The Nobel Prize in Chemistry 1988

Johann Deisenhofer

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 on September 30, 1943 in Zusamaltheim, Bavaria, now Federal Republic of Germany, as the first son of Thekla and Johann Deisenhofer. After my father's return from military service my parents ran the family farm. In 1948, our family grew to its final size with the birth of my only sister, Antonie. My early youth was influenced by the environment provided by a little village that, after World War II, tried to find its way back to some kind of a normal life. Nevertheless, it was a most enjoyable place for a little boy. In 1949, I entered elementary school at Zusamaltheim, and continued to attend until 1956. According to the local custom, the oldest son was designated to take over the family's farm. However, to their great dissapointment, my parents early noticed my lack of interest in farming, and made the diffcult decision to send me away to school. My way to higher education started in 1956 at the "Knabenmittelschule HI. Kreuz", Donanwoerth, and continued 1957 to 1959 at the "Staatliche Realschule Wertingen" and 1959 to 1963 at the "Holbein Gymnasium", Augsburg. There, in 1963, I underwent the "Abitur" examination that allowed me to go to a university. I was awarded the "Stipendium f?r besonders Begabte" of the "Bayerische Staatsministerium f?r Unterricht und Kultus" which helped to lower the financial burden on my parents for my education.

In the fall of 1965, after 18 months of military service in the German Bundeswehr, where I did not exceed the rank of private, I began to study physics at the Technische Universit?t M?nchen. A major reason for choosing physics was an interest in physical, especially astronomical problems, aroused by popular books on this subject. The book I most clearly remember was a popular review of the state of astronomy by Fred Hoyle, describing the impact of modern physics on astronomy, and the recent achievements and open questions in that field. The Technische Universit?t M?nchen (TUM) was the obvious choice because Rudolf L. Moessbauer had just accepted a professorship at the TUM; moreover Munich is only about 100 km from Zusamaltheim.

During my time at the TUM, I learned that physics was quite different from what I expected; also, my interest slowly shifted to solid state physics. Together with a couple of colleagues I started my Diplomarbeit in this field in the laboratory of Klaus Dransfeld. As it turned out, Klaus Dransfeld was a person almost as shy as myself, so that we could not establish a good personal contact at the time. Nevertheless, the experimental work I did under the supervision of Karl-Friedrich Renk in Dransfeld's lab was very successful, and led to a publication in Physical Review Letters in 1971; this was my first scientific publication. During my time in his lab, Klaus Dransfeld transmitted his interest in biophysical problems to many students. This had direct consequences for my career because it made me look for a suitable institution to get a Ph. D. in this field. From a friend I heard about a new group at the Max-Planck-Institut f?r Eiweiss- und Lederforschung whose head, Robert Huber, was looking for students. After a brief interview with Robert Huber, it was agreed that I could start my work in June 1971, following the final examination for my physics diploma at the TUM. In 1972, the institute moved a few kilometers from Munich to Martinsried, and became the "Max-Planck-Institut f?r Biochemie". The work I did together with Wolfgang Steigemann (also one of Huber's Ph.D. students at that time) on the crystallographic refinement of the structure of bovine pancreatic trypsin inhibitor was a success, and our 1975 paper in Acta Crystallographica has been cited ever since.

At the end of 1974, when I had obtained my Ph.D. degree, Robert Huber offered me a postdoctoral position for two years which I accepted. This position was converted into a permanent position in 1976. I joined Peter M. Colman, then a postdoctoral fellow in Huber's lab, and Walter Palm from the University of Graz, Austria, in their work on the human myeloma protein Kol. After the solution of this interesting structure, I continued, together with Robert Huber, Peter Colman's work on the human Fc-fragment, and its complex with an Fc-binding fragment from protein A from Staphylococcus aureus. The refinement of these structures was finished in 1980. In the following two years I joined several projects in Robert Huber's lab: human C3a, citrate synthase, and alpha-1 -proteinase inhibitor. During all my time in Martinsried I enjoyed working with computers, and developing and maintaining crystallographic software.

In 1982, Hartmut Michel, who had come to Martinsried together with Dieter Oesterhelt, reported in one of Huber's group seminars about his spectacular success with the crystallization of the photosynthetic reaction center from Rhodopseudomonas viridis. After discussions between Hartmut and myself, and after Robert Huber had given his agreement, I joined the reaction center project in order to determine the three-dimensional structure of this molecule. Shortly afterwards Kunio Miki, a post-doctoral fellow from Osaka University, arrived in Martinsried, and helped us until September 1983. Later Otto Epp, a colleague and friend since I joined the Max Planck-Institute, made most valuable contributions to the project.

In a surprisingly short time, at the end of 1983, we came to a point where the success of the project was at the horizon. It still took almost two years until we had worked out the complete structure, and two more years to refine the model at 2.3? resolution.

The work on the photosynthetic reaction center changed my life in many ways. It was a special privilege to belong to the very small group of people who saw the structural model of this molecule grow on the screen of a computer workstation, and it is hard to describe the excitement I felt during this period of the work. Soon after the news of our success spread through the interested scientific community, we received many invitations to report our results during scientific meetings, in seminars, and even in TV shows. The wide recognition of our work also opened the possibility for me to move to a new place, and to build a research group of my own. The best of several opportunities was an offer from the University of Texas Southwestern Medical Center at Dallas which I joined in March 1988 to become Professor of Biochemistry, and Investigator in the Howard Hughes Medical Institute. Almost immediately after my arrival I fell in love with Kirsten Fischer Lindahl, Professor of Microbiology and Biochemistry and Investigator in the Howard Hughes Medical Institute; we got married in 1989.

For the determination of the three-dimensional structure of the reaction center Hartmut Michel and I received the 1986 Biological Physics Prize of the American Physical Society, and the 1988 Otto Bayer Prize. The 1988 Nobel Prize in Chemistry was followed by several non-scientific honors such as honorary citizenships of my home town Zusamaltheim and of my current residence Dallas, and a high order of the Federal Republic of Germany. I am a member of the Academia Europaea, and a Fellow of the American Association for the Advancement of Science.

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