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Photosynthesis Research 73: 63–71, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 63 Minireview Research on photosynthetic reaction centers from 1932 to 1987 Roderick K. Clayton Liberty Hyde Bailey Professor Emeritus of Biophysics, Section of Plant Biology, Cornell University, Ithaca, New York; Mailing address: 176 Mountain Vista Circle, Santa Rosa, CA 95409, USA (e-mail: [email protected]) Received 4 July 2001; accepted in revised form 14 September 2001 Key words: W. Arnold, R.K. Clayton, J. Deisenhofer, L.N.M. Duysens, isolation of reaction centers, H. Michel, photosynthetic bacteria, Rhodobacter sphaeroides, Rhodopseudomonas viridis, C.B. van Niel Abstract The history of research on photosynthetic reaction centers is outlined, starting with the implication of their existence through the discovery of the photosynthetic unit, as reported by R. Emerson and W. Arnold in 1932, and culminating in the crystallization and X-ray analysis of the anoxygenic bacterial reaction centers, reported by J. Deisenhofer, H. Michel, and coworkers, over the period 1982–1987. Reaction centers of purple photosynthetic bacteria have received the most attention because they have been well purified and characterized. Structures of cyanobacterial reaction centers of Photosystems I and II are now available from the laboratories of H. Witt and W. Saenger. Background In 1932, R. Emerson and W. Arnold published two landmark papers on the yield of photosynthetic oxygen evolution in the green alga Chlorella in response to brief flashes of light (Emerson and Arnold 1932a, b). The yield per flash was maximal if the flashes, about 10 µs in duration, were separated by more than about 40 ms. The yield amounted to no more than one O2 evolved for every 2400 molecules of chlorophyll (Chl), even though the quantum efficiency under optimal conditions must have been one O2 per eight quanta absorbed (Emerson 1958). Thus most of the Chl is not involved directly in the photochemistry of photosynthesis. Instead, it acts as an antenna for absorbing light and delivering energy to a relatively small number of reaction sites where the photochemistry of photosynthesis begins. Emerson and Arnold called this system of ca. 2400 Chls a ‘photosynthetic unit’. In contemporary terms of one quantum driving a single photochemical act (electron transfer), the size of the photosynthetic unit in Chlorella is 2400/8 or about 300 Chls since 4 electrons are transferred twice from water to CO2 . A quantum absorbed any- where in an antenna of 300 Chls, or collectively by the entire ensemble, could drive a single act of electric charge separation, presumably at a single photochemical site. Thus, the concept of a reaction center served by a light-harvesting antenna emerged in 1932, but lay fallow for lack of specific knowledge for decades. The literature of the 1930s and 1940s was burdened by much speculation about ‘water splitting’ (formation of H and OH radicals, or the equivalent, from H2 O) and by the great quantum efficiency controversy: one O2 evolved per four quanta according to O. Warburg and his adherents and one O2 per eight quanta according to R. Emerson and others. Eventually, the value of one O2 per eight quanta became accepted by ‘the majority’ and the polemics dried up (see Govindjee 1999). Meanwhile, C. B. van Niel (see van Niel 1941) developed a formulation for the overall chemistry of photosynthesis, which unified the description for green plants and photosynthetic bacteria and gave a new view of the role of water: In bacteria, CO2 + 2H2 A + light → (CH2 O) + 2A, where H2 A can be H2 S or any of a variety of oxidizable organic substrates and (CH2 O) represents carbohydrate. In green plants, 64 H2 A is H2 O. Water serves as the oxidizable substrate. This formulation emphasized the recognition of photosynthesis as an oxidation–reduction process, and put ‘water splitting’ in a new perspective (see Clayton 1980). During this period, every well-equipped laboratory had a Warburg–Barcroft apparatus for measuring gas exchanges related to life processes. In the 1950s, a well-equipped laboratory had a spectrophotometer, which could monitor changes in the optical absorption spectra of biologically significant molecules. This could give a far greater variety of specific information than could the measurements of gas exchange. Optical techniques flourished and the Warburg–Barcroft apparatus began to gather dust. Students of photosynthesis could record small