FEBS 21004 FEBS Letters 438 (1998) 5^9

Minireview Photosynthetic reaction centers

J.P. Allen*, J.C. Williams

Department of Chemistry and Biochemistry and Center for the Study of Early Events in , Arizona State University, Tempe, AZ 85287-1604, USA

Received 23 July 1998

thetic organisms with an emphasis on the lessons learned from Abstract The reaction center is the key component for the primary events in the photochemical conversion of light into reaction centers isolated from purple bacteria. chemical energy. After excitation by light, a charge separation that spans the cell membrane is formed in the reaction center in a 2. A reaction center motif few hundred picoseconds with a quantum yield of essentially one. A conserved pattern in the cofactors and core proteins of reaction Photosynthetic reaction centers can be classed into two cat- centers from different organisms can be defined based on egories based upon the nature of the electron acceptors (for a comparisons of the three dimensional structure of two types of review see [6]). Purple bacteria, green ¢lamentous bacteria, reaction centers. Different functional aspects of the reaction and II belong to the -quinone type, center are discussed, including the properties of the bacterio- while green sulfur bacteria, heliobacteria, and or chlorophyll dimer that constitutes the primary belong to the iron-sulfur type (Fig. 1). While anoxygenic bac- electron donor, the pathway of electron transfer, and the different functional roles of the electron acceptors. The teria have only one photosystem, and plants implication of these results on the evolution of the reaction contain both types of . A structure for each center is presented. type of reaction center has been determined by X-ray di¡rac- z 1998 Federation of European Biochemical Societies. tion, and generalizations can be drawn from these structures since sequence comparisons indicate that all the reaction cen- Key words: Purple bacterium; Green bacterium; Plant; ters within each type are homologous. The reaction center Photosystem II; Photosystem I; Evolution from purple bacteria has four , two bac- teriopheophytins, two quinones, a , and one non- iron arranged into two symmetrically-related branches 1. Introduction (for reviews see [7^9]). Photosystem II has a large number of cofactors and protein subunits, although complexes can be In every photosynthetic organism, the primary events in- isolated containing only six , two pheophytins, volve an integral membrane pigment- called two and three protein subunits. Although there the reaction center that creates a charge separation across the is no three-dimensional structure of photosystem II, the co- cell membrane after light excitation. Coupling to secondary factors are thought to be arranged in a similar fashion to that electron donors and acceptors allows the electrons and accom- of purple bacteria with the additional presence of the oxygen panying protons to be transferred to other components of the evolving complex (for a review see [10]). The structure of photosynthetic apparatus ultimately to be converted into photosystem I reveals a large number of chlorophylls also chemically rich compounds such as ATP and NADP. This arranged with a two-fold symmetry axis with three iron-sulfur general pattern is remarkably conserved, although the speci¢c clusters [11]. While most of the in the iron-sulfur pathway utilized for these processes varies depending upon type of reaction center function in light harvesting, six central the organism. In recent years there have been excellent reviews tetrapyrroles and three iron-sulfur complexes appear to con- on all aspects of photosynthesis [1^4] including an engaging stitute the photochemical heart of this reaction center. personal account of bacterial reaction centers [5]. In this re- A common feature in the structures of the two types of view, we compare the structures of di¡erent reaction centers reaction centers is the presence of a two-fold symmetry axis and discuss some of the current questions, including those relating both the cofactors and protein subunits. This feature pertaining to the evolution of the reaction center. Many of is particularly striking when considering the portion of the the presented aspects are based upon extensive mutagenesis reaction centers that contains the components essential for studies, in particular on the reaction center from purple bac- . Surrounding the cofactors is a hydrophobic teria, for which the in£uence of speci¢c protein-cofactor in- environment provided by amino acid residues from two core teractions can be probed due to the availability of the high- subunits, for example L and M in purple bacteria, and PsaA resolution X-ray structures. Overall, the focus is on the fea- and PsaB in photosystem I. Just as the cofactors are arranged tures common to reaction centers from di¡erent photosyn- in two symmetrical branches, the two core subunits are sym- metrically located about the central two-fold axis. In reaction centers from purple bacteria each core subunit is largely com- prised of ¢ve long transmembrane helices. The subunits of photosystem I are signi¢cantly larger but the pattern of ¢ve *Fax: (1) (602) 965 2747. centrally located and symmetrically related transmembrane E-mail: [email protected] helices from each core subunit is still present. The outermost

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FEBS 21004 2-11-98 6 J.P. Allen, J.C. Williams/FEBS Letters 438 (1998) 5^9

