins can be prepared a t a sligh t ly acid pH orin the presence of the chaot ropic anions th iocyana teor perch lora te (MacColl, 1983; MacColl et al., 1971,1980, 1981). The chaot ropes are more universa llyapplicable. For a thermophilic protein , a h igherchaot rope concent ra t ion is needed to produce mono-mers (Edwards et al., 1997). Monomers of phycoeryth-rocyanin have been prepared using urea or chao-t rope (Siebzenr ubl et al., 1989). Monomers can beproduced, without a dissocia t ing agent near neut ra lpH, a t a very low protein concent ra t ion (MacColl andGuard-Fr ia r, 1987). Biliproteins can be completelydissocia ted in to the individua l a and b polypept ides

by var ious dena tur ing agents (MacColl and Guard-Fr ia r, 1987).The determina t ion of the amino acid sequences of

var ious biliproteins has been an impor tan t step inunder st anding their proper t ies. Zuber and co-workers obta ined the first complete amino acid se-quences of any biliprotein using cyanobacter ia l C-phycocyanin and allophycocyanin (Frank et al., 1978;Sidler et al., 1981), and Troxler and co-workers didlikewise for red a lga l C-phycocyanin and a llophyco-cyanin (Offner et al., 1981; Troxler et al., 1981; Offnerand Troxler, 1983). There is sign ificant homologyamong the var ious biliproteins. There is very out -standing conserva t ion of the placement of the bilinsin the amino acid sequence.The complete sequence of the amino acids is a lso

known for phycoerythrocyanin (F uglista ller et al.,1983). The bilins are loca ted a t a84, b84, and b155.There is a 21% homology between the two polypep-t ides, 63% homology of a and 67% homology of b tothe a and b polypept ides of C-phycocyanin , respec-t ively. Sidler et al. (1986) have obta ined the aminoacid sequence for C-phycoerythr in . For th is cyanobac-ter ia l biliprotein there are five bilins, two on the apolypept ide and three on b. The bilins are bound tocysteines a t a84 and a143, and there is a bilin doublybound to b50 and b61. The remain ing two bilins area t tached a t b84 and b155. A CU-phycoerythr in hasa lso been sequenced (Wilbanks et al., 1991).The bilins are open-cha in tet rapyr roles (F ig. 3),

which are cova len t ly a t tached to apoprotein byth ioether bonds to pa r t icu la r cyst eine residues(Fig. 4). In the many cyanobacter ia , there appear tobe four differen t bilins (Table I): phycocyanobilin ,phycoerythrobilin , phycourobilin , and phycoviolobi-lin (a lso ca lled phycobiliviolin ). They are a t tachedthrough their A r ings or have join t a t tachments totheir A and D rings (Fig. 3) (MacColl and Guard-Fr ia r, 1987; Bishop et al., 1987). C-Phycocyanin andallophycocyanin have phycocyanobilins and C-phyco-erythr in has phycoerythrobilins (Table II). Meta lions are not associa ted with the bilins.Besides C-phycocyanin , a llophycocyanin , and C-

phycoerythr in , cer ta in cyanobacter ia possess dua l-bilin biliproteins, including the CU-phycoerythr ins,phycoerythrocyanin , R-phycocyanin II, and a uniquephycocyanin processing one urobilin and two phyco-cyanobilins (ca lled phycocyanin WH8501) (Hoffmanet al., 1990; Swanson et al., 1991; Stadnichuk, 1993;Ong and Glazer, 1987; Fujita and Schimura , 1974;Rippka et al., 1974; Bryant et al., 1981; Kursar et al.,1981; Ong et al., 1984; Stadnichuk et al., 1985; Cox etal., 1985; Larkum et al., 1987; Par ry, 1988; Ong andGlazer, 1991; Hirose et al., 1969; Bryant et al., 1976).The CU-phycoerythr ins conta in phycourobilin in ad-dit ion to phycoerythrobilin , a common bilin combina-

313PHYCOBILISOMES

t ion in red a lga l phycoerythr ins. Phycoerythrocya-n in conta ins phycoviolobilin and phycocyanobilin .R-Phycocyanin II has both phycocyanobilin and phy-coerythrobilin (Table II), and the phycocyanobilin isa t b84, and the phycoerythrobilins are a t b155 anda84, which are the same bilin loca t ions as C-phycocyan in and phycoeryth rocyan in (Ong andGlazer, 1987). Stadnichuk (1995) has reviewed stud-ies on bilin conten t .The CU-phycoerythr ins, first observed in cyanobac-

ter ia l ext ract s by Hirose et al. (1969), a re a verydiverse group of biliproteins. The C indica tes acyanobacter ia l source for the biliprotein , and C-phycoerythr in conta ins phycoerythrobilin . A CU-phycoerythr in has phycourobilin in addit ion to phy-coerythrobilin . Their mult ifaceted diversity definesthem as a group. The first aspect of th is diversity istha t the ra t io of the two bilins var ies widely fromprotein to protein (Ong and Glazer, 1991). For ex-ample, one par t icu la r CU-phycoerythr in has fourphycourobilins and one phycoerythrobilin on a mono-mer and another has four phycoerythrobilins andtwo phycourobilins (Table III). The number of bilinsper monomer differs, being either 5 or 6. Those withfive bilins have three on the b polypept ide and two ona, and those with six have an addit iona l phycourobi-

lin on a. There may be two differen t phycoerythr insper cyanobacter ium. In some cases, a CU-phycoery-thr in may be found with a C-phycoerythr in (Ong andGlazer, 1991; Stadnichuk, 1993), and in other cyano-bacter ia there may be two differen t CU-phycoery-thr ins. A prepara t ion of CU-phycoerythr in had themolecular mass of a typica l disk, a6b6g, of a phycobili-some rod (Ong et al., 1984). When the polypept ideswere separa ted, in addit ion to a and b, there were

FIG. 3. Structures of selected bilins. FIG. 4. Posit ion ing of bilins in the amino acid sequences ofcer ta in biliproteins: C-phycoerythr in , C-phycocyanin , and allophy-cocyanin . PEB and PCB are phycoerythrobilin and phycocyanobi-lin , respect ively.

TABLE IBilins of Cyanobacter ia l Biliproteins

Bilin a Conjuga ted double bonds b

Phycocyanobilin 8Phycoerythrobilin 6Phycoviolobilin 7Phycourobilin 5

a Bishop et al. (1987) for st ructures.b More conjuga t ion for a bilin produces lower energy for the first

excited sta te. For example, C-phycocyanin monomers have onlyphycocyanobilins and usua lly have a 614-nm absorpt ion maxi-mum. The phycourobilins in CU-phycoerythr in have an absorp-t ion maximum at 495 nm.

314 ROBERT MACCOLL

th ree red bands of around 29 000 molecula r massthe expected va lue of rod-rela ted linkers. Whenlinkers from the rods bear bilins, they are named gpolypept ides. Linkers from rods of C-phycocyaninand C-phycoerythr in are probably color less. Onelinker from a CU-phycoerythr in has been sequencedand shown to have a single phycourobilin (Wilbanksand Glazer, 1993). Such bilin diversifica t ion signifiesligh t harvest ing as an impor tan t aspect of the ecol-ogy for these organisms.Biliproteins funct ion in photosynthesis by harvest -

ing sola r energy in regions where chlorophyll aabsorbs poor ly, causing th is energy to migra te withgrea t efficiency from the poin t of absorpt ion toward areact ion center. It is impor tan t tha t biliproteinsabsorb photons over wide ranges of energies. Thevar ious bilins achieve a diversity of ligh t absorpt ionin it ia lly because of the var ia t ions in their systems ofconjuga ted double bonds (Fig. 3). Phycocyanobilinhas more conjuga t ion than phycoerythrobilin so solu-t ions of phycocyanin near neut ra l pH absorb a t lowerenergy (610- to 620-nm maximum) than phycoery-thr in (545- to 565-nm maximum). Phycourobilin hasst ill less conjuga t ion (st ructure not shown) andabsorbs maximally around 495 nm, a higher energy than phycoerythrobilins (Table I). Besides bilipro-teins having a par t icu la r bilin for ligh t harvest ing, a

biliprotein may have two differen t bilins, whichdramat ica lly increase the range of photon absorp-t ion . So far, no single cyanobacter ia l biliprotein hasbeen discovered having the ligh t -ha rvest ing a t -t r ibu te of three differen t bilins, a lthough th is occursfor cryptomonad biliproteins (Sidler, 1994; MacColland Guard-Fr ia r, 1987).Sta r t ing from the chemica l st ructure of the bilins,

fine-tun ing is applied to the bilins to achieve im-proved ligh t harvest ing and energy migra t ion . Thefactors changing the absorpt ivit ies and spect ra ofbilins can be considered in six ca tegor ies. F ir st ,Scheer and Kufer (1977) have shown tha t the apopro-tein causes the bilins to be main ta ined in an ex-tended sta te, which maximize absorpt ion in thevisible region of the spect rum. Without the influenceof the apoprotein , bilins would tend to be more cyclicand have low visible absorpt ion .The ana lysis of t he cryst a l st r uctu r es of C-

