Photosynthetic Antennas and Reaction Centers: Current Understanding and Prospects for Improvement
Abstract
A brief introduction to the principles, structures and kinetic processes that take place in natural photosynthetic reaction center complexes is presented. Energy is first collected by an antenna system, and is transferred to a reaction center complex where primary electron transfer takes place. Secondary reactions lead to oxidation of water and reduction of CO2 in some classes of organisms. Antenna systems are highly regulated to maximize energy collection efficiency while avoiding photodamage. Some areas that are presently not well understood are listed.
Introduction
Nature's most sophisticated and important solar energy storage system is found in photosynthetic organisms, including plants, algae and a variety of types of bacteria. All these organisms utilize sunlight to power cellular processes and ultimately derive most or all of their biomass through chemical reactions driven by light. In this short report I will give an introduction to the basic principles of how natural photosynthetic systems work, including structural, mechanistic and regulatory aspects. This complex subject cannot be adequately explained in such a short space, so the interested reader is referred to some recent books in which various aspects of photosynthesis are explained in more detail (1-3).
Photosynthesis begins when light is absorbed by an antenna pigment. This pigment can be a (bacterio)chlorophyll, carotenoid or bilin (open chain tetrapyrrole) depending on the type of organism. A wide variety of different antenna complexes are found in different photosynthetic systems (4). Antennas permit an organism to increase greatly the absorption cross section for light without having to build an entire reaction center and associated electron transfer system for each pigment, which would be very costly in terms of cellular resources. More details of antenna structure and function are given below. Energy transfer processes that may involve transfers to many intermediate pigments eventually results in the electronic excitation of a closely coupled pair of (bacterio)chlorophyll molecules in the photochemical reaction center (Figure 1). The reaction center is an integral membrane pigment-protein that carries out light-driven electron transfer reactions. The excited (bacterio)chlorophyll molecule transfers an electron to a nearby acceptor molecule, thereby creating an ion pair state consisting of the oxidized chlorophyll and reduced acceptor.
After the initial electron transfer event, a series of electron transfer reactions takes place that eventually stabilizes the stored energy in forms that can be used by the cell. Some types of photosynthetic organisms have two different reaction center complexes that work together in tandem, with the reduced acceptors of one photoreaction (photosystem 2) serving as the electron donor for the other center (photosystem 1). In these organisms, the eventual electron donor is water, liberating molecular oxygen, and the ultimate electron acceptor is carbon dioxide, which is reduced to sugars. Other types of photosynthetic organisms contain only a single photosystem, which in some cases is more similar to photosystem 2 and in other cases to photosystem 1 of the oxygen-evolving organisms. Oxygen is not produced in any of the naturally occurring single photosystem organisms, which are therefore called anoxygenic. Figure 2 shows comparative electron transfer diagrams of the oxygen evolving and anoxygenic photosystems.
Reaction Center Structure and Function
The reaction center complexes from the anoxygenic purple photosynthetic bacteria are the best understood of all photosynthetic reaction centers, from both a structural and a functional point of view (1,2). These were the first reaction center complexes to be purified, the first to be studied by picosecond kinetic methods and the first to have X-ray structures solved. Much of the molecular level understanding of the early events in photosynthesis is based on the information derived from these systems. The structure of the reaction center from Rhodobacter sphaeroides is shown in Figure 3.
Figure 1. Basic concept of photosynthetic antenna and reaction center function. (Figure courtesy of Judy Zhu).
Figure 2. Electron transport diagrams for photosynthetic reaction centers. Vertical arrows indicate energy input by photon absorption, lines indicate preferred electron transfer pathways. Carriers in parentheses indicate alternate species in some organisms. Question marks indicate carriers or electron transfer steps that are likely but have not been unambiguously established. The cytochrome bc 1 and b 6f complexes are boxed, and the details of the electron flow in these complexes are omitted. Figure adapted from reference 5, which includes a complete list of abbreviations.
