QuarterlyReviewsofBiophysics35,1(2002),pp.1–62. "2002CambridgeUniversityPress 1 DOI:10.1017/S0033583501003754 PrintedintheUnitedKingdom Photosynthetic apparatus of purple bacteria Xiche Hu1, Thorsten Ritz2, Ana Damjanovic!2, Felix Autenrieth2 and Klaus Schulten2,* 1DepartmentofChemistry,UniversityofToledo,Toledo,OH43606,USA 2BeckmanInstituteandDepartmentofPhysics,UniversityofIllinoisatUrbana-Champaign,Urbana, IL61801,USA 1. Introduction 2 2. Structure of the bacterialPSU 5 2.1 Organization of the bacterial PSU 5 2.2 The crystal structure of the RC 9 2.3 The crystal structures of LH-II 11 2.4 Bacteriochlorophyll pairsinLH-II and the RC 13 2.5 Models of LH-Iand the LH-I–RCcomplex 15 2.6 Model for the PSU 17 3. Excitation transfer in the PSU 18 3.1 Electronicexcitationsof BChls 22 3.1.1 Individual BChls 22 3.1.2 Rings of BChls 22 3.1.2.1 Excitonstates 22 3.1.3 Effective Hamiltonian 24 3.1.4 Optical properties 25 3.1.5 The effect of disorder 26 3.2 Theoryof excitation transfer 29 3.2.1 General theory 29 3.2.2 Mechanismsof excitation transfer 32 3.2.3 Approximation for long-rangetransfer 34 3.2.4 Transferto excitonstates 35 3.3 Ratesfor transferprocesses inthe PSU 37 3.3.1 CarUBChltransfer 37 3.3.1.1 Mechanismof CarUBChltransfer 39 3.3.1.2 Pathwaysof CarUBChltransfer 40 3.3.2 Efficiencyof CarUBChltransfer 40 3.3.3 B800–B850transfer 44 3.3.4 LH-IIULH-II transfer 44 3.3.5 LH-IIULH-Itransfer 45 3.3.6 LH-IURC transfer 45 3.3.7 Excitation migrationinthe PSU 46 3.3.8 Geneticbasis of PSU assembly 49 * Author towhomcorrespondenceshould be addressed.E-mail: kschulte!ks.uiuc.edu 2 X. Hu et al. 4. Concludingremarks 53 5. Acknowledgments 55 6. References 55 1. Introduction Lifeasweknowittodayexistslargelybecauseofphotosynthesis,theprocessthroughwhich light energy is converted into chemical energy by plants,algae, and photosynthetic bacteria (Priestley, 1772; Barnes, 1893; Wurmser, 1925; Van Niel, 1941; Clayton & Sistrom, 1978; Blankenship et al. 1995; Ort & Yocum, 1996). Historically, photosynthetic organisms are groupedintotwoclasses.Whenphotosynthesisiscarriedoutinthepresenceofairitiscalled oxygenicphotosynthesis(Ort&Yocum,1996).Otherwise,itisanoxygenic(Blankenshipet al. 1995). Higher plants, algae and cyanobacteria perform oxygenic photosynthesis, which involves reduction of carbon dioxide to carbohydrate and oxidation of water to produce molecular oxygen. Some photosynthetic bacteria, such as purple bacteria, carry out anoxygenicphotosynthesisthatinvolvesoxidationofmoleculesotherthanwater.Inspiteof thesedifferences,thegeneralprinciplesofenergytransductionarethesameinanoxygenicand oxygenic photosynthesis (Van Niel, 1931, 1941; Stanier, 1961; Wraight, 1982; Gest, 1993). Theprimaryprocessesofphotosynthesisinvolveabsorptionofphotonsby light-harvesting complexes (LHs), transfer of excitation energy from LHs to the photosynthetic reaction centers(RCs),andtheprimarychargeseparationacrossthephotosyntheticmembrane(Sauer, 1975;Knox,1977;Fleming&vanGrondelle,1994;vanGrondelleetal.1994).Inthisarticle, we will focus on the anoxygenic photosynthetic process in purple bacteria, since its photosynthetic system is the most studied and best characterized during the past 50 years. The photosynthetic apparatus of purple bacteria is a nanometric assembly in the intracytoplasmic membranes and consists of two types of pigment–protein complexes, the photosynthetic RC and LHs (Kaplan & Arntzen, 1982; Zuber & Brunisholz, 1991). The functionofLHsistocapturesunlightandtotransfertheexcitationenergytotheRCwhere it serves to initiate a charge separation process (Fleming & van Grondelle, 1994; Lancaster etal.1995;Parson&Warshel,1995;Woodbury&Allen,1995;Huetal.1998).Depictedin