phycocyanins provided deta iled informat ion on thebilins (Schirmer et al., 1985, 1987; Duerr ing et al.,1991). All th ree bilinsa84, b84, and b155arefound to be extended and have simila r geomet r ies.The bilin a t b155 differs in the configura t ion of it s Dr ing. The in teract ion with apoprotein establishes thebilin geomet ry.Second, the bilins are observed to be singly or

doubly a t tached by th ioether bonds to cysteine resi-dues of the apoprotein (Fig. 3).Third, changes occur through in teract ions involv-

ing the bilins and their near sur roundings, resu lt ing

TABLE IICyanobacter ia l Biliproteins

Biliprotein Bilin

Suggestedbiliproteinrenaminga

C-Phycocyanin PhycocyanobilinC-Phycoerythr in PhycoerythrobilinAllophycocyanins PhycocyanobilinsCU-Phycoerythr ins Phycoerythrobilin ,

phycourobilinPhycoerythrocyanin Phycocyanobilin , phy-

coviolobilinCV-phycocyanin

R-Phycocyanin II Phycocyanobilin , phy-coerythrobilin

CE-phycocyanin

Phycocyanin WH8501 Phycocyanobilin , phy-courobilin

CU-phycocyanin

a Since the prefix R or igina lly indica ted a red a lga l biliprotein ,the name R-phycocyanin II is ambiguous. It would be in terest ingto consider renaming cer ta in cyanobacter ia l biliproteins to reflecttheir bilin conten ts. R-phycocyanin II might bet ter be designa tedCE-phycocyanin , indica t ing the phycoerythrobilin , and likewisephycoerythrocyanin would be CV-phycocyanin . It can be notedtha t C-phycocyanin , CE-phycocyanin , and CV-phycocyanin a llhave three bilins loca ted a t a84, b84, and b155. The cyanobacte-r ia l phycocyanin WH8501 isola ted by Swanson et al. (1991),having urobilin and phycocyanobilin , would fit th is nomencla turen icely as CU-phycocyanin , where the C indica tes a cyanobacter ia lsource and the U a phycourobilin in addit ion to the phycocyanobi-lin . The CU-phycoerythr ins a lready use th is protocol. Bilin names,like phycocyanobilin , a re the same for free and protein-boundbilin even though the binding to protein elimina tes one doublebond. The prefix B indica tes a cer ta in type of red a lga , theBangiophyeans.

TABLE IIIBilin Loca t ions and Conten ts for Severa l

Cyanobacter ia l Biliproteins

BiliproteinBilin loca t ion a

a75 a84 a140 b50/61 b84 b155Allophycocyanin PCB b PCBC-Phycocyanin PCB PCB PCBPhycoerythrocyanin c PVB PCB PCBR-Phycocyanin II c PEB PCB PEBPhycocyanin WH8501 c PUB PCB PCBC-Phycoerythr in PEB PEB PEB PEB PEBCU-Phycoerythr in (1) PUB PUB PUB PEB PUBCU-Phycoerythr in (2) PEB PUB PUB PEB PEBCU-Phycoerythr in (3) PUB PUB PUB PUB PEB PEBCU-Phycoerythr in (4) PUB PEB PEB PUB PEB PEB

a Ong and Glazer (1987); Frank et al. (1978); F uglista ller et al.(1983); Swanson et al. (1991); Sidler et al. (1981, 1986). The bilina t b84 was determined to be the lowest -energy bilin for thesephycocyanins and phycoerythr ins (Ong and Glazer, 1987, 1991).

b PCB, phycocyanobilin ; PVB, phycoviolobilin ; PEB, phycoeryth-robilin ; PUB, phycourobilin .

c See Table II for suggest ions on renaming some of thesecyanobacter ia l biliproteins.

315PHYCOBILISOMES

in grea t modula t ion of bilin energies. Examplesof th is var iability of energy are the individua l phyco-cyanobilins of C-phycocyanin , C-phycocyanin /a llo-phycocyanin , and the 545- and 565-nm forms ofphycoerythrobilin . Even though C-phycocyanin anda llophycocyanin have only phycocyanobilins, theirabsorpt ion spect ra are sa lien t ly differen t . Allophyco-cyanin t r imers have a 650-nm maximum. The absorp-t ion maximum of C-phycocyanin t r imers is about620 nm. Even with in C-phycocyanin , the three phy-cocyanobilins on an ab unit have very differen tenergies (Siebzehnr ubl et al., 1987; Debreczeny etal., 1993; Demidov and Mimuro, 1995).Four th , the aggrega t ion of biliproteins affect s the

spect ra of the biliproteins. Monomers and t r imers ofa llophycocyanin and C-phycocyanin have very differ-en t absorpt ion (Table IV) and fluorescence spect ra .F ifth , the loca l environment of a bilin may have a

specia l factor, another nearby bilin . Two bilins inclose proximity and proper or ien ta t ion may undergoexciton split t ing. The energy of these bilins will besplit in to h igh- and low-energy levels, and th ismodula t ion of the energy levels may have a profoundeffect on ligh t harvest ing and energy migra t ion .Allophycocyanin t r imers have been discussed aspossible candida tes for exciton split t ing (MacColl etal., 1980; Edington et al., 1995, 1996). There is nogenera l agreement tha t exciton split t ing occurs forany biliprotein .Sixth , another factor tha t causes modifica t ions of

bilin absorpt ion energies is the linkers. When color-less linkers isola ted from cyanobacter ia in teractwith C-phycocyanin , the energies of the bilins can beaffected (Yu et al., 1981; Lundell et al., 1981a;

Got t scha lk et al., 1991; Schneider et al., 1995). Thelinker-free and linkerbiliprotein complexes had dif-feren t opt ica l spect ra as did differen t linkers plusbiliprotein (Table IV). A red a lga l biliprotein exhibit ssimila r behavior (Watson et al., 1986). P icosecondfluorescence kinet ics a lso showed differences in thebehavior of C-phycocyanin with or without linker.The biliprotein with linker had faster energy t rans-fer (Wendler et al., 1986). It seems reasonable tha tthese numerous modifica t ions of the energy levelsresu lt in bet ter ligh t harvest ing or improved effi-ciency in energy migra t ion . Bhalerao et al. (1991),however, have shown tha t rod linker polypept idesmay have only a minor influence on energy t ransferin a par t icu la r situa t ion . There is informat ion tha tdoes suggest tha t the linker next to the core, LRC, hasred-sh ifted the spect rum of it s phycocyanin disk toopt imize rod to core energy t ransfer (Sidler, 1994).There are two lower-energy forms of a llophycocya-

n in , aB and Lcm, both of which fluoresce near 680 nm,which is simila r to emission from intact phycobili-somes and is a t lower energy than the more commonform of a llophycocyanin . It is unknown what combi-na t ions of the above factors cause the bilins of aB andLcm to possess th is cr it ica lly impor tan t fea ture. It hasbeen repor ted tha t each of these polypept ides hasone phycocyanobilin (Glazer and Bryant , 1975; Lun-dell et al., 1981b).In addit ion to the studies ment ioned a lready,

biliproteins have been invest iga ted by a number ofperspect ives and techniques. Biliproteins have beenexamined in monolayers (Almog and Berns, 1983),mult ilayers (Yamazaki et al., 1988), films (Fracko-wiak et al., 1986; J uszczak et al., 1991), and inphotochemica l and elect rochemica l studies (Berns,1976; He et al., 1996; Evst igneev and Bekasova ,1970). They have been studied in immunochemica lresearch (Berns, 1967), as fluorescent labels in immu-noassays (Kronick, 1986), and as deutera ted pro-teins (Berns and MacColl, 1989). The biliproteins donot normally conta in meta l ions, bu t meta l ion can becomplexed to biliproteins (Park and Sauer, 1991;Berns, 1976; Cheng et al., 1990; MacColl et al., 1994).Bryant (1991) has reviewed the extensive and highlyproduct ive applica t ions of molecula r genet ics to thestudy of phycobilisomes. Hole burn ing techniqueshave been used in the study of biliproteins (Koh ler etal., 1988a ,b; Feis et al., 1992).An ext remely in terest ing aspect of biliprotein re-

search is the observa t ion of reversible photochemis-t ry in some situa t ions. There were a number of ear lykey cont r ibu t ions in the study of th is type of photo-chemist ry (Bjorn , G. S., 1979; Bjorn , L. O., 1979;Ohki and Fujita , 1979; Bjorn and Bjorn , 1980; deKok et a l., 1981; Murakami and Fujita , 1983; Scheer,1987). More recent ly, Scheer and co-workers (Schmidt

TABLE IVAbsorpt ion Maxima of Phycocyanobilins

in Differen t Situa t ionsProtein Condit ion Absorpt ion maximum (nm)

Allophycocyanin Monomer (ab) 614Tr imer (a3b3) 650

C-Phycocyanin ab 614a3b3 621

C-Phycocyanin a a3b3 1 27-kDalinker

638

a3b3 1 32.5-kDalinker

629

Allophycocyanin b a3b3 1 8.9-kDalinker (Lc8.9)

652

C-Phycocyanin 3 bilins on ab b155 600 c 596 d 598600 ea84 624 618 616618b84 628 625 622624

a Yu et al. (1981).b Fuglista ller et al. (1987).c Debreczeny et al. (1993).d Demidov and Mimuro (1995).e Siebzehnr ubl et al. (1987).