X-ray structure of the reaction center from Rhodobacter sphaeroides. Left, cofactors; right protein. The complex is buried in the plasma membrane of the cell, with the helical regions of the protein spanning the lipid bilayer. The cofactor abbreviations are: P, P870 special pair bacteriochlorophyll; B, accessory bacteriochlorophyll; H, bacteriopheophytin; Q, ubiquinone. The A and B subscripts refer to the active and inactive branches of the electron transfer pathways, respectively. (Figure courtesy of James Allen).
The reaction centers from purple photosynthetic bacteria contain a core protein complex consisting of two related yet distinct integral membrane proteins, known as L (Light) and M (Medium). Most also contain a third protein, known as H (Heavy), and some contain a fourth subunit known as C (cytochrome). The C subunit is a four-heme containing c-type cytochrome.
In addition to the protein complement, these reaction centers contain several additional cofactors, that are not covalently attached to the protein (Figure 3). These include bacteriochlorophyll a (in some cases b), the corresponding metal-free bacteriopheophytins, two quinones (either ubiquinone or menaquinone), a non-heme Fe, and in most cases a molecule of carotenoid. The cytochrome subunit contains four heme c groups, covalently bound to cysteine residues (not shown in Figure 3).
The reaction center protein forms a scaffolding upon which the cofactors are arranged. The part of the protein that crosses the lipid bilayer is almost purely alpha helical in secondary structure, and contains predominantly nonpolar amino acids, with almost no charged amino acids. There are 11 transmembrane helices, with 5 each from L and M, and one from the H subunit. The L and M proteins have a pseudo-2 fold axis of symmetry, running approximately perpendicular to the plane of the membrane. The symmetry is broken by the H subunit, which has no symmetry-related counterpart, and also by the fact that the L and M subunits have only about 60% sequence identity.
A large number of different techniques have been utilized on the bacterial reaction center system, including almost every imaginable kind of spectroscopy, as well as a wide range of biochemical and genetic manipulations. Here it is only possible to give a brief summary of some of the results. The technique of picosecond absorbance transient difference spectroscopy has been especially informative with respect to elucidating the pathway of electron flow in these complexes (6). Figure 4 summarizes the photochemical and early secondary reactions that take place in isolated reaction centers. A variety of evidence indicates that the electron transfer pathway and kinetics in isolated reaction centers are not significantly altered from their behavior in vivo.
Function and Regulation of Antenna Systems
The vast majority of the pigments in a photosynthetic organism are not chemically active, but function primarily as an antenna (1,4). The photosynthetic antenna system is organized to collect and deliver excited state energy by means of excitation transfer to the reaction center complexes where photochemistry takes place. The antenna system increases the effective cross section of photon absorption by increasing the number of pigments associated with each photochemical complex. The intensity of sunlight is sufficiently dilute so that any given chlorophyll molecule only absorbs at most a few photons per second. By incorporating many pigments into a single unit, the biosynthetically expensive reaction center and electron transport chain can be used to maximum efficiency. A remarkable variety of antenna complexes have been identified from various classes of photosynthetic organisms. There seems to be little doubt that there have been multiple evolutionary origins of antenna complexes, as there is no common structural theme evident. Excitation transfer must be fast enough to deliver excitations to the photochemical reaction center and have them trapped in a time short compared to the excited state lifetime in the absence of trapping. Excited state lifetimes of isolated antenna complexes, where the reaction centers have been removed, are typically in the 1-5 ns range. Observed excited state lifetimes of systems where antennas are connected to reaction centers are generally on the order of a few tens of picoseconds, which is sufficiently fast so that under physiological conditions almost all the energy is trapped by photochemistry.
Antenna systems are often viewed as being "on" all the time, with the regulation of photosynthesis in response to different conditions taking place primarily in the reaction centers and carbon metabolism enzymes. Clearly, this is not the case, and the modern view is of a much more actively regulated system at all stages of energy storage. The advantages of "directional signals" or "volume controls" to regulate either the distribution between the photosystems or the number of excitations delivered by the entire antenna network are easy to appreciate.
One of the most interesting and important of these regulatory mechanisms is the phenomenon of "nonphotochemical quenching" (qN) of chlorophyll excited states in chloroplasts (7). During periods of high irradiance such as midday, or under certain stress conditions, a substantial fraction of the excited state energy is dissipated by quenching before it is ever transferred to the reaction center. The basic idea is that it is much easier and safer for cells to dispose of this energy before it initiates the photochemical processes in reaction centers than it is for them to try to repair the substantial photooxidative damage that can result from excess light. This process is now thought to be a major regulatory mechanism and understanding it is likely to have great economic significance.