Fig. 1 schematically is the intracytoplasmic membrane of purple bacteria with its primary photosynthetic apparatus. This apparatus, one of the simplest of its kind, feeds through excitation bysunlight acyclic flowof electrons and protons that leads to synthesisof ATP. Over the past few decades, extensive biochemical and spectroscopic studies of bacterial photosynthetic systems have revealed the following structural and dynamical principles for bacterial light harvesting: (1) Bacterial photosynthetic membranes contain thousands of pigment molecules (bacteriochlorophyllsandcarotenoids)thatarenon-covalentlyboundtoproteinstoform well organized pigment–protein complexes (Stoll, 1936; Smith, 1938; Rees & Clayton, 1968;Thornberetal.1983;Zuber,1986;Scheer,1988;Zuber&Brunisholz,1991;Zuber &Cogdell,1995).Outofallthesepigments,onlyveryfewbacteriochlorophylls(BChls) in the primary reaction site, termed the photosynthetic RC, directly take part in photochemical reactions; most BChls serve as light-harvesting antennae capturing the Photosynthetic apparatus of purple bacteria 3 Fig.1. Schematicrepresentationofthephotosyntheticapparatusintheintracytoplasmicmembraneof purplebacteria.Thereactioncenter(RC,red)issurroundedbythelight-harvestingcomplexI(LH-I, green) to form the LH-I–RC complex, which is surrounded by multiple light-harvesting complexes (LH-IIs)(green),formingaltogetherthephotosyntheticunit(PSU).PhotonsareabsorbedbyLHsand excitationistransferredtotheRCinitiatingacharge(electron-hole)separation.Electronsareshuttled backbythecytochromec#complex(blue)fromtheubiquinone-cytochromebc"complex(yellow)tothe RC. The electron transfer across the membrane produces a large proton gradient that drives the synthesisofATPfromADPbytheATPase(orange).Electronflowisrepresentedinblue,protonflow in red,quinone flow, likely confinedtotheintramembranespace,in black. sunlightandfunnelingelectronicexcitationtowardstheRC(Emerson&Arnold,1932; Arnold&Kohn,1934;Duysens,1952,1964).Byconvention,thephotosyntheticRCand the associated LHs are collectively named the photosynthetic unit (PSU) (Mauzerall &Greenbaum,1989;Francke&Amesz,1995;Freiberg,1995;Cogdelletal.1996).Asa matteroffact,theorganizationofpigmentsintoPSUsinwhichmultiplelight-harvesting antennae serve the RC has been adopted by all photosynthetic organisms (Duysens, 1988;Mauzerall&Greenbaum,1989;Grossmanetal.1995;Fromme,1996;Gantt,1996; Green & Durnford, 1996; Hankamer et al. 1997). With it they can collect light from a broaderspectralrangeanduseenergymuchmoreefficiently.Light-harvestingantennae enlarge the cross-section for capturing sunlight by the RC. The latter possesses light- absorbing chlorophylls itself, but photons absorbed by the RC chlorophylls are not sufficient to saturate its maximum turnover rate. When exposed to direct sunlight, chlorophylls absorb at a rate of at most 10Hz and, in dim light, at a rate of 0–1Hz (Borisov&Godik, 1973). However,thechemical reactionof theRCcan ‘turn over’at 1000Hz.LHsfuelexcitationenergytotheRC,whichkeepstheRCrunningatanoptimal rate. (2) The light-harvesting antenna is composed of multiple LHs with varying spectral characteristics and a particular structural organization in the whole antenna. In most purplebacteria,thePSUscontaintwotypesofLHs,commonlyreferredtoasB875(LH- I) and B800–850 (LH-II) complexes according to their in vivo absorption maxima in the near-infrared (Thornber et al. 1983; Zuber & Brunisholz, 1991; Hawthornthwaite & Cogdell, 1991). LH-I is found surrounding directly the RCs (Miller, 1982; Walz & Ghosh, 1997), while LH-II is not in direct contact with the RC, but transfers energy to it via LH-I (Monger & Parson, 1977; Sundstro$m & van Grondelle, 1991, 1995; van Grondelle et al. 1994). For some bacteria, such as Rhodopseudomonas (Rps.) acidophila and 4 X. Hu et al. Fig.2. EnergylevelsoftheelectronicexcitationsinthePSUofBChlacontainingpurplebacteria.The diagramillustratesafunnelingofexcitationenergytowardsthephotosyntheticRC.Theverticaldashed lines indicate intra-complex excitation transfer; the diagonal solid lines inter-complex excitation transfer.LH-Iexistsinallpurplebacteria;LH-IIexistsinmostspecies;LH-IIIarisesincertainspecies only and itis usuallyregulatedbyambiencelight. Rhodospirillum (Rs.) molischianum strain DSM 120 (Germeroth et al. 1993), there exists a thirdtypeoflight-harvestingcomplex,LH-III.ThenumberofLH-IIsandLH-IIIsinthe PSU varies according to growth conditions such as light intensity and temperature (Aagaard & Sistrom, 1972). (3) Photosyntheticbacteriaevolvedapronouncedenergetichierarchyinthelight-harvesting system.Purplebacteriaabsorblightinaspectralregioncomplementarytothatofplants and algae, mainly at wavelengths of approximately 500nm through carotenoids and above800nmthroughBChls.ShowninFig.2aretheenergylevelsforthekeyelectronic excitationsinthePSU.PigmentsofouterLHsabsorbatahigherenergythandotheinner ones. For example, the LH-II, which surrounds LH-I, absorbs maximally at 800 and 850nm; LH-I, which in turn surrounds the RC, absorbs at a lower energy (875nm) (Thornberetal.1983;Zuber&Brunisholz,1991;vanGrondelleetal.1994;Sundstro$m &vanGrondelle,1995).Theenergycascadeservestofunnelelectronicexcitationsfrom the LH-IIs through LH-I to the RC. (4) Time-resolvedpicosecondandfemtosecondspectroscopyrevealedthatexcitationenergy transferwithinthePSUoccursonanultrafasttimescaleandatnearunit(95%)efficiency (Pullerits&Sundstro$m,1996;Fleming&vanGrondelle,1997).Ittakeslessthan100ps for the energy of the excited LHs to reach the RC. Timescales for elementary energy transfer steps range from femtoseconds to picoseconds. Tremendousprogressinourunderstandingofbacterialphotosynthesishasbeenachieved during the last 15 years with the determination of the atomic structures of the bacterial photosynthetic RC, followed by two high-resolution crystal structures of LH-II. Structures Photosynthetic apparatus of purple bacteria 5 oftheRCfor Rps.viridis(Deisenhofer etal.1985) aswellasfor Rhodobacter(Rb.) sphaeroides (Allen et al. 1987; Ermler et al. 1994) were determined by X-ray crystallography. Recently, high-resolution crystal structures of LH-IIs from two species (Rps. acidophila and Rs. molischianum) have been determined (McDermott et al. 1995; Koepke et al. 1996). The I structure of LH-I is not yet known to atomic resolution, although an 8.5A resolution projection map observed by electron microscopy was reported for LH-I of Rs. rubrum (Karrasch et al. 1995). The structures mentioned provide detailed knowledge of the organization of chro- mophores in the photosynthetic membrane and stimulated a new wave of more focused theoretical and experimental investigations of bacterial photosynthesis in an already active field of research (Clayton, 1973; Clayton & Sistrom, 1978; Govindjee, 1982; Scheer, 1991; Deisenhofer&Norris,1993;Fleming&vanGrondelle,1994;Blankenshipetal.1995;Fyfe & Cogdell, 1996; Hu & Schulten, 1997; Hu et al. 1998; Sundstro$m et al. 1999; Krueger et al. 1999a; Cogdell et al. 1999). In this review, we will look at our current understanding of structureand dynamics of bacterial light harvestingand highlight some pressing issues that meritfurtherinvestigation.Thescopeofthisreviewwillbelimitedtothemolecularmodel of the bacterial PSU and structure-based theoretical studies of excitation energy transfer mechanisms. Spectroscopic probe of the excitation transfer dynamics in the PSU, when relevant,willbediscussed,butwillnotbeemphasized.Readersinterestedinthesubjectare referredtootherreviews(Sauer,1975;Borisov,1978;Hunteretal.1989;Sundstro$m&van Grondelle, 1991; van Grondelle et al. 1994; Pullerits & Sundstro$m, 1996; Fleming & van Grondelle, 1997; Sundstro$m et al. 1999; Krueger et al. 1999a: van Amerongen et al. 2000). 