Fig. 1. Comparison of the properties of the pheophytin-quinone (left) and iron-sulfur (right) type of reaction centers. The arrangement of the cofactors in electron transfer for purple bacteria, photosystem II, photosystem I, and green sulfur bacteria is depicted at the top of the ¢gure. The dotted line represents the absorption of light by the primary electron donor (BChl2 or Chl2). The solid line shows the energy transfer steps in the reaction center, from the tyrosine residue (YZ) in photosystem II, through the monomer (BChl), monomer bacterio- pheophytin (BPhe) or pheophytin (Phe) and quinones (QA and QB) in the pheophytin-quinone type of reaction center, and through the mono- mer chlorophyll (Chl), quinone (Q), and iron-sulfur centers (FX, FA and FB) in the iron-sulfur reaction centers. The speci¢c nature or role has not been established for two components shown in italics: the primary donor of photosystem II [10,13], and the quinone of iron-sulfur reaction centers in green sulfur bacteria [28]. The dashed line indicates electron transfer events that take place outside of the reaction center. Views of the essential cofactors and central transmembrane helices are depicted in the middle and bottom of the ¢gure. Shown are the bacteriochloro- phyll dimer (bold), monomer bacteriochlorophylls, monomer bacteriopheophytins, quinones, and iron atom of the reaction center from the pur- ple bacterium Rhodobacter sphaeroides, and the chlorophyll dimer (bold), central chlorophyll monomers, and the FX iron-sulfur center of the photosystem I reaction center from Synechococcus elongatus. The symmetry axis spans the membrane and is in the plane of the paper in the middle view and rotated by 90³ so that it is perpendicular to the paper in the bottom view. The phytyl and isoprenoid chains have been trun- cated. The structure ¢gures were created using the program MOLSCRIPT [36] and coordinates from the Brookhaven Databank ¢les 4RCR and 2PPS. of the ten central helices are packed in a similar pattern in the two types of reaction centers look similar, but the details of two types of reaction centers. The four innermost helices are the binding sites for the cofactors di¡er. For example, histi- most closely associated with the cofactors, and have di¡erent dine residues from the inner helices coordinate the non-heme relative positions and orientations re£ecting the di¡erences in iron in pheophytin-quinone reaction centers, but in iron-sulfur the nature of the electron acceptors. Thus on a broad scale the reaction centers the central helices are below the central metal