Considerable evidence indicates that a cycle involving carotenoids known as xanthophylls plays a role in this regulation, with zeaxanthin associated with the quenched state and violaxanthin associated with the nonquenched state (Figure 5). (8). Enzymes interconvert these carotenoids in response to energetic signals generated in the chloroplast. The chemical mechanism of the quenching effect is not yet understood in molecular detail. One proposal under investigation is that the zeaxanthin molecule with its more extensive conjugation has a lower excited state energy than does violaxanthin. The excited state energies are proposed to be such that violaxanthin lies above chlorophyll and therefore acts as an energy donor while zeaxanthin lies below chlorophyll and therefore acts as an excited state quencher (9). Other factors are also known to be important, including the state of aggregation of the antenna complex and the pH of the interior lumen region of the thylakoid membrane (10). The chlorophyll a/b-containing antenna complex known as LHC II is thought to be the site of much of this quenching reaction. This complex is well characterized structurally (11). While the linkage of the quenched state to the presence of zeaxanthin in the membrane is clear, it is mostly based on evidence showing that the two are correlated, rather than an unambiguous cause and effect relationship
I was born in Ludwigsburg, W?rttemberg, in the southwestern part of the Federal Republic of Germany on July 18, 1948, as the elder son of Karl and Frieda Michel. My ancestors lived in that area for generations, mainly as farmers. There the inherited land is equally divided among sisters and brothers, and not enough land was left for one family's living during my grandparents' generation. During the day my father worked in a factory as a joiner, my mother at home as a dressmaker, in the evenings and on Saturdays care had to be taken of the huge gardens.
As a child I liked to play outside, to stroll through the fields, and I was an active member of the local children's gang, frequently being chased by field guards and building supervisors. Nevertheless, my performance at school was very good, and mainly due to the influence of my mother I was allowed to attend high school. At age eleven I became a member of the circulating library of my home town. From there on I was rarely seen outside, but was reading two to four books per week, the subjects ranging from archaeology over ethnology and geography to zoology. Needless to say that I did not do much homework. At school my favorite subjects were history, biology, chemistry and physics. Especially the teaching in physics was excellent. Most of my understanding of it I got at high school, not at the university.
In parallel, my interest in molecular biology rose. In 1969 - after the obligatory military service - I applied to study biochemistry at the University of T?bingen. At that time T?bingen was the only place in Germany, where one could study biochemistry from the first year, and I was happy to be accepted. Studying biochemistry meant that one had to take part in nearly the same amount of lectures and courses as chemistry students in addition to numerous lectures and courses in biology. The atmosphere between senior teachers and students was impersonal, and the only time I talked to the full professor of biochemistry was during the final examination. However, the possibility existed to work for one year in the various biochemistry labs at the University of Munich and the Max-Planck-Institut f?r Biochemie instead of attending lab courses in T?bingen. I took that chance in 1972/1973, and at the end I was convinced that academic research was what I wanted to do.