2. Structure of the bacterial PSU The PSU combines in the intracytoplasmic membrane of purple bacteria a nanometric assemblyofthephotosyntheticRCandanarrayofLHs.Duringthelast15years,structural detailsofmanyoftheseindividualpigment–proteincomplexeshaveemerged,albeitnotfrom the same species. All the known crystal structures of RCs and LH-IIs are from BChl a- containingpurplebacteriaexceptthatofRCfromRps.viridis(Deisenhoferetal.1985)which contains BChl b as the major pigment. Consequently, we will describe the structural organizationofPSUbasedonBChla-containingpurplebacteria.Atfirst,wewillintroduce the overall organization of the bacterial PSU. We will then describe structural features of individualpigment–proteincomplexes.Inparticular,wewillpresentthecrystalstructureof the RC from Rb. sphaeroides (Allen et al. 1987; Ermler et al. 1994), the modeled structure of LH-I from Rb. sphaeroides, and the crystal structure of LH-II from Rs. molischianum with a comparison to the crystal structure of LH-II from Rps. acidophila. 2.1 Organization of the bacterial PSU It has been firmly established that the bacterial PSU consists of multiple pigment–protein complexes,includingtheRCs,LH-IsandLH-IIs.However,theinter-complexorganization ofRCs,LH-IsandLH-IIsinsidethePSUiscurrentlyamatterofhotdebate(Papizetal.1996; Nagarajan & Parson, 1997; Westerhuis et al. 1998). Figure 3 depicts two proposed models for the bacterial PSU, denoted model A and model B, that are based on low-resolution 6 X. Hu et al. (a) (b) Fig.3. ProposedmodelsofthebacterialPSU.(a)ModelA:PSUaccordingtoNiederman(Westerhuis et al. 1998) and Parson (Monger & Parson, 1977; Nagarajan & Parson, 1997). A pair of RCs, each surroundedbyanopencircleLH-I,associatewiththecytochromebc"complex(bc")toformthecore ofthePSU.Thecoreisinturnsurroundedbyperipheralantennacomplexes(LH-IIs).Thesmallcircles representtransmembraneheliceswithBChlssandwichedinbetween.AlsoshownisthePufXprotein (soliddot)locatedbetweenRCandbc".(b)ModelB:PSUasproposedbyCogdellandcolleagues(Papiz etal.1996).AnisolatedRCissurroundedbyaclosed-circleLH-IcomplextoformthecoreofthePSU that is in apoolofLH-IIs. Also shownis thecytochrome bc" complex(bc"). Photosynthetic apparatus of purple bacteria 7 Fig.4. ProjectionmapofnegativelystainednativetubularflatmembranefromRb.sphaeroidesat20AI resolution after processing and averaging (adapted from fig. 4B of Jungas et al. 1999). The unit cell (afl198 AI , bfl120AI and cfl103(cid:176)) is outlined in black. Positive density representing the protein is shownas solid linesand negative density as dottedlines. electronmicroscopicprojectionmapsandspectroscopicanalyses(Papizetal.1996;Nagarajan & Parson, 1997; Westerhuis et al. 1998). The two models of bacterial PSU in the figure displaysignificantdifferencesintwomajoraspects:(1)themonomericLH-I–RCcomplexis completelysurroundedbyLH-IIsinmodelB,whereasinmodelApairsofRCsareclustered together;(2)inmodelA,LH-Iformsanopen-ringstructurethatallowsshufflingofquinone between the RC and the cytochrome bc" complex, while in model B LH-I forms a closed circular structure. Model A is based on fluorescence yield and singlet–singlet annihilation measurements of phospholipid-enrichedRb.sphaeroideschromatophores(Westerhuisetal.1998).Thismodelis