316 ROBERT MACCOLL

et al., 1988; Siebzehnr ubl et al., 1989; Maruth i Sa i etal., 1992, 1993; Hong et al., 1993; Schneider et al.,1994, 1996; Zhao et al., 1995; Zhao and Scheer, 1995)have focused successfu l effor t s on phycoerythrocya-n in and it s phycoviolobilin on the a polypept ide.Siebzenr ubl et al. (1989) have studied phycoeryth-

rocyanin and found the phycoviolobilin to be thephotoact ive par t of the protein . The photoeffect wasmaximal when the protein was dissocia ted to mono-mers. Conversely, the photot ransformat ion a lso af-fected the aggrega t ion of protein . The phycoviolobi-lin is obta ined with a 570-nm absorpt ion maximum;ir radia t ion a t 570 nm causes the bilin to lose absor-bance a t 570 nm and simultaneously to increaseabsorbance a t 510 nm. The effect could be reversedwith 510-nm light . F luorescence measurements wereper formed on the two spect ra l forms of the bilin(Maruth i Sa i et al., 1992). The 510-nm absorbingform had lit t le fluorescence and energy t ransferwould occur efficien t ly only from the 570-nm form.The isomer iza t ion of phycoviolobilin responsible forthe photochemist ry has been invest iga ted (Zhao andScheer, 1995; Zhao et al., 1995).In agreement with the resu lt s of Siebzehnr ubl et

al. (1989), Kufer and Bjorn (1989) isola ted the apolypept ide of phycoerythrocyanin and showed tha tit was photoreversible. Theoret ica l ca lcu la t ions havebeen per formed on the reversible photochemist ry ofthe a polypept ide from phycoerythrocyanin (Schar-nagl and Fischer, 1993).

Evolu tion and BiliproteinsCyanobacter ia are procaryotes conta in ing bilipro-

teins and chlorophyll a. There have been repor t s ofbiliproteins discovered in procaryotes with differen tch lorophylls. Phycocyanin and a llophycocyanin havebeen observed in an organism conta in ing chlorophylld (Marquardt et al., 1997). A phycoerythr in wasfound in a mar ine organism also having chlorophylla, b, and c (Hess et al., 1996). This phycoerythr inmay have both phycourobilin and phycoerythrobilinand may be another member of the diverse CU-phycoerythr ins. These organisms were discussed interms of their implica t ions in the evolu t ion of photo-synthet ic life.Phycobilisomes and their biliproteins have pro-

vided usefu l clues concern ing the evolu t ion of photo-synthet ic organisms. Berns (1967) made the firstcont r ibu t ion to the rela t ionsh ips among biliproteinsfrom the procaryot ic and eucaryot ic phycobilisomes.Using an immunochemica l technique, phycocyaninsfrom cyanobacter ia were demonst ra ted to be closelyrela ted to phycocyanins from red a lgae, and thesame was discovered for the red a lga l and cyanobac-ter ia l phycoerythr ins. This was an impor tan t ear lymolecula r demonst ra t ion tha t the red a lgae evolved

from a cyanobacter ium or a cyanobacter ium-likephotosynthet ic organism. There is evidence tha teucaryot ic red a lgae arose from endosymbiosis be-tween a procaryot ic cyanobacter ium and a nonphoto-synthet ic organism, with the cyanobacter ium becom-ing the chloroplast of the resu lt ing red a lga . A reda lga then underwent a second endosymbiot ic eventto become the chloroplast of a cryptomonad (e.g.,Pa lmer and Delwiche, 1996; McFadden et al., 1994;Ludwig and Gibbs, 1985; Gibbs, 1990). Immunochem-ist ry was used to suggest tha t the biliproteins ofcyanobacter ia , red a lgae, and the cryptomonads werea ll rela ted (Berns, 1967; Guard-Fr ia r et al., 1986;MacColl and Guard-Fr ia r, 1987). Amino acid se-quences established firmly tha t extensive homolo-gies existed among the var ious biliproteins. Sidler etal. (1990) have shown from the amino acid sequenceof a cryptomonad biliprotein tha t the b polypept idehas a 73% homology to the b polypept ide of red a lga lB-phycoerythr in and a 63% homology to cyanobacte-r ia l C-phycoerythr in . Cryptomonads, however, lackphycobilisomes (Gant t et al., 1971); their biliproteinsare loca ted in the in t ra thylakoid space, and theypossess ch lorophyll c in addit ion to chlorophyll a.While the b polypept ide of the cryptomonad bilipro-teins is closely rela ted to cyanobacter ia l and reda lgae biliproteins (Sidler et al., 1990), the a polypep-t ide is of unknown or igin (Sidler, 1994). Sidler (1994)refers to the amino acid sequence of the cryptomonada polypept ide as the most in t r igu ing mysteryin theunderstanding of biliprotein phylogeny. An unusua lN-methyla ted asparagine residue is conserved andsuggests a close rela t ionsh ip among all these bilipro-teins (Wilbanks et al., 1989). Cyanelles might a lso beder ived from cyanobacter ia l endosymbionts (e.g.,Bryant et al., 1985; Lamber t et al., 1985).The amino acid sequences of the biliproteins have

been ana lyzed for homologies (Apt et al., 1995;Ducret et al., 1994). Phycoerythrocyanins belong tothe same family as the phycocyanins. They may be asepa r a t e group pla ced wit h in t he phycocyan inbranch. Ducret et al. (1994) proposed that phycoeryth-rocyanins are specia lized phycocyanins adapted forgreen-ligh t absorpt ion .Schirmer et al. (1985) discussed some aspects of

evolu t ion rela t ing to the x-ray crysta llographic st ruc-ture of C-phycocyanin . The a- and b-polypept ideshave very simila r ter t ia ry st ructures. In addit ion ,each polypept ide of C-phycocyanin has a resem-blance to the globin fold. Par t s of globin proteins, likemyoglobin , have ter t ia ry st ructures simila r to C-phycocyanin. The authors suggested that C-phycocya-n in and the globins may have evolved to a simila rst ructure from differen t ancestors or might havesome distan t rela t ionsh ip.

317PHYCOBILISOMES

Complem entary Chrom atic AdaptationThe ability of phycobilisomes to exhibit var iability

in the harvest ing of ligh t is most advantageous ina llowing a good match between the absorpt ion spec-t ra of a par t icu la r cyanobacter ium and the ava ilableligh t of it s habita t . An example is a cyanobacter iumliving on land and another which grows a t somedepth in the ocean . The sunligh t st r iking the ear thssur face is br igh t and white in color ; as it moves downthe water column the ligh t dims and usua lly be-comes blue-green . A cyanobacter ium on land re-quires less ligh t harvest ing from its phycobilisomes.Typica lly, they will have the a llophycocyanin coreand rods of C-phycocyanin , bu t perhaps no phycoery-thr in . Phycoerythr ins, however, a re per fect ly su itedto blue-green ligh t absorpt ion and extend the abilityof the cyanobacter ium to harvest more of a gradua llydiminish ing ligh t as it proceeds down the watercolumn. A phycobilisome having C-phycoeythr in ,C-phycocyanin , and a llophycocyanin will fill mostof the window of small ch lorophyll a absorpt ion .Therefore, mar ine cyanobacter ia frequent ly havephycobilisomes conta in ing C-phycoerythr in or CU-phycoerythr in in their rods. The urobilins of CU-phycoerythr ins are excellen t absorbers of blue ligh t .In addit ion to a cyanobacter ium seeking a su itable

loca t ion for successfu l growth , some cyanobacter ia

can acclimate in var ious ways to a range of habita t s.One acclimat ion ava ilable to cer ta in cyanobacter ia iscomplementary chromat ic adapta t ion . In th is pro-cess, the rods of the phycobilisomes change to achievebet ter ligh t harvest ing in var ious circumstances.The rods are composed of stacked biliprotein disks(a6b6), and the disk closest to the core will a lways bephycocyanin . The remain ing disks can be eitherphycocyanin or phycoerythr in in many combina-t ions.When a cyanobacter ium exhibit s complementary

chromat ic adapt ion , the rods of the phycobilisomeschange with growth ligh t (F ig. 5). The change in-cludes one or both biliproteins and linkers (Tandeaude Marsac and Cohen-Bazire, 1977). Red growthligh t may produce C-phycocyanin , which absorbs redwell, and green growth ligh t produces C-phycoery-thr in , which absorbs green well (F ig. 5). The key toth is adapta t ion is the linkers tha t vary between27 000 and 36 000 molecula r mass. It was observedhow, for a par t icu la r cyanobacter ium, the linkersresponded to growth ligh t (F ig. 6). Rod linkers forcyanobact er ia , except for cer t a in CU-phycoery-thr ins, have no bilins. For red a lgae, linkers ofR-phycoerythr ins do have bilins.Many cyanobacter ia have been studied for t rends

in their responses to complementary chromat ic adap-

FIG. 5. Model for complementary chromat ic adapta t ions of two cyanobacter ia . In one case (A), C-phycocyanin increases in red growthligh t and in the other (B), C-phycocyanin remains constan t in varying growth ligh t . Adapted from Siegelman and Kycia (1982) andGingr ich et al. (1982a).