FEBS 21004 2-11-98 J.P. Allen, J.C. Williams/FEBS Letters 438 (1998) 5^9 7 cluster FX that is coordinated by cysteine residues from ex- cult for these large complex systems. The delocalization of the tramembrane loops of the core subunits. electrons throughout the conjugated system and hence the In general, both types of reaction centers contain a dimer of limited electron density on the central Mg atoms is re£ected molecules £anked by four monomeric tetrapyr- in the limited e¡ect of substitutions to the ligands. Replace- role molecules, and an iron-binding site on the opposite ment of the histidine serving as a ligand to the central Mg can side. Both the dimer and the iron atoms are situated near result in an altered cofactor composition [15,16], but when the the central two-fold axis of symmetry, with the ligands to coordination of the Mg is retained very little is changed in the the Mg atoms of the dimer and for the metal complex pro- properties of the donor [17^19]. In contrast, for heme systems vided by amino acid residues from both of the core subunits. substitution of the ligand to the metal results in signi¢cant From these comparisons emerges a common reaction center alteration of the protein properties, such as a change in the motif of two symmetric branches of cofactors that are potential by several hundred mV [20]. bounded by a dimeric tetrapyrrole and a metal site and sur- Both the di¡erences of the chemical properties of the tetra- rounded by ¢ve helices each from two symmetric core sub- pyrroles and the interactions with the surrounding protein units (Fig. 1). contribute to the observed range in the properties of the do- nors in di¡erent reaction centers. The wavelength of the opti- 3. Primary electron donor cal band of the dimer in purple bacteria ranges from 860 nm to 900 nm, with reaction centers containing bacteriochloro- All photosynthetic systems have bacteriochlorophyll or phyll b having the longer wavelength transition compared to chlorophyll containing complexes that serve to collect the those containing bacteriochlorophyll a. For chlorophylls the light over a large surface area and focus that energy to the energy di¡erence between the excited and ground states is reaction center. In principle any of the tetrapyrroles in these larger than for bacteriochlorophylls (Fig. 1), so the absorption systems could be oxidized and thereby serve as the primary spectrum is shifted to the higher energy at 680 nm. In general, electron donor. However, the light energy is always trans- mutations that do not alter the cofactor composition have ferred to a uniquely de¢ned pair of bacteriochlorophylls or been found not to signi¢cantly alter the optical spectrum, chlorophylls. In reaction centers from purple bacteria, a wide presumably due to the neutrality of the ground and excited variety of measurements has established that the two overlap- states. The large range of types of tetrapyrroles found in dif- ping bacteriochlorophylls evident in the three-dimensional ferent organisms enables a precise tuning of the absorption structure serve as the primary electron donor (Fig. 1). These and hence maximum utility of the light spectrum available in two bacteriochlorophylls are separated by approximately 3 Aî each environment of photosynthetic organisms. In solution, and overlap at the ring A position. The presence of the dimer chlorophylls are more di¤cult to oxidize than bacteriochloro- is not required for assembly of the reaction center but is phylls by about 0.2 V, contributing to di¡erences in the values necessary for functional photochemistry to occur [12]. In pho- of the midpoint potentials of the primary electron donors (for tosystem I, two chlorophylls located in a comparable position reviews see [21]). Interactions with the protein also contribute are thought to serve as the primary donor. The relative ori- substantially to the observed range of over 0.5 V in midpoint entation of the chlorophylls in photosystem I is unknown but potentials. Hydrogen bonds between amino acid residues and the separation is larger at approximately 4 Aî . Models of pho- the conjugated carbonyl groups of the dimer are one type of tosystem II vary considerably, but a chlorophyll dimer is gen- interaction that has been identi¢ed as signi¢cantly a¡ecting erally thought to be at a location similar to that of purple the midpoint potential. In purple bacteria, loss of the existing bacteria [13]. Based upon the structures and mutagenesis stud- hydrogen bond results in a V0.1 V decrease in potential while ies, the tetrapyrroles forming the dimer in both types of re- addition of up to 3 hydrogen bonds results in additive in- action centers have histidine ligands, although coordination to creases in midpoint potential up to 0.25 V [22]. Also contrib- other residues is possible and has been observed in the struc- uting to a lesser extent are electrostatic interactions from tures of light harvesting complexes. non-ionized but polar amino acids, such as tyrosines, and Some characteristic features of the primary electron donor van der Waals contacts involving aromatic side chains. Since result from the dimeric organization. For example, the pres- the highly hydrophobic environment of the dimer is unfavor- ence of a distinct optical absorption band in the near infrared able for charges, it is unlikely that any of the nearby residues region for the donor of purple bacteria arises from the exciton are ionized, so that electrostatic interactions from charged interaction generated by the close overlap of the bacterio- amino acid residues may not play a major role in adjusting chlorophylls. For photosystem II the donor absorption band the energy levels of the dimer. The electron density distribu- overlaps with the monomer bands and hence the dimer is tion over the two halves of the oxidized dimer is primarily usually modeled as having a greater separation with a more dependent on di¡erences in the protein surroundings. For limited overlap. The distribution of the electron wavefunc- example, the addition of each hydrogen bond to the dimer tions over both tetrapyrroles creates electronic states that of the reaction center from purple bacteria stabilizes the bac- are intrinsically di¡erent from a monomer and can be de- teriochlorophyll containing the proton accepting carbonyl, scribed by simple Huëckle models, which can be used to predict and this preferential stabilization of one side of the dimer the oxidation/reduction midpoint potential and the electron results in both an increase in the midpoint potential and a density of the donor [14]. In principle, all of the precise prop- change in the asymmetry of the donor [14]. The spin density erties of the donor and other pigments, including the electron ratio in these types of mutants ranges from almost completely transfer rates, could be explicitly calculated by molecular or- asymmetric to nearly symmetric, depending on the arrange- bital theory, but in practice the highly interacting nature of ment of hydrogen bonds. The relative contribution of protein the electron donors and acceptors and the critical contribution interactions to the properties of donors continues to be ac- of the surrounding protein makes such calculations very di¤- tively investigated in di¡erent photosystems.