inapparentagreementwiththesupercomplexmodelofthephotosyntheticelectrontransfer chainintermsofthedimericassociationoftheRCs(Joliotetal.1989).Evidenceinfavorof Model A appeared in newly reported electron micrographs of purified tubular membranes from Rb. sphaeroides (Jungas et al. 1999; Vermeglio & Joliot, 1999). It was found that the tubular membrane contains all the components of the photosynthetic apparatus, with a relativeratioofonecytochromebc"complex:twoRCs:C48LH-IBChls(Jungasetal.1999). I Shown in Fig. 4 is the 20 A resolution electron microscopic projection map of a negatively stainednativetubularmembranefromRb.sphaeroidesasreportedinJungasetal.(1999).The unit cell contains an elongated S-shaped supercomplex with a pseudo-twofold symmetry. Jungasetal.(1999)interpretedthemaptomeanthateachsupercomplexiscomposedofLH- IsthattaketheformoftwoC-shapedstructuresofC112AI inexternaldiameter,facingeach otherontheopensideandenclosingthetwoRCs.Suchamodelallowsshufflingofquinone betweentheRCandcytochromebc"complex.Unfortunately,thelocationofthebc"complex can not be positively identified in this projection map due to a weak signal arising from a 8 X. Hu et al. Fig. 5. The measured 8–5AI electron microscopic projection map (black) of LH-I of Rs. rubrum (reproducedfromKarraschetal.1995).Contoursareinstepsof0–3‹r.m.s.density;scalebarrepresents 20AI . The overall dimensionofthecell is 120‹120AI . technicaldifficulty:thestainuseddoesnotpenetratetotheperiplasmicsideofthemembrane wheremostoftheextramembranousparts(RieskeproteinandcytC")ofthebc"complexare located (Jungas et al. 1999). Model B is based on spectroscopic observations (Deinum et al. 1991) and an 8.5AI resolutionelectronmicrographforLH-I,whichhasbeendeterminedfromatwodimensional (2D) crystal of the reconstituted LH-I of Rs. rubrum by Karrasch and colleagues (Karrasch et al. 1995; Papiz et al. 1996; Pullerits & Sundstro$m, 1996; Hu et al. 1997). The electron microscopicelectrondensityprojectionmap,reproducedinFig.5,showedLH-Iasaringof I I 16subunits.Theringhasadiameterof116Awitha68Aholeinthecenterwhich,aspointed outbyKarraschetal.(1995),islargeenoughtoincorporateaRCinvivo.Arecentreportof an electron micrograph of a 2D crystal of the LH-I–RC complex from Rs. rubrum further confirmsthelocationoftheRCinthecenterofLH-I(Walz&Ghosh,1997).Questionshave been raised as to whether the dissociation and reconstitution process employed, e.g. in Karraschetal.(1995), introducesartifacts that renderthereconstituted LH-Is of Rs.rubrum (Karraschetal.1995)anartificialvariantofnativeLH-Is.Theseconcernswerealleviatedby the observation that the 2D crystal of the LH-I–RC complex, formed under much milder crystallization conditions under which no dissociation of the LH-I–RC complex can be detected,displayedthesameLH-IringsizeasthereconstitutedLH-I(Walz&Ghosh,1997). Furthermore, the gross morphology of the core PSU, which consists of a central core RC Photosynthetic apparatus of purple bacteria 9 surrounded by an LH-I ring, is consistent with earlier models of the PSU for both BChl b- and BChl a-containing bacteria based upon electron microscopy and image processing (Miller,1982;Starketal.1984;Engelhardtetal.1986;Meckenstocketal.1992;Boonstraet al.1994).RecentlyreportedelectronmicrographsoftheLH-I–RCcomplexfromRp.viridis andRb.sphaeroidesalsoindicatedasingleRCinsideaclosedringoftheLH-I(Ikeda-Yamasaki etal.1998;Walzetal.1998).However,thelatestanalysisofelectronmicroscopicprojection mapsof2DcrystalsoftheLH-I–RCcomplexfromRs.rubrumbytheGhoshgroupsuggested that, in carotenoid-less mutants, the LH-I–RC complex may have a non-circular symmetry (Stahlberg et al. 1998). 