318 ROBERT MACCOLL

t a t ion (Tandeau de Marsac, 1977). Three responsesto growth ligh t a re observed. No effect occurs on therods; more phycoerythr in is synthesized in greengrowth ligh t and less in red growth ligh t , bu t C-phycocyanin is invar ian t ; more phycoerythr in isproduced in green growth ligh t and less in redgrowth ligh t , and C-phycocyanin is a lso ligh t con-t rolled, being produced more in red than in greengrowth ligh t (F igs. 5 and 6). It would seem tha t sucha highly developed response to a ligh t habita t mustbe an impor tan t mechanism for the surviva l ofcyanobacter ia in sh ift ing environments. However,there are differ ing opin ions (Saffo, 1987).Since the in it ia l observa t ion (Tandeau de Marsac

and Cohen-Bazire, 1977), linkers and complemen-ta ry chromat ic adapt ion have been well invest iga ted(Zilinskas and Howell, 1983; Bryant , 1981, 1991;Bryant and Cohen-Bazire, 1981; Tandeau de Mar-sac, 1977, 1991; Westermann and Wehrmeyer, 1995;Tandeau de Marsac and Houmard, 1993; Glauser etal., 1992b; Grossman and Kehoe, 1997; Ohki andFujita , 1991, 1992; Westermann et al., 1993). Aspecific linker is required for the addit ion of apar t icu la r disk to the rod. One phycobilisome linker,LRC, is invar ian t since it connects the rod to the core,and the first disk in the rod is a lways phycocyanin .In green growth ligh t , the next disk, LR, might be aC-phycoerythr in disk, which would require synthe-sis of a completely specific linker. At tach ing a secondC-phycoerythr in disk to the first C-phycoerythr indisk would likewise require synthesis of anotherunique linker. If growth is turned to red ligh t , thespecific linkers for addit ion of disks of C-phycoery-thr in are no longer synthesized and new linkers tha tcan begin to add C-phycocyanin disks may or may

not appear. Again each disk added to the rod requiresa new and unique linker. Effects other than comple-mentary chromat ic adapta t ion , e.g., responses toligh t in tensity, can a lso occur in varying types ofgrowth ligh t (Fujita et al., 1994; Sidler, 1994).A mar ine cyanobacter ium has provided a unique

type of complementary chromatic adaptat ion (Wester-mann and Wehrmeyer, 1995; Westermann et al.,1993; Ohki and Fujita , 1992). In th is cyanobacte-r ium, red-ligh t growth produced hemidiscoida l phy-cobilisomes and green ligh t resu lted in hemiellipsoi-da l phycobilisomes. This change in phycobilisometype occur red together with the usua l change inphycoerythr in and phycocyanins in the rods of thesephycobilisomes. In a ll previous cases, complemen-tary chromat ic adapta t ions did not affect the hemidis-coida l st ructure.Red a lgae apparen t ly do not exhibit complemen-

tary chromatic adaptat ion (Gant t , 1990). Gant t (1990)has reviewed issues of photoregula t ions as theyper ta in to the red a lgae, as well as cover ing impor-tan t aspects of their biliproteins, phycobilisomes,and photosynthet ic act ivit ies. In terms of how reda lgae respond to ligh t (Gant t , 1990), it might bein terest ing to observe whether the macrophyte reda lga l seaweeds have very differen t modes of acclima-t ion when compared to the unicellu la r types.

C-Phycocyanin and Gene DuplicationAs discussed above, red ligh t can produce more

C-phycocyanin to be added to the rods of cer ta incyanobacter ia . Bryant and Cohen-Bazire (1981) havestudied one of these cyanobacter ia and found tha t asecond set of a and b polypept ides of C-phycocyaninwere produced in red ligh t , in addit ion to the usua l aand b polypept ides of th is biliprotein . The aminoacid sequences of the two C-phycocyanins provedtha t the proteins were products of separa te genes.Mazel and Mar lie`re (1989) grew a cyanobacter ium

in a medium conta in ing minimal su lfur. The usua lC-phycocyanin was replaced by a new C-phycocya-n in having fewer su lfur-conta in ing amino acids,meth ionine and cysteine. As with cer ta in red-ligh tgrown cyanobacter ia , phycoerythr in was elimina ted.The resu lt was a more efficien t use of the smallamount of su lfur ava ilable. The bilin binding siteswere not deleted by th is th ird type of C-phycocyaningene.

Rod LinkersThe roles of linkers in causing phycobilisome rod

assembly and spect ra l modifica t ions of biliproteinshave a lready been presented, but more deta ils can beadded. Lundell et al. (1981a) pur ified C-phycocyaninand four linkers from a cyanobacter ium. Three of thelinkers (27 000, 30 000 and 33 000 Da) are rod

FIG. 6. The dist r ibu t ion of linkers in phycobilisomes of apar t icu la r cyanobacter ium exhibit ing complementary chromat icadapt ion . L indica tes linker, based on the findings of Zilinskas andHowell (1983).

319PHYCOBILISOMES

associa ted. Each linker was reacted with C-phycocya-n in as were mixtures of linkers and the productsana lyzed. The 27 000-Da linker and C-phycocyaninproduced disks, a complex of a6b6, and a 27 000-Dalinker. The 30 000- and 33 000-Da linkers, LR, be-haved simila r ly, and they could produce both disks(a6b6 plus one linker ) and la rge rod-like st ructures.The disks could be readily pur ified. It was estab-lished tha t the 27 000-Da linker, LRC, was associa tedwith the core, and therefore, it s disk is the oneimmedia tely adjacent to the core (Yamanaka et al.,1980; Lundell et al., 1981a). This linker is specia lsince it serves to be involved in rod to core assemblyand to adjust the spect rum of C-phycocyanin to helpt ransfer energy from rod to core.The funct ions of rod linkers have a lso been studied

by methods other than complementary chromat icadapta t ion (Yamanaka et al., 1980; Yamanaka andGlazer, 1981; Williams et al., 1980; Anderson et al.,1984; Gingr ich et al., 1982a ,b; de Lor imier et al.,1990b), and the findings of these studies suppor t thepresence of a specific linker with each disk a t apar t icu la r rod loca t ion (Table V).There is a 9000 molecula r mass linker, LR9 . It s

funct ion is involved with termina t ing the length ofrods, and it may be complexed with per iphera l disks(F uglista ller et al., 1986; de Lor imier et al., 1990a ,b).

CORE

The phycobilisome core conta ins linkers and thea llophycocyanins. Zilinskas et al. (1978), Zilinskas(1982), Relinger and Gant t (1981, 1982), and a ser iesof definit ive papers by Glazer and co-workers (e.g.,Glazer and Bryant , 1975; Gingr ich et al., 1983;Lundell and Glazer, 1983a ,b,c; Lundell et al., 1981b;Yamanaka et al., 1982) produced the basis for anexcellen t understanding of th is complex and st ra tegi-ca lly placed st ructure.Two components of the core possess low-energy

fluorescence simila r to the phycobilisome it self.

Glazer and Bryant (1975) isola ted a complex conta in-ing the aB allophycocyanin-like polypept ide and notedtha t it could be the biliprotein to t ransfer energy tochlorophyll a. The amino acid sequence of aB isknown (Suter et al., 1987). Then from a red a lga(Relinger and Gant t , 1981) and from a cyanobacte-r ium (Lundell et al., 1981b), la rge molecula r masspolypept ides of 95 000 and 75 000 Da, respect ively,were isola ted. Both these polypept ides (the anchor orLcm polypept ides) had low-energy fluorescence simi-la r to aB. So now, there were two candida tes forenergy migra t ion out of the phycobilisome and in tothe thylakoid membrane.For th is review, it will be usefu l to focus on a

publica t ion by Gingr ich et al. (1983), and otherpublica t ions should be consulted for more deta il(Lundell and Glazer, 1983a ,b,c; Andersen and Eiser-ling, 1986; Ducret et al., 1998). These phycobilisomesconta in cores of three cylinders, two of which areadjacent to the thylakoid membrane, the basa l cylin-ders (F ig. 2). The cylinder not lying on the membranesur face, the top cylinder, conta ins four t r imers of thea llophycocyanin having a 650-nm maximum, andthese biliproteins receive energy from the rods andt ransfer it to the lower-energy a llophycocyanins.Each of the two cylinders on the thylakoid sur facehas the same overa ll composit ion : each has twotr imers of allophycocyanin (650 nm) and two tr imeric-like st ructures each including one of the lower-energy a llophycocyanin (Table VI). The lower-energya llophycocyanins appear to per form the pivota l roleof funneling energy out of the core and in to thethylakoid membrane to chlorophyll a.A 16.2- to 18.3-kDa polypept ide, b16, has been

isola ted from the phycobilisome cores of cyanobacte-r ia (Yamanaka et al., 1982; Lundell and Glazer,1983c; F uglista ller et al., 1984; Rumbeli et al., 1987).

