By: Robert E. Blankenship
Department of Chemistry and Biochemistry
Arizona State University
Tempe, AZ 85287-1604
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.
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.
Figure 3. 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.
Figure 4. Energy-kinetic diagram for reaction centers from purple photosynthetic bacteria.
Perhaps the most striking aspect of the comparison of the structural and kinetic data on reaction centers is the apparent two-fold symmetry of the structure, contrasted with the clear evidence for asymmetry in the electron transfer pathway. Estimates of the branch ratio for electron transfer down the A branch in Figure 3, compared to the B branch are as high as 200:1. A considerable effort, including analysis of many site-directed mutants as well as detailed theoretical calculations has gone into trying to understand the factors that facilitate electron transfer down the A branch instead of the B branch. A molecular understanding of this observation has so far been elusive, and forms the basis of much current research.
Another interesting feature of the rate constants shown in Figure 4 is that the rates of recombination processes, in which the electron simply returns from one of the acceptors directly to the oxidized special pair, are invariably a factor of fifty or more slower than are the rate constants for the forward reaction. It is because of this kinetic control of the rates of wasteful processes that the quantum yield for photochemistry is so high. Again, the molecular reasons for this remarkable behavior have proven difficult to elucidate. One of the major factors thought to be involved in the slow recombination rates is the large energy gap involved, placing these reactions in the Marcus inverted region of the rate vs free energy curve. See the contribution by John Miller for more details of this issue.
Other types of photosynthetic bacteria contain reaction center complexes that are structurally quite different from those of the purple bacteria, and our understanding of them is at a much more primitive state. Similarly, structural information on the photosystems from oxygenic organisms is less detailed than that from purple bacteria. It is clear that all photosynthetic reaction centers operate on similar overall principles, so the general lessons learned from the purple bacteria are almost certainly largely applicable to other systems. However, some systems include activities that are not found in the purple bacteria, such as oxygen evolution by photosystem 2. Therefore, study of more than one type of organism is necessary to understand the wide scope of processes that are carried out by photosynthetic reaction centers.
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.
Figure 5. Molecular structures of zeaxanthin and violaxanthin.
2. Deisenhofer, J.; Norris, J.R. (Eds.) The Photosynthetic Reaction Center, Academic: San Diego, 1993.
3. Scheer, H. (Ed.) Chlorophylls, CRC Press: Boca Raton, 1991.
4. van Grondelle, R.; Dekker, J.P.; Gillbro, T.; Sundstrom, V. Biochim. Biophys. Acta (1994), 1187,1.
5. Blankenship, R.E. Photosynth. Res. (1992), 33, 91.
6. Woodbury, N. W.; Allen, J. P. In: Blankenship, R.E..; Madigan, M.T.; Bauer, C.E. (Eds). Anoxygenic Photosynthetic Bacteria, Kluwer: Dordrecht, 1995, pps 527-557.
7. Krause, G.H.; Weiss, E. Ann. Rev. Plant Physiol. Plant Mol. Biol. (1991) 42, 313.
8. Demmig-Adams, B.; Adams, W.W. Ann. Rev. Plant Physiol. Plant Molec. Biol. (1992) 43, 599.
9. Frank, H.A.; Cua, A.; Chynwat, V.; Young, A.; Gosztola, D.; Wasielewski, M.R. Photosynth. Res. (1994), 41, 389.
10. Phillip, D.; Ruban, A.V.; Horton, P.; Asato, A.; Young, A.J. Proc. Nat'l. Acad. Sci. USA (1996) 93, 1492
11. Kühlbrandt, W.; Wang, D.N.; Fujiyoshi, Y. Nature (1994) 367, 614.
12. Asada K. In: Foyer C.H. and Mullineaux P.M. (Eds.) Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants, (1994) CRC Press: Boca Raton, pp 77-104.
February 5--8, 1996
Estes Park, CO
U.S. Department of Energy
Office of Energy Research
Office of Basic Energy Sciences
Division of Chemical Sciences
National Renewable Energy Laboratory
The full text of the workshop is available on-line.
Revised 4 June 1996
copyright ©1996 Robert E. Blankenship
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Arizona State University
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Tempe, AZ 85287-1604
13 February 2006
phone: (480) 965-1963
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