2.2 The crystal structure of the RC ThebestknownstructuralcomponentsofthebacterialPSUareLH-IIandtheRC.Wewill brieflyoutline thecrystalstructureof theRCfromRb.sphaeroideswhich istheonlyBChla- containingspecies for which a high-resolution crystal structure of the RC is known (Chang etal.1986;Allenetal.1987;Ermleretal.1994). Theotherknown bacterialRCstructureis from a BChl b-containing species Rps. viridis (Deisenhofer et al. 1985). As shown in Fig. 6, theRCfromRb.sphaeroidescontainsthreeproteinsubunits,knownasL(light),M(medium), and H (heavy), respectively. The L- and M-subunits are homologous and are related by a pseudo-twofoldcircularsymmetry.Multiplepigmentmolecules(cofactors)areboundtothe L- and M-subunits, and are arranged accordingly in two symmetric branches, commonly referred to as A branch and B branch: two BChls which form a strongly interacting dimer constitutingtheso-calledspecialpair(P ,P ),twoaccessoryBChlsincloseproximitytothe A B special pair (B ,B ), two bacteriopheophytins (H ,H ), and a pair of quinones (Q ,Q ) A B A B A B (Chang et al. 1986; Allen et al. 1987; Ermler et al. 1994). UponexcitationoftheRCspecialpair,amulti-stepchargeseparationprocessisinitiated. Sincewearemainlyconcernedwiththelight-harvestingprocessandnofurtherexplanation oftheelectrontransferprocesswillbeattempted,weseeitfitheretogiveabriefdescription oftheelectrontransferprocess.Forreadersinterestedinthesubject,thisprimaryprocessof charge separation has been studied extensively and was the subject of numerous review articles (Clayton, 1966, 1973; Deisenhofer & Michel, 1991; Breton & Verme!glio, 1992; Deisenhofer&Norris,1993;Fleming&vanGrondelle,1994;Lancasteretal.1995;Parson & Warshel, 1995; Woodbury & Allen, 1995; Michel-Beyerle, 1996; Hoff & Deisenhofer, 1997; Bixon & Jortner, 1999). It has been determined through X-ray crystallographic analysis and spectroscopic measurement that electron flow in the photosynthetic bacteria is cyclic. In addition to the photosyntheticRC,otherelectroncarriersareinvolvedinthecyclicflow,includingamobile quinonepool,acytochromebc"oxidoreductase,andamobilecytochromecmolecule.Upon excitation, an electron within the special pair (P ,P ) is promoted to an excited state. This A B electron is transferred, through the accessory BChl (B ), to the bacteriopheophytin (H ) in A A 2–3 ps. The reduced bacteriopheophytin (H−") donates an electron to the adjacent quinone A molecule (Q ) in about 200ps. The Q in turn passes an electron to the Q molecule in A A B 200ls, converting Q to a semiquinone radical. In the mean time, the positively charged B special pair is neutralized by the transfer of an electron from a reduced cytochrome c# moleculeontheperiplasmicsideofthemembrane.Asecondphotonisthenabsorbedbythe specialpair,andtheflowofasecondelectrontoQ takesplace.Withtheacceptanceoftwo B 10 X. Hu et al. (a) (b) Fig. 6. Structure of the photosynthetic RC of Rb. sphaeroides (Ermler et al. 1994). (a) The protein subunitsarerepresentedasC traceswiththeL-,M-andH-subunitsoftheRCinyellow,redandgray, a respectively.(b)Chromophoresarerepresentedinalicoricerepresentationwiththefollowinglabeland colorcoding:RCspecialpair(P ,P ,green),accessoryBChls(B ,B ,green),bacteriopheophytins(H , A B A B A H ,cyan),quinones(Q ,Q ,white),andacarotenoidmolecule(magenta).ThevanderWaalsspheres B A B showthepositionofthecentralMg#+atomsofBChls.ThephytoltailsofBChlsandbacteriopheophytin are omitted for clarity. electrons by QB, the quinone molecule is reduced to the quinol form (H#Q) by the simultaneousuptake oftwo protonsfromthecytoplasmicside of themembrane. This H#Q moleculeisthenreleasedfromtheRCintoamobilepoolofquinones.Theelectrontransfer