TABLE VLinker Assembly Funct ions

in a Par t icu la r Cyanobacter ium a

Linker(molecula r mass) Funct ion

27 000 J oins C-phycocyanin disk to core33 500 J oins second C-phycocyanin disk to first

C-phycocyanin disk31 500 J oins first C-phycoerythr in disk to second

C-phycocyanin disk30 500 J oins per iphera l C-phycoerythr in disk to

end of roda In white growth ligh t , th is cyanobacter ium has phycobilisome

rods consist ing of two C-phycocyanin disks and two C-phycoery-thr in disks (Gingr ich et al., 1982a ,b).

TABLE VICore St ructures of Hemidiscoida l PhycobilisomesCore type Cylinders a Composit ion

Bicylindr ica l A Both A cylindersa3b3a3b3Lc8.9a2b2b16LcmaBa2b3Lc8.9

Tr icylindr ica l A Same as bicylindr ica lB a3b3 (2 copies)

a3b3Lc8.9 (2 copies)Pentacylindr ica l A Same as bicylindr ica l

B Same as t r icylindr ica lC Both C cylinders

a3b3 (1 copy)a3b3Lc8.9 (1 copy)

a A represents two basa l cylinders, which are adjacent tomembrane, B is the top cylinder (F ig. 1), and C are per iphera lha lf-cylinders, which are situa ted on either side of B.

320 ROBERT MACCOLL

This polypept ide is another biliprotein and possessesa single phycocyanobilin . A color less linker is a lsofound in the core. It s molecula r mass is 890010 500da . It is designa ted Lc8.9, where L indica tes linker, cmeans core, and 8.9 indica tes kiloda ltons. The effectof th is linker on the spect rum of a llophycocyanint r imer s ha s been st udied (Bet z et al., 1993;F uglista ller et al., 1987; Got t scha lk et al., 1993;Lundell and Glazer, 1983b; Holzwar th et al., 1990;Schneider et al., 1995). Another linker-rela ted poly-pept ide, which is der ived from Lcm, can a lso inducespect roscopic changes in allophycocyanin (Got tschalket al., 1994).The Lcm linker is so named because it is associa ted

with both the phycobilisome core and the thylakoidmembrane. Lcm is the la rgest polypept ide in thephycobilisomes of cyanobacter ia and red a lgae andmay vary in molecula r mass from about 70 000 to128 000, depending on the source. It is mult ifunc-t iona l having: (1) involvement in the organiza t ion ofthe core; (2) a low-energy bilin tha t probably isresponsible for energy t ransfer in to the thylakoidmembrane; (3) the funct ion of anchor ing the phyco-bilisome core to the thylakoid membrane; and (4) theability to modify the spect roscopic proper t ies ofassocia ted biliprotein (Rusckowski and Zilinskas,1982; Redlinger and Gant t , 1981, 1982; Tandeau deMarsac and Cohen-Bazire, 1977; Lundell and Glazer,1983a ,b,c; Lundell et al., 1981b; Mimuro et al.,1986b; Gant t et al., 1988; Isono and Katoh , 1987;Reuter and Wehrmeyer, 1990; Houmard et al., 1990;Gant t , 1990; Capuano et al., 1991, 1993; Bryant ,1991; Gindt et al., 1992; Zhao et al., 1992; Got t scha lket al., 1994).The Lcm polypept ide has been loca ted in both the

core of the phycobilisome and the thylakoid mem-brane (Tandeau de Marsac and Cohen-Bazire, 1977;Redlinger and Gant t , 1981, 1982; Ruskowski andZilinskas, 1982) in both cyanobacter ia and red a lgae.It is suggested tha t it funct ions by a t tach ing thephycobilisome core to the thylakoid membrane. It isrefer red to as the anchor polypept ide or the coremembrane linker. Studies on cyanobacter ia l phyco-bilisomes indica ted tha t the Lcm polypept ide mightbe the likely pa th for the impor tan t energy migra t ionout of the phycobilisome core and in to the photosys-tem II receptors (Mimuro et al., 1986b). The role of aBwas suggested to be a bypass.Isono and Katoh (1987) discovered a hemidiscoida l

phycobilisome having a five-cyclinder core in a par-t icu la r cyanobacter ium. They noted tha t the Lcmpolypept ide from this core has a molecula r mass of115 000, which is grea ter than tha t found in two orth ree cylinder cores. When they prot eolyt ica llycleaved a small piece from this Lcm, the smaller Lcmproduced a t r icylindr ica l core. Based on th is decisive

discovery and other da ta , they proposed tha t Lcm wasst rongly involved in core assembly. This ana lysisproved to be ext remely insigh t fu l.It is known from its amino acid sequence how Lcm

can fill differen t funct ions in the phycobilisome core.Bryant (1991) and Capuano et al. (1991) have pro-posed models tha t give Lcm a paramount role in theassembly of the core. The amino acid sequences ofvar ious Lcm polypept ides are known; see Sidler (1994)for references on biliprotein and linker sequences.Bryant (1991) and Capuano et al. (1991) note tha tthe N-termina l amino acids resemble the sequence ofamino acids for the biliproteins and the bilin will beloca ted in th is region . A loop, which prot rudes fromthis por t ion of the polypept ide, could serve as theanchor of the phycobilisome to the thylakoid mem-brane. The rest of the sequence is of two types: aregion tha t resembles the linkers of the phycobili-some rods, and another region . The linker-like re-gion is ca lled Rep and the other Arm. The Repregions are repea ted once for hemidisoida l phycobili-somes having a two-cylinder core and twice for thosewith a three-cylinder core, having Arm regions be-tween them. The proposa l is tha t the Reps organizethe a llophycocyanin t r imers the way the rod linkersorganize disks of phycocyanin , phycoerythr in , andphycoerythrocyanin . The Arms twist among the coreto posit ion the Reps for th is task. The molecula rmass of Lcm in the two-cylinder cores is about 70 00075 000 Da and for the three-cylinder cores it is about92 00099 000 Da. Each Rep would bind two allophy-cocyanin t r imers. There are two Lcm polypept ides ineach core, and, therefore, for a three-cylinder corethe two Lcm polypept ides would have six Rep do-mains, which could bind 12 t r imers (F ig. 2). Capuanoet al. (1993) successfu lly tested aspects of th is pro-posa l.The th ird type of hemidiscoida l core has five

cylinders. Three of the cylinders are the same incomposit ion and loca t ion as the three-cylinder cores.The remain ing two are ha lf-cylinders and are com-posed of a llophycocyanin t r imers, a3b3Lc8.9 and a3b3.They are loca ted above the two basa l cylinders tha ta re cont iguous to the thylakoid membrane and oneither side of the top cylinder (Sidler, 1994). The Lcmpolypept ide is about 115 000 to 128 000 Da and hasfour Rep domains. The four th Rep may funct ion toorganize the two-ha lf cylinders, ca lled the C orper iphera l cylinders, of the core. The two Lcm polypep-t ides in th is core would tota l eigh t Reps and couldposit ion the 16 t r imers. Ducret et al. (1996, 1998)have studied pentacylindr ica l a llophycocyanin coresin deta il and have presented much evidence concern-ing their st ructure. They propose tha t the rods area t tached to the core as follows: the basa l core cylin-ders adjacent to the thylakoid membrane have one

321PHYCOBILISOMES

rod a t tached to each , and the top cylinder and twoper iphera l ha lf-cylinders bind two rods each .Cyanobacter ia l mutan ts tha t lack phycobilisome

rods have been const ructed (Bryant , 1991; Bhaleraoet al., 1995; Anderson et al., 1984). The organismsst ill possess funct iona l a llophycocyanin cores.