changes in the optical absorption spectra of chlorophylls, cytochromes, and other substances, induced by illumination of plant or microbial materials. Such measurements, combined with information from biochemical analyses, and the discovery of the Emerson enhancement effect (reviewed in Govindjee 2000 and see later) culminated in the renowned ‘Z-scheme’ for oxygenic photosynthesis, first proposed in 1960 by R. Hill and F. Bendall (1960) and given compelling experimental support by L. N. M. Duysens, J. Amesz and B. M. Kamp (1961). In this scheme there are two kinds of photosystems in green plants, each performing a specific act: a single electron transfer driven by one quantum. System I makes a strong reductant, able to mediate the assimilation of CO2 , and a weak oxidant, an oxidized cytochrome (Cyt). System II makes a strong oxidant, able to mediate the evolution of O2 from water, and a weak reductant, a reduced quinone. The two systems are connected by electrons flowing from reduced quinone to oxidized cytochrome. Eight quanta can thus form the four oxidizing equivalents that can release one O2 from water, and the four reducing equivalents needed for assimilation of one CO2 . We were thus ready to visualize and begin to characterize the specific photochemical reaction centers (RCs)1 implied by Emerson and Arnold’s discovery of the photosynthetic unit. Each type, I and II, of RC in green plants mediates the transfer of a single electron at the expense of one quantum. From optical absorbance changes suggesting reversible lightinduced oxidation of Chl a, it could be interpreted [first for System I (B. Kok 1956); later for System II (Döring et al. 1969) in H. Witt’s laboratory] that each involves Chl a in the specialized context of a RC, acting as the primary electron donor. Green plants, algae, and cyanobacteria (‘bluegreen algae’) all show the same pattern of two distinct photochemical systems with two types of RC. The green and purple photosynthetic bacteria fall into several categories, but each type has but one kind of RC that engages in one kind of primary photochemistry. The RCs of the non-sulfur purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis have proven to be especially easy to extract and purify and have been the most rewarding sources of detailed information on chemical patterns, composition, structure, and physical mechanisms. Accordingly, I shall outline briefly much of what we have come to know about the RCs of green plant Systems I and II, and then examine in more detail what we have learned about the bacterial photosynthetic RCs. Early accounts are reviewed in Clayton (1978, 1980), and Breton and Vermeglio (1988). The reaction centers of green plant Photosystems I and II In the 1950s, information leading to the recognition of two types of photosystems in green plants came largely from action spectra: wavelength dependence of an effect caused by illumination. Such studies yielded some surprising results. First, there was the ‘red drop’ effect, discovered by Emerson and Lewis (1943): Light beyond 680 nm, well within the absorption band of Chl a in vivo, was peculiarly ineffective for photosynthesis. This deficiency could be corrected by mixing in some shorter wave light (the Emerson enhancement effect; see Emerson et al. 1957). The enhancing light was most effective with light absorbed in accessory pigments (such as Chl b) as well as Chl a absorbing around 670 nm (Emerson and Rabinowitch 1960; Govindjee and Rabinowitch 1960). Second, light absorbed by accessory pigments (such as phycobilins) was more effective in promoting Chl a fluorescence than was light absorbed by Chl a itself (Duysens 1952; a review by Blinks 1954). These phenomena could be explained by proposing the presence of two forms of Chl a in vivo: ‘long wave,’ effective beyond 680 nm, and ‘shorter wave,’ ineffective beyond 680 nm (see Govindjee and Rabinowitch 1960 for a ‘shorter wave’ Chl a 670; also see French 1961). The shorter wave form is the more strongly fluorescent 65 and is served preferentially by phycobilins where such pigments are present. The longer wave or ‘far red’ forms, Chl a 680 and beyond, were found to mediate oxidation of endogenous Cyt or of an externally supplied electron donor, and did not promote O2 evolution, explaining the ‘red drop.’ The shorter wave Chl a 670 favored reduction of oxidized Cyt or of an externally supplied electron acceptor, coupled with O2 evolution (‘Hill reaction’). These observations became crystallized in the Z-scheme, and we could begin to inquire into the natures of the two photosystems. Meanwhile a wealth of information began to come from the development of new instruments that could detect changes of optical absorbance with unprecedented sensitivity. L.N.M. Duysens first applied his newly constructed instrument to the study of photosynthetic bacteria (Duysens 1952, 1953); subsequently he and others, including B. Kok (1956), H.T. Witt and coworkers (1961), B. Chance (Chance and Smith 1955, Chance and Strehler 1957) and others detected minute light-induced changes in the absorption of light by green plant tissues, as well as by photosynthetic bacteria. These changes reflected oxidation of Chl a and Cyt, reduction of quinones, and other events including shifts in the wavelengths of absorption maxima attributed to electric fields arising from charge separation (oxidation–reduction). The optical measurements often became more informative when they were correlated with changes in the fluorescence emitted by Chl in the material, since fluorescence and photochemical utilization are alternative fates of excitation energy in Chl. Information has also come from increasingly refined measurements of electron paramagnetic resonance (EPR). In broken cell preparations, another dimension could be added to all of the foregoing observations by poising the material at specific ambient oxidation–reduction potentials. This could reveal the midpoint potential of an electron transfer event and also the number of electrons involved. In time, against a background of much more decisive information obtained with photosynthetic bacteria, many of these observations came to be associated with photochemical activities in or in proximity to the RCs. These developments came mainly in the 1960s and 1970s. The results can be summarized as follows. The RC of System II is analogous to those of purple bacteria insofar as singlet excited Chl a 680, P680, transfers an electron to pheophytin a (Pheo a) and on to plastoquinones, QA and QB . But the elec- tron donor to oxidized Chl a 680 is not a Cyt but ‘Z’ (now called YZ ), identified with a tyrosine residue in a polypeptide ‘D1’ that is a part of the RC complex. The Z, in turn, interacts with a manganese complex that cycles through four progressive states and results in the liberation of O2 from water (for references and details, see Ke 2001). The RC of System I resembles that of the purple bacteria insofar as P700, a Chl a dimer (or ‘special pair’; see later about P870), gives up an electron when excited, and the oxidized P700 is re-reduced by a Cyt (Cyt f) through the copper-containing plastocyanin. Excited P700 delivers electrons not to Pheo and quinones, but to iron–sulfur complexes (ferredoxins). So the reducing side of the System I RC is not like that of the purple bacteria; rather it resembles that of the green sulfur bacteria, making a reductant (reduced Fe–S complexes) more electronegative than reduced quinone (see, e.g., a review by Fromme 1999). Perhaps the RCs of purple bacteria diverged in two directions, becoming able to make a stronger reductant, as in green plant System I and in green sulfur bacteria, and a stronger oxidant, as in green plant System II. The latter development would have had enormous survival value, making water accessible as oxidizable substrate. This development altered the planetary environment drastically and changed the nature of all subsequent life on Earth. For a recent study of reaction center evolution, see Xiong et al. (2000). The reaction centers of anoxygenic photosynthetic bacteria My own odyssey in photosynthesis research began in collaboration with William Arnold (see my ‘Personal perspective,’ Clayton 1988; at the suggestion of the editors, pictures of the author are shown in Figure 1). Chance and M. Nishimura (1960) had just shown that in the purple bacterium Chromatium vinosum, light-dependent oxidation of a Cyt proceeds at 77 K. Wondering if drying acted like low temperature, in limiting reactions that depend on diffusion, we prepared dried films of membrane fragments from Rhodobacter sphaeroides and looked for photochemical activity as evidenced by light-induced changes of optical absorbance. We found, as had L.N.M. Duysens earlier in cells of Rhodospirillum rubrum (Duysens 1952, 1953; Duysens et al. 1956), that light caused changes, suggesting the photochemical oxidation of 66 Figure 1. Four images of the author. Top left: from the Student Yearbook, California Institute of Technology, 1941, age 19. Top right: at the C.F Kettering Research Laborotory, 1964. The instrument is part of a helium cryostat. Bottom left: at Cornell University, ca. 1972. Bottom right: self portrait, 2000. 