FEBS 21004 2-11-98 8 J.P. Allen, J.C. Williams/FEBS Letters 438 (1998) 5^9

4. Electron transfer of protonatable residues in the electron transfer process re- quires more detailed studies. Open areas of investigation are A critical aspect of the photochemistry of reaction centers is delineation of the mechanism of electron transfer from the their ability to perform electron transfer with a quantum yield primary to the secondary quinone and the role of quinone of almost unity. This high quantum yield is achieved by the movement during electron transfer [29,30]. Unlike the quinone utilization of a number of intermediate electron acceptors type of reaction centers, the iron-sulfur reaction centers could (Fig. 1). Within 30 ps after excitation a stable charge sepa- ideally be well served by having two functional branches of rated state is formed in all photosystems. Although the nature cofactors. The iron-sulfur clusters are more functionally sym- of the spectral changes with time is complex, the role of many metric than the quinone electron acceptors re£ective of their factors driving the initial electron transfer process has been role of single electron carriers to an external protein [31]. established in purple bacteria (reviewed in [23]). For other Whether electron transfer in the iron-sulfur type of reaction types of reaction centers, the larger number of tetrapyrroles centers is symmetric or asymmetric remains a subject of cur- and the highly overlapping nature of the optical bands have rent investigation. hindered interpretation of the optical changes, and research continues to delineate the electron transfer processes. In pur- 5. Implications for evolution ple bacteria, the rate is sensitive to the free energy di¡erence between the excited state and the charge-separated state but The features common to all types of photosystems can be not to the relative distribution of electrons over the two mac- taken as a re£ection of a primordial reaction center. The rocycles of the donor. After extensive studies, the rate is now consistency of the overall motif indicates that structural or established to be critically coupled to the properties of the assembly needs dictate a particular packing arrangement of bacteriochlorophyll monomer that lies between the donor transmembrane helices that embed the cofactors. The conser- and bacteriopheophytin acceptor (Fig. 1). The involvement vation of a tetrapyrrole dimer as the primary electron donor of the bacteriochlorophyll monomer may give rise to multiple suggests that this is an essential requirement for any function- pathways for electron transfer [24] and has been shown to al photosystem. Within this constraint, the individual charac- partially determine the asymmetry of the electron transfer teristics of the donor can be modi¢ed to adjust to changes in along one branch [25]. A set of elegant experiments has dem- the photosynthetic pathways and to di¡erent light conditions. onstrated the coupling of speci¢c vibrational modes with the The elasticity of the dimer properties contributes to the ease donor [26]. However, the involvement of protein vibrational with which these types of modi¢cations can evolve. The pho- modes or other additional factors that give rise to the ob- tosystems were able to retain the general donor structure of served functional asymmetry remains an open question. two coupled macrocycles and adjust the functionality by both Electron transfer in the reaction center culminates at either changing the protein-donor interactions and varying the quinone acceptors or iron-sulfur centers depending upon the chemical nature of the macrocycle from bacteriochlorophylls type of reaction center. Although a metal atom, usually iron, to chlorophylls. For example, in order to gain the functional is coupled to the quinones in the pheophytin-quinone reaction capability to oxidize water, it was necessary for the primary centers, it is not required for electron transfer. The two qui- donor to both become highly oxidizing and coordinate elec- none acceptors in this type of reaction center have di¡erent tron and proton transfer with a metal complex [32]. functional properties, with the primary quinone being a tran- Although the evolutionary pathways cannot be established sient one electron acceptor and the secondary quinone being a uniquely, the process giving rise to the two core subunits that two electron acceptor coupled to the exogenous quinone pool are related by an approximate two-fold symmetry axis can be of the cell membrane [27]. Electron transfer in the iron-sulfur traced by alterations in the structural genes (reviewed in [6]). type of reaction center proceeds from the chlorophyll acceptor Comparison of the biosynthetic pathways of the various tet- A0 to the iron-sulfur center FX (Fig. 1). A quinone is thought rapyrroles found in the reaction centers can also be used to to serve as an intermediate acceptor in photosystem I, but the track the evolutionary path. Consideration of both the ener- role of quinones in reaction centers from heliobacteria or getic requirements and the pigment composition needed for green sulfur bacteria is not settled [28]. The electron is then photosynthetic capability has led to speci¢c scenarios for the transferred to an external protein, , by way of the stages in the evolution of photosynthesis (reviewed in [33]). two iron-sulfur clusters FA and FB that are located in the Additional clues should be provided by characterization of protein subunit PsaC. newly discovered photosynthetic organisms that contain novel Despite the striking symmetry of the cofactors into cofactors [34,35]. 2 branches (Fig. 1), electron transfer in the pheophytin-qui- none reaction centers proceeds only along one branch with at Acknowledgements: We wish to thank Robert Blankenship for many least a 10:1 ratio. The functional asymmetry is served by the helpful conversations. This work was supported by the United States Department of Agriculture 97-35306-4524. signi¢cantly di¡erent properties of the two quinones, which arise from protein-quinone interactions. For example, the pri- References mary quinone environment is much more hydrophobic than that of the secondary quinone and the hydrogen bonding is [1] Blankenship, R.E., Madigan, M.T. and Bauer, C.E. (Eds.) (1995) more asymmetric. The protonation states of nearby amino Anoxygenic Photosynthetic Bacteria, Kluwer, Dordrecht. acid residues, in particular carboxylate groups, has been [2] Bryant, D.A. (Ed.) (1994) The Molecular Biology of Cyanobac- clearly established to be crucial to the function of the second- teria, Kluwer, Dordrecht. [3] Ort, D.R. and Yocum, C.F. (Eds.) (1996) Oxygenic Photosyn- ary quinone, which becomes fully protonated and leaves the thesis: The Light Reactions, Kluwer, Dordrecht. reaction center. The quinones of photosystem II are thought [4] Ho¡, A.J. and Deisenhofer, J. (1997) Phys. Rep. 287, 1^248. to bind in homologous sites although the possible involvement [5] Feher, G. (1998) Photosynth. Res. 55, 3^40.

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