Phycobilisom es and Photosystem IThe associa t ion of phycobilisomes to photosystem

II is quite st rong, and complexes of the two havebeen isola ted and studied (Clement -Met ra l et al.,1985; Chereskin et al., 1985). It has been long knowntha t under cer ta in growth-ligh t condit ions tha t en-ergy absorbed by the phycobilisomes could be usedby photosystem I, but only recent ly has it becomeapparen t tha t phycobilisomes could direct ly t ransferenergy to photosystem I. Studies on a wild-type anda mutant of a cyanobacter ium yielded da ta consis-ten t with direct t ransfer of energy from the phycobili-somes to photosystem I (Mullineaux 1992, 1994). Ina mutant lacking photosystem II, phycobilisomest ransfer red excita t ion energy to photosystem I (Mul-lineaux, 1994).Using mutants of aB and Lcm, it was demonst ra ted

(Maxson et al., 1989; Bryant , 1991; Zhao et al., 1992;Gindt et al., 1992) tha t the Lcm polypept ide a lone issufficien t to media te extensive energy migra t ion tophotosystem II. Addit iona l resu lt s st rongly indica tedtha t a fundamenta l role of aB is energy t ransfer tophotosystem I. The aB direct ion of excita t ion energyto photosystem I would occur under growth ligh ttha t st rongly favored photosystem II.An enzyme, fer redoxin-NADP1 oxidoreductase, is

found to have an N-termina l amino acid sequencetha t is 78% simila r to the rod-termina t ing linker, LR9 .It is, therefore, specula ted tha t the enzyme, forwhich there is evidence of phycobilisome associa t ion ,could bind to a phycobilisome rod a t the disk fur thestfrom the core (Schuchter and Bryant , 1992). Thisobserva t ion suggests tha t phycobilisomes serve afunct ion in providing molecules with access to thethylakoid membrane sur face. In par t icu la r, th is en-zyme should be associa ted with photosystem I.Bald et al. (1996) and Sidler (1994) have exten-

sively reviewed the deta ils of the in teract ion amongphycobilisomes and receptors in the thylakoid mem-brane. Phycobilisomes pr imar ily t ransfer excita t ionenergy to photosystem II receptors, bu t under cer-ta in ligh t condit ions direct t ransfer to photosystem Imay occur. Actua l docking of phycobilisomes to photo-system I is discussed (Bald et al., 1996). Dockingmight be accomplished through the aB-conta in ingcore t r imer, by fer redoxin-NADP1 oxidoreductase, orby some other mechanism. Fujita et al. (1994) hasreviewed the cases for and aga inst direct t ransfer of

excita t ion energy to photosystem I from the phycobili-somes.A cyanobacter ia l mutan t tha t has no phycobili-

some core has been prepared (Su et al., 1992). Thephycocyanin aggrega tes tha t were present t rans-ferred their energy primarily and direct ly to photosys-tem I.

ENERGY TRANSFER BETWEEN BILINS

It has been shown tha t absorbed energy migra testhrough the rods in to the core and then to thechlorophyll a in the thylakoid membrane. Theseevents are nonradia t ive, very efficien t , and direc-t iona l. Methods have been employed, par t icu la r ly forlinker-free C-phycocyanin , phycoerythrocyanin , anda llophycocyanin (650-nm maximum), to develop anunderstanding of the individua l steps in th is com-plex process. These approaches are based on theana lysis of da ta obta ined by fast laser kinet ics andx-ray diffract ion studies on crysta ls together withvar ious opt ica l spect ra . The arch itecture of the phy-cobilisome and the organiza t ion of it s many bilinsprovides efficien t energy migra t ion from the highest -energy bilin to the lowest , from which energy ist ransfer red to chlorophyll a. It st ill needs to beunderstood how the bilin organiza t ion establishesth is efficiency.Crysta ls for var ious cyanobacter ia l biliproteins

have been prepared by Huber and co-workers andsubjected to x-ray diffract ion ana lysis (Schirmer etal., 1985, 1986, 1987; Brejc et al., 1995; Duerr ing etal., 1990, 1991). All the t r imer ic and hexamer icbiliproteins have very simila r genera l st ructures(F ig. 7). The cent ra l channel is common in a ll cases,having a 3.5- to 4.5-nm diameter. Cer ta in of thebilins (b84) are in close proximity to th is opening.Both polypept ides have nine a-helices connected byir regula r turns and have simila r ter t ia ry st ructures.Allophycocyanin when pur ified is composed of threeprotein monomer (ab) un it s. Each a and each bpolypept ide has one phycocyanobilin . The two clos-est bilins (a84 and b84) are on adjacent monomers20.6 nm apar t , while bilins on the same monomer are49.6 nm apar t (F ig. 8). These distances might indi-ca te tha t the energy t ransfer events differ sharplyfor monomers and tr imers. For C-phycocyanin , mono-mers, t r imers, hexamers, and rod assemblies havebeen studied by x-ray crysta llography. The distancesbetween the a84 and the b84 bilins of C-phycocyaninmonomers and t r imers (F ig. 9) change in rela t iveposit ion as found for a llophycocyanin .How is energy transferred in photosynthesis? There

is apparen t ly one predomina te mechanism, F or sterresonance t ransfer in the weak coupling limit (F or-ster, 1965), tha t provides much of the chromophore-to-chromophore t ransfer. In th is method, somet imes

322 ROBERT MACCOLL

ca lled very weak dipoledipole coupling, the indi-vidua l chromophores tend to reta in their spect ra . Asecond mechanism, exciton coupling, may occur whenchromophores are brought in to closer contact (Can-tor and Schimmel, 1980). In th is method, a pa ir ofchromophores may share deloca lized energy, behav-ing as if they are one unit . In exciton coupling, theexcited sta tes of the individua l chromophores aresplit in to h igh and low energies (F ig. 10). In terna lconversion provides for the movement of energy from

the high- to low-energy sta tes of the coupled chromo-phore pa ir. This situa t ion may be observed by a shiftin absorpt ion maximum.C-PhycocyaninThe energy migra t ion process for isola ted bilipro-

teins was first ana lyzed by Dale and Teale (1970) andTeale and Dale (1970) using fluorescence pola r iza-t ion . Since then many studies have been car r ied outon th is topic. For C-phycocyanin , the more recentresu lt s will be discussed in which x-ray crysta llogra-phy da ta , femtosecond laser resu lt s, and the mostsoph ist ica t ed spect r a l deconvolu t ion s were em-ployed.Debreczeny et al. (1993, 1995a , 1995b) have ana-

lyzed the energy t ransfer proper t ies of C-phycocya-n in monomers and t r imers and determined tha tF or ster resonance energy t ransfer is the methodused for the radia t ion less t ransfer of energy betweenthe var ious pa ir s of bilins on these two oligomers. A

FIG. 7. Diagram of the x-ray diffract ion resu lt s for a llophyco-cyanin t r imer. The genera l shape and cent ra l channel a re simila rfor a ll the biliproteins studied so far as t r imers and hexamersfrom phycobilisomes. Bilin loca t ions are indica ted by ar rows. Thenumbers 1, 2, and 3 refer to the three monomer ic unit s. Eachmonomoner is composed of an a (ligh t ) and a b (dark) polypept ide.The b84 bilins are poin t ing in to the cent ra l channel, and the a84bilins are loca ted on the per ipheny of the st ructure.

FIG. 8. Rela t ive bilin ar rangement for a llophycocyanin t r imers. The prefix is the number of the monomer, and the suffix (st ructure onthe r igh t ) is the posit ion in the amino acid sequence of the cysteine to which a bilin is a t tached.

FIG. 9. Rela t ive bilin ar rangement in C-phycocyanin t r imers.The prefix is the number of the monomer, and the suffix is theposit ion in the amino acid sequence of the cysteine to which a bilinis a t tached. The ar row links the two bilins tha t a re closest andmost st rongly coupled.

323PHYCOBILISOMES

mutant of C-phycocyanin was used extensively inwhich the b155 cysteine was replaced by a ser ine.The resu lt ing biliprotein had bilins a t b84 and a84,but of course not a t b155. Using the mutant andwild-type proteins, the absorpt ion and fluorescencespect ra of the three bilins were determined (TableIV). These spect ra are necessary because in Forsterresonance theory the modified spect ra l over lap be-tween the fluorescence of the donor and the absorp-t ion of the acceptor is key da ta . From these resu lt s,other spect ra l da ta , the refract ive index, and thedistance and or ien ta t ions of the bilins from x-raycrysta llography (Schirmer et al., 1987; Duerr ing etal., 1990, 1991), they determined, using Forstertheory, the ra te constan ts for energy t ransfer for thevar ious bilin pa ir s. For monomer, energy t ransfersfrom b155 to b84 and a84 to b84 were found to occurrapidly (Table VII). The Forster-ca lcu la ted va luesagreed well with the exper imenta l resu lt s. Rela -t ively slow energy t ransfer was found for the t rans-fer from b155 to a84, and th is lifet ime was producedby the unfavorable or ien ta t ion of these bilins. Theradia t ive fluorescence lifet imes for these bilins areabout 12 ns, and nonradia t ive product ive energyt ransfer must be much faster. The b84 bilin is thelowest -energy bilin in the monomers and will serveas the bilin from which energy is t ransfer red out ofthe protein . The other two higher-energy bilins, b155and a84, extend the range of ligh t harvest ing. Theb155 bilin absorbs a t the highest energy.For t r imers (in the absence of linkers), there are

more possibilit ies for energy t ransfer, especia lly

between the closely spaced a84 and b84 bilins oncont iguous protein monomers in the t r imer (Fig. 9).A decay t ime of 1.0 ps was ca lcu la ted from theForster ra te constan t for th is in termonomer ic t rans-fer. The exper imenta l resu lt for t ransfer betweenth is pa ir of bilins was 1.4 ps, which agreed verynicely with the Forster ca lcu la t ions in the weakcoupling limit . For the in t ramonomer ic t ransfer fromb155 to b84, the Forster ca lcu la t ion gave 49 ps. Theauthors suggested tha t these resu lt s were the firstdeta iled evidence for Forster resonance energy t rans-fer between chromophores on a protein .Other groups using C-phycocyanin t r imers have