67 Figure 2. Absorption spectrum (a) and spectra of light-induced absorbance changes (b, c) of reaction centers isolated from Rb. sphaeroides. Trace b, reaction centers alone. Trace c, reaction centers plus an electron donor to prevent accumulation of oxidized bacteriochlorophyll during illumination. Changes due to oxidation of the donor have been discounted. The increase at 1250 nm (trace b) shows formation of oxidized bacteriochlorophyll. The features at 275 nm and 320 nm reflect reduction of ubiquinone. Reproduced from Clayton (1980). bacteriochlorophyll (BChl) manifested mainly as a small partial bleaching of the long wave absorption band near 870 nm. The presumptive species responsible for the small change had already been termed P870 by Duysens (1952). Similar changes were caused by chemical oxidation and the effects of light and oxidants were complementary. We found the lightinduced change to be reversible, even at 1.3 K (W. Arnold and R.K. Clayton 1960). Other changes suggested that ubiquinone (UQ) is involved as an electron acceptor (see Figure 2). At the time, we had no way of knowing whether the effects we were seeing reflected a small change of absorbance in the antenna or a large change in a specific minor component: BChl in a reaction center. This question was answered through a fortuitous and remarkably fortunate choice of material. We had developed a carotenoidless mutant of Rb. sphaeroides, designated R26 (Clayton and Smith 1960), in order to study the irreversible photooxidative destruction of BChl, already known to occur in carotenoidless Rsp. rubrum. (Incidentally, I had selected a constitutive high-catalase mutant in isolating R26 because I wondered if that enzyme would protect against the destruction of BChl. It did not.) In strain R26, the major absorption bands at 800 and 850 nm were missing, leaving a large band at 870 nm and much smaller ones at 760 and 800 nm. The reversible bleaching amounted to about four per cent of the total absorbance at 870 nm, reflecting an unusually small antenna for a photosynthetic unit. When I accidentally left some mature cultures of strain R26 in illuminated bottles for two weeks, I saw that their slate blue color had changed to pink. Companion cultures kept anaerobically in the dark 68 did not show this change, nor did illuminated cultures of the parent strain that contained carotenoid pigments. In the pink cultures nearly all of the BChl had been converted, in an irreversible light-dependent process, to bacteriopheophytin (Bpheo, BChl with the magnesium atom removed). In this pheophytinized material, the 870 nm band of BChl was much attenuated, and what remained showed almost complete reversible light-induced bleaching. The magnitude of this effect was the same as in the companion cultures kept in the dark and in which the change amounted to four per cent of the total absorbance. This showed that the reaction occurred in a specific minor component in which the BChl had resisted conversion to BPheo and not in the antenna as a whole. I identified this minor component hypothetically as part of the photosynthetic RC (Clayton 1963). Most of the BPheo could be washed away from broken cell preparations of pheophytinized strain R26, leaving an absorption spectrum showing maxima at 760 nm (residual BPheo), 800 nm (BChl a), and 870 nm (photoactive BChl a). I obtained a similar spectrum by illuminating cell membrane fragments in the presence of the detergent Triton X-100. In this treatment the antenna BChl was degraded without harm to the photoactive part. When D. Reed came to my laboratory in 1967, he and I attacked the problem of separating the RC component from the rest of the photosynthetic membrane. We succeeded in this, using broken cell preparations treated with Triton X-100 in two ways: (1) centrifugation through a sucrose density gradient and (2) fractional precipitation with ammonium sulfate. We thus had our first relatively crude RC preparation (Reed and Clayton 1968). G. Feher then obtained much cleaner preparations by using lauryl dimethyl amine oxide instead of Triton X-100 as the detergent (see Feher 1971; and the Personal perspective of Feher 1998). In Feher’s laboratory and in mine the following picture emerged (see Feher 1971; and G. Feher and M. Okamura 1978), with Feher’s group providing the greater