After the examination in T?bingen in 1974 I did the experimental part of my biochemistry diploma in Dieter Oesterhelt's lab at the Friedrich Miescher-Laboratorium of the Max-Planck-Gesellschaft in T?bingen. In cooperation with Walter Stockenius, Dieter Oesterhelt had discovered bacteriorhodopsin in halobacteria and later proposed that it acts as a lightdriven proton pump in the framework of Peter Mitchell's chemiosmotic theory. During my diploma work I characterized the ATPase-activity of halobacteria. In 1975, Dieter Oesterhelt moved to W?rzburg. I joined him, and as a thesis I correlated the intracellular levels of adenosine di- and triphosphate with the electrochemical proton gradient across the halobacterial cell membrane. Having received the doctorate in June 1977 I tried to fuse delipidated bacteriorhodopsin with bacterial vesicles in order to achieve light-driven amino acid uptake. Upon storage in the freezer the delipidated bacteriorhodopsin yielded solid, glass-like aggregates. On the basis of this observation I was convinced that it should be possible to crystallize membrane proteins like bacteriorhodopsin, which was considered to be impossible at that time. With Oesterhelt's help I started the experiments, and already four weeks later we obtained a new two-dimensional membrane crystal of bacteriorhodopsin. It was not the three-dimensional crystal we wanted, but allowed me to travel to the MRC at Cambridge, England, and to do electron microscopical studies together with Richard Henderson. Back in W?rzburg, we observed the first real three-dimensional crystals of bacteriorhodopsin in April 1979. The success led me to cancel my plans to do post-doctoral studies with Susumu Ohno, Duarte, California, on sexual differentiation in mammals. Instead of this, I moved with Dieter Oesterhelt again, this time to the Max-Planck-Institut f?r Biochemie at Martinsried near Munich, where he became a department head and director. Before moving to Munich, Ilona Leger became my wife. Her understanding and patience helped me a lot.
A promising aspect of the move to Martinsried was the possibility of a cooperation with Robert Huber and colleagues, who at the Max-PlanckInstitut had established a very productive department for X-ray crystallographic protein structure analysis. Our bacteriorhodopsin crystals were found to diffract X-rays, but to be too small and too disordered for a structural analysis. We tried to improve size and quality of the crystals. Since all the X-ray crystallographers had beautifully diffracting crystals of soluble proteins, I, understandably, had very limited access to the X-ray equipment at Martinsried. As a consequence, I spent four months at the MRC in Cambridge, England, together with Richard Henderson in 1980, in order to perform X-ray experiments. This period was essential for improving the crystallization method. After my return Dieter Oesterhelt decided to buy an X-ray generator for the ongoing work with bacteriorhodopsin. The generator was installed in Robert Huber's department and guaranteed us continued access to the equipment, and the know how, of the X-ray crystallographers. Later on, I used this generator for the work with the reaction centres.
Frustrated from the lack of the final success with bacteriorhodopsin, I tried to crystallize several other membrane proteins, mainly photosynthetic ones. After developing a new isolation procedure I obtained the first crystals of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis at the end of July 1981. One week later our daughter Andrea was born. During September 1981 the first reaction centre crystal was X-rayed by Wolfram Bode and myself, and turned out to be of excellent quality. Therefore 1981 was the happiest and most successful year of my life.
Dieter Oesterhelt immediately agreed that the reaction centre should be a project of the young people. In February 1982, I started the data collection for the X-ray structure analysis. In April or May I gave a seminar in Robert Huber's department and asked officially for collaboration. After some internal discussions Robert Huber agreed that Johann ("Hans") Deisenhofer, who was the partner of my choice, should take part in the reaction centre project. During the work Hans and I became the best friends. In August 1982, Hans and Kunio Miki, a Japanese post-doctoral research associate in Robert Huber's department, started to evaluate the pile of X-ray films. I continued with the experimental work, occasionally helped by Robert Huber, who showed me how the diffraction pattern of a promising derivative should look like. Not only the X-ray work, but also the entire biochemical characterization and sequence determination had to be done. After the preliminary tracing of the peptide chains by Johann Deisenhofer, the sequence determination, which was performed by Karl A. Weyer, Heidi Gruenberg and myself with Dieter Oesterhelt's support and help, turned out to be the bottle neck for our progress. During that period of heavy work our son Robert Joachim was born in 1984.
As one of the results of the success I received many offers. I accepted the one to become a department head and director at the Max-Planck-Institut f?r Biophysik in Frankfurt/Main, West Germany, where I am since October 1987.
For the success with the crystallization of membrane proteins and the elucidation of the three-dimensional structure of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis I received various prizes and awards. Among these are the Biophysics Prize of the American Physical Society (together with d. Deisenhofer), the "Chemiedozentenstipendium" of the "Fonds der Chemischen Industrie", the "Otto Klung-Preis" for chemistry, the Leibniz-Preis of the Deutsche Forschungsgemeinschaft, the "Otto-Bayer-Preis" (together with J. Deisenhofer) and now the Nobel Prize (together with J. Deisenhofer and R. Huber).
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