a lso exper imenta lly studied energy t ransfer froma84 to b84 (Gillbro et al., 1993; Xie et al., 1992).Gillbro et al. (1993) found a 500-fs energy t ransferand suggested the resu lt s suppor t F or ster resonancet ransfer. Xie et al. (1992), however, repor ted tha ttheir resu lt s were inconsisten t with F orster induc-t ive resonance energy t ransfer theory. This conclu-sion was based on an ana lysis of the wavelengthdependence of the fluorescence depolar iza t ion kinet -ics.Less is known about energy migra t ion with in

hexamers and between hexamers of C-phycocyanin .When hexamers form, new pathways for energymigra t ion are opened (Schirmer et al., 1986; Duer-r ing et al., 1991). For efficien t energy t ransfer be-tween hexamers, the crysta l st ructures suggest tha tthe b84 bilins provide the pa thways.Demidov and co-workers have produced insigh ts

on energy t ransfer, fluorescence pola r iza t ion , anddeconvolu t ion of spect ra for C-phycocyanin (Demi-dov and Mimuro, 1995; Demidov and Andrews, 1995;Demidov and Bor isov, 1993, 1994; Demidov, 1994a ,b).The monomer absorpt ion spect rum of C-phycocyaninwas deconvolu ted using a procedure involving theirtheoret ica l t rea tment of spect ra (Table IV). After thetwo bilins on the b polypept ide were resolved, the apolypept ide spect rum was added, and the fluores-cence, fluorescence pola r iza t ion , and fluorescencequantum yield of the monomer were est imated. Theca lcu la ted and exper imenta l spect ra showed accept -able agreements (Demidov and Mimuro, 1995). Therewere a lso ear lier studies on chromophore assign-ments in C-phycocyanin (Siebzehnr ubl et al., 1987;Mimuro et al., 1986a).Demidov and Mimuro (1995) noted tha t their

deconvolu ton of the monomer C-phycocyanin absorp-t ion spect rum differed by 46 nm (Table IV) fromthose obta ined by Debreczeny et al. (1993). Theysuggested these resu lt s could be expla ined by thefact tha t the C-phycocyanin used in the two studiescame from differen t cyanobacter ia . This idea mayhave mer it . C-Phycocyanin , isola ted from a par t icu-la r thermophilic cyanobacter ium, had a significant ly

FIG. 10. Diagram of energy levels for a chromophore dimerinvolved in exciton coupling. Arrow poin ts to increasing energy.

TABLE VIIEnergy Transfer between Pairs of Bilins in

C-Phycocyanin Monomers

Bilin pa irDistance()

Or ien ta t ion(degrees)

Exper imenta laenergy

t ransfer (ps)

Calcu la tedenergy

t ransfer (ps)a

b155 to a84 48.0 62 .500 815890b155 to b84 34.2 47 52 4649a84 to b84 50.5 16 149 111158

a Debreczeny et al. (1995a).

324 ROBERT MACCOLL

blue-sh ifted absorpt ion spect rum compared to otherC-phycocyanins (Edwards et al., 1996). Monomershad an absorpt ion maximum at 608 nm compared toother monomers a t about 614 nm. It was proposedtha t one or more phycocyanobilins on th is proteinwere affected differen t ly by apoprotein than for otherC-phycocyanins. Schneider et al. (1993), a lso, foundspect roscopic differences among t r imers of C-phyco-cyanins from differen t cyanobacter ia .

PhycoerythrocyaninPhycoerythrocyanin is found in cer ta in cyanobac-

ter ia , which lack phycoerythr in . It s amino acid se-quence (F ugista ller et al., 1983) and three-dimen-siona l st ructure (Duerr ing et al., 1990) are known.Like C-phycocyanin it has three bilins per monomera t a84, b84, and b155 (Table III). It s un iqueness is init s bilin a t a84, where it possesses a phycoviolobilin .Parbel et al. (1997) have shown tha t the b84 phyco-cyanobilin is a t the lowest energy and the phycoviolo-bilin is the highest -energy bilin . In th is study, theopt ica l spect ra of the individua l polypept ides, mono-mers, and t r imers were obta ined. Monomers andt r imers had differences in their spect ra , and excito-n ic coupling between adjacent monomers of thet r imer was discussed. Femtosecond absorpt ion stud-ies have been car r ied out on th is biliprotein (Huckeet al., 1993).

AllophycocyaninEar ly on , there were two proposa ls to expla in why

allophycocyanin t r imers would have a 650-nm maxi-mum and C-phycocyanin t r imers have their absorp-t ion maximum at 621 nm. One suggest ion was tha tone of the bilins on a llophycocyanin t r imers wouldexper ience a differen t apoprotein in teract ion (Mu-rakami et al., 1981). The other was tha t a llophycocya-n in t r imers might have two in teract ing bilins, withclose proximity on adjacent monomers tha t producedthe rela t ively sharp 650-nm absorpt ion maximum(Csa torday et al., 1984; Huang et al., 1987; MacCollet al., 1980). In cont rast , the monomer (ab) spect ra ofa llophycocyanin and C-phycocyanin are similar (Mac-Coll et al., 1980; Murakami et al., 1981).The difference between the absorpt ion spect ra of

monomers and t r imers of a llophycocyanin is st r ik-ing, and it was thought th is change might a llow astudy of the unusua l 650-nm maximum of t r imers.Homogenous monomers for a llophycocyanin werefound to have an absorpt ion maximum at 614 nm,while t r imers had a sharp maximum at 650 nm and aprominent shoulder a t about 610620 nm. The fluo-rescence (excita t ion) pola r iza t ion spect ra of mono-mers and dimers were likewise differen t (MacColl etal., 1980). The t r imer spect rum had a polar iza t ion of10.05, while monomers approached 10.4 a t the red

edge. There have been two quite differen t proposa lsto expla in the spect roscopic differences. In one case,one of the bilins on the monomer, either a84 or b84,is changed by in teract ion with apoprotein whent r imers are formed. The changed bilin then pos-sesses the 650-nm maximum. The bilin might have aunique conformat ion (Sugimoto et al., 1984), or be ina differen t environment . The other bilin reta ins aspect rum simila r to what it had in the proteinmonomer, and it now appears as the 610- to 620-nmshoulder in the t r imer spect rum. Energy t ransfer inth is scenar io would be by Forster resonance from oneindividua l donor bilin a t 610620 nm to the secondacceptor bilin a t 650 nm. Schneider et al. (1995)reviewed deta ils of how the energy levels could beaffected from monomer to t r imer and to t r imer withlinker. The second proposa l suggested tha t the bilinsmight be close together and proper ly or ien ted in thet r imer oligomer across the monomermonomer in ter-face. These bilins would engage in exciton split t ingand the two bilins, a84 and b84, would share excita -t ion deloca liza t ion . The 650-nm maximum and 610-to 620-nm shoulder would be the two exciton sta tes.Instead of F or ster resonance t ransfer, in th is modelin terexciton-sta te relaxa t ion would a llow the t rans-fer of energy.Studies on the monomert r imer equilibr ium were

car r ied out using kinet ics techniques (Huang et al.,1987). When the change from tr imers to monomerswas studied using ligh t sca t ter ing, a single exponen-t ia l decay was observed. Since ligh t sca t ter ing mea-sures the changes in molecula r mass, th is resu lt wasa direct measure of the change from tr imers tomonomers. The ident ica l exper iment was a lso per-formed using absorpt ion changes as the monitor ingmethod. In th is case, there were two events. Thefaster was near ly simultaneous with the light sca t ter-ing kinet ics, and the second absorpt ion change wasmuch slower. The faster change would be expected inthe case of exciton coupling since the pa ir of bilins ont r imers would lose their split spect rum at the samera te tha t monomers were formed. The slower changecould be a change in the bilins as newly formedallophycocyanin monomers rear ranged to form theirmost stable ter t ia ry and secondary st ructures. Thisresu lt suggests tha t both factorsexciton split t ingand bilin environment or conformat ioncont r ibu teto spect roscopic differences between monomers andt r imers, bu t ten ta t ively poin ts to exciton split t ing asestablish ing the energy t ransfer mechanism in t r im-ers.The x-ray crysta llography resu lt s on a llophycocya-

n in t r imers (Brejc et al., 1995) show tha t there seemsto be a bilin with an unusua l conformat ion , but theya lso show a somewhat small distance between bilinson adjacent monomer ic unit s (F ig. 8). Brejc et al.

325PHYCOBILISOMES

(1995) favor the former factor for the cause of the650-nm absorpt ion maximum. It would be of in terestfor a theoret ica l ana lysis to be per formed on th isbilin conformat ion to determine whether it wouldgenera te the 650-nm absorpt ion maximum.Fast laser kinet ics studies (Homoelle et al., 1998;

Sharkov et al., 1992, 1994; Edington et al., 1995,1996; Gillbro et al., 1993; Xie et al., 1992) have notyielded a consensus decision on how energy is t rans-fer red between the two closest bilins of a llophycocya-n in t r imers. Some data appear to st rongly favor theidea tha t two independent bilins having differen tspect ra t ransfer energy from the high-energy tolow-energy bilin by the Forster resonance mecha-n ism. Other da ta indica te a very differen t mecha-n ism: tha t th is pa ir of bilins are sufficien t ly closetogether and share deloca liza t ion energy.Sharkov et al. (1992) used a femtosecond absorp-

t ion method and found a 440-fs (0.44 ps) lifet imehaving a 0.4 anisot ropy dur ing the first few picosec-onds. They propose tha t these resu lt s suppor t amodel of an a llophycocyanin t r imer in which thereare two uncoupled bilins, having maxima at 600 or650 nm. Energy is t ransfer red from the 600-nm bilinto the 650-nm bilin by the Forster resonance mecha-n ism. Sharkov et al. (1994) likewise found agree-ment between their femtosecond spect roscopy dataand Forster resonance t ransfer.In cont rast , Edington et al. (1995) found two-color

pump probe exper iments on a llophycocyanin t r imersgave an in it ia l an isot ropy of 0.580.70. They notedtha t an isola ted dipole would have a maximumanisot ropy of 0.4, bu t a coupled pa ir of chromophorescould have a va lue up to 0.7. In addit ion , they founda fastest decay component of 1030 fs and main ta intha t th is is too shor t for F or ster resonance energyt ransfer. Their da ta are best fit by in terna l conver-sion between the two exciton sta tes of a st ronglycoupled bilin pair (Fig. 10). They propose that cluster-ing of chromophores would provide for more rapidmovement of energy to the lowest -energy chromo-phore from which energy would be t ransfer red out ofone protein and in to another pigment . Also, F or sterresonance t ransfer to other h igher-energy chromo-phores would be decreased by very rapid in terna lconversion . After the in terna l conversion , excita t ionwould loca lize on a par t icu la r bilin . Beck and Sauer(1992) and Edington et al. (1996) studied the a llophy-cocyanin t r imer by a var iety of spect roscopic meth-ods and concluded tha t energy t ransfer was notproduced by Forster resonance, but ra ther resu ltedfrom exciton sta tes produced by in teract ion of thebilins a t a84 and b84. Homoelle et al. (1998) cont in-ued the study of a llophycocanin , using advancedtechniques, and t ime constan ts of 56 and 220 fs wereassigned to radia t ion less decay between exciton

sta tes in the t r imers. These t ransit ions were notobserved in prepara t ions of the a polypept ide fromC-phycocyan in . Other t ime-resolved fluorescencestudies have appeared on the separa ted a and bpolypept ides of C-phycocyanin (Fischer et al., 1990),and on the aB-conta in ing t r imer (Yamazaki et al.,1994).The sum of these studies suggests tha t a ll t rans-

fers on C-phycocyanin and a llophycocyanin mono-mers may occur by Forster resonance, as well assome of the t ransfers on t r imers, especia lly thoseinvolving the b155 bilin of C-phycocyanin . The closelyspaced a84/b84 pair on t r imers of both biliproteins issubject to fur ther inquir ies, bu t there are sign ificantresu lt s for a llophycocyanin suppor t ing deloca liza -t ion . Highly sophist ica ted femtosecond laser resu lt s(Homoelle et al., 1998) provide compelling suppor tfor exciton sta tes in t r imers of a llophycoyanin . Delo-ca liza t ion between pa irs of bilins has a lso beensuggested for cryptomonad biliproteins based onresu lt s using opt ica l spect roscopy (MacColl et al.,1995, 1998).Ear lier t ime-resolved energy t ransfer studies us-

ing isola ted biliproteins, biliprotein complexes, andphycobilisomes have been reviewed (Holzwarth, 1986,1991). These cita t ions include pioneer ing work doneon biliproteins from red a lgae and cryptomonads, aswell as cyanobacter ia .

CYANOBACTERIA AND HABITATS

Previously, it was noted tha t mar ine cyanobacter iamight u t ilize the ligh t harvest ing proper t ies of C-phycoerythr in and the CU-phycoerythr ins, as wellas complementary chromat ic adapta t ion in somecases, to thr ive in habita t s of changing and cha lleng-ing ligh t . Cyanobacter ia a lso live in such diversity asthe pola r regions and thermal hot spr ings. Phycobili-somes have been observed in a cyanobacter ium,which grows in hot spr ings above 70C (Edwards andGant t , 1971). Berns, Edwards, and co-workers haveshown tha t C-phycocyanins pur ified from thermo-philes have differen t ways to meet th is cha llenge.For one thermophilic cyanobacter ium, the C-phyco-cyanin is tempera ture resistan t as fa ir ly la rgepieces of rod were stable from 10 to 80C (Edwards etal., 1996, 1997). For another thermophile, the C-phycocyanin is cold dissocia ted as the la rger rodpieces fa ll apar t a t low tempera ture, bu t a re associ-a ted near growth tempera tures (Berns and Scot t ,1966). Recent ly, a thermophilic cyanobacter ium waslysed and yielded a C-phycocyanin aggrega te ofthree disks (Hayashi et al., 1997). This isola te maybe the in tact rods of the organism.There have been thermodynamic studies on these

thermophilic C-phycocyanins (Chen et al., 1994).Result s a re in terpreted to suggest tha t en t ropy

326 ROBERT MACCOLL

changes are quite sign ificant in the unfolding ofC-phycocyanin , and for thermophilic protein theent ropy cont r ibu t ion is even grea ter.

EPILOGUE

Much has been accomplished since Gant t andco-workers began to establish a st ructura l model forphycobilisomes, which so nicely produced under-standing of how they work. The discovery of linkersand the ensuing research on biliproteins and linkersproduced leaps of knowledge which made a complex,but en ligh ten ing story. Studies on such a la rge andvar iable st ructure isola ted from the diverse cyanobac-ter ia and the red a lgae are never rea lly finished, butwhat a re the key fea tures yet to be established?One issue may stand out : the linkers have been

nicely studied, but what is known about the molecu-la r na ture of their in teract ion with the biliproteins,or how they accomplish their exquisite specificity ofdisk to rod associa t ion? Next to noth ing is the bestanswer. There are published drawings showing thelinker occupying the cent ra l hole in the biliproteinst ructure. Yes, it is clear they should be there, bu t isthere proof? X-ray crysta llography studies have beencar r ied out on b-phycoerythr in and B-phycoerythr infrom red a lgae (Ficner et al., 1992; Ficner and Huber,1993). The former lacked a linker and the la t terpossessed one. The linker st ructure was not obta inedin these studies, bu t the linker by compar ison of thetwo biliprotein st ructures is in the cent ra l hole of thebiliprotein and does not prot rude from it . Kessel etal. (1973) have produced elect ron micrographs ofC-phycocyanin disks from a cyanobacter ium show-ing a cent ra l st ructure (Fig. 11). This object could belinker.Some quest ions: how do linkers in teract with

biliproteins; since they do not prot rude, how do theycause a specific biliprotein disk to add to the rods;does one linker in teract with another ; is there aconformat iona l change when linker joins disk or diskjoins rods; would linker from a thermophile conferthermostability on mesophilic C-phycocyanin ; howare the bilins near the cent ra l hole affected bylinkers; how does the Lcm in nonhemidiscoida l phyco-bilisomes funct ion ; does Lcm in some version exist forcryptomonads?All of these poin ts could be the subject of specula -

t ion , but let us take on just one. The b84 bilins areloca ted very near the cent ra l channel in the disks.The bilins of C-phycocyanin are affected by linkersas indica ted by the effect s of linkers on their spect raand energy t ransfer kinet ics. These observa t ionsmay suggest tha t bilins are spa t ia lly displaced bylinkers. If th is is t rue, what is the sta tus of ourunderstanding of the routes of energy migra t ion?Changes in bilin -to-bilin distances and or ien ta t ionswill sa lien t ly change the ca lcu la t ion of how energy ist ransfer red. It may be tha t ca lcu la t ions using linker-free biliproteins are usefu l but give an incompleteview of the actua l energy migra t ion system. Theopen-cha in st ructure of the tet rapyr roles may makethem flexible in responding to changes tha t resu ltfrom modifica t ion of their nearest neighbor environ-ment . The na ture of linkers cont r ibu tes to the diffi-cu lty in their study. A phycobilisome conta in ing fourdisks, even if the disks are ident ica l, will have fourdifferen t rod-rela ted linkers. Remember linkers varywith each disk moving among the rod, even if thedisks themselves are ident ica l. A phycobilisome diskconta in ing one specific linker, in par t icu la r LRC,should be studied for rea l progress. Analogously,examining the st ructure of a llophycocyanin withlinker a t tached would be of in terest .

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