part of the definitive characterization. The RC in Rb. sphaeroides is a protein consisting of three subunits H, M, and L (for heavy, medium, and light) weighing 28, 24, and 21 kilodaltons, respectively. Photochemical activity resides in M and L; H can be removed without loss of activity. Bound to this protein are two molecules of Bpheo a and four of BChl a. The latter are responsible for the absorption bands at 800 and 870 nm. The bound BPheo a contributes the band at 760 nm. Other components are two molecules of UQ and an iron atom. One of these quinones, UQ1 , is tightly bound; the other, UQ2 , can be removed along with the Fe atom by chemical treatments. J.R Norris and J.J. Katz (1978) proposed the term ‘special pair’ rather than ‘dimer’ for the two molecules of P870 to emphasize that the exact nature of their interaction was not known. Water appears to be involved in the structure and function of RCs: exhaustive dessication causes loss of photochemical activity, restored on rehydration (Clayton 1966). The other two BChl molecules, responsible for the 800 nm band, appeared to be mere voyeurs of the photochemistry, but evidence has emerged for their participation in the earliest phases of the photochemical electron transfer. One of the two BPheos is definitely implicated as an early electron acceptor in the process. Strictly speaking, the bands at 800 and 870 nm should be regarded as joint properties of all four BChl, describable by a single wave function. However, it is useful, and has not been misleading, to regard the two pairs as separate entities, each with its own properties and function. For a time the view was defended that the primary event is oxidation of Cyt coupled to reduction of BChl, with oxidation of P870 regarded as an aberrant reaction; perhaps a safety valve for dissipation of excess energy. Apart from the lack of any evidence for the photochemical formation of reduced BChl, this view was laid to rest in two ways. First, W. Parson (1968, 1978), exposing subcellular preparations of Chr. vinosum to brief laser flashes, observed that the oxidation of P870 was complete within 0.2 µs and the oxidation of Cyt then paralleled the return of P870 to its reduced form, with a half-time of about 2 µs. Second, a mutant of Rb. sphaeroides (strain PM-8, isolated by W.R. Sistrom, B.M. Ohlsson and J. Crounce in 1963) possesses a normal content of antenna BChl but cannot grow photosynthetically. It must be grown aerobically. This strain lacks P870; there is no evidence that it has RCs (Clayton et al. 1965; Clayton and Sistrom 1966). More has been learned from the fluorescence of BChl, especially in isolated RCs. In RCs, fluorescence accompanies the return of singlet-excited P870 to its ground state. This process competes with other fates of the excited state, mainly the photochemistry of photosynthesis: oxidation of P870 coupled with reduction of UQ by way of earlier electron acceptors, BPheo and perhaps BChl 800. In photochemically active RCs, the photochemical efficiency is close to unity and the efficiency (quantum 69 yield) of fluorescence is 3 × 10−4 (Clayton 1967; Zankel et al. 1968; Wraight and Clayton 1973). If fluorescence were the only pathway for dissipation of singlet-excited P870, this excited state would have its ‘intrinsic’ lifetime τ 0 . With other pathways operating, in this case photochemical utilization, the lifetime is shorter, in proportion to the efficiency of the competing pathway. Then the ratio of actual lifetime τ to intrinsic lifetime τ 0 will equal the quantum yield of fluorescence; in this case τ /τ 0 = 3 × 10−4 . Because the probability of an upward transition (P + hν → P∗ ; absorption) is proportional to that of the corresponding downward transition (P∗ → P +hν, fluorescence), the intrinsic lifetime can be estimated from the integrated area under the absorption band, in this case the 870 nm band. This gives τ 0 =2 × 10−8 s and τ = 2 × 10−8 × 3 × 10−4 or 6 × 10−12 s. We could thus estimate that the first step in the photochemistry of photosynthesis, with conversion of singlet-excited P870 to oxidized P870, occurs in about 6 ps (Zankel et al. 1968; Clayton 1978). The validity of this estimate was confirmed when the picosecond time domain became accessible to spectroscopists. The new technology also showed BPheo to be an electron carrier between P870 and UQ. It could be seen that BPheo becomes reduced within less than 10 ps after a flash, and passes its electron to the tightly bound UQ1 with a half-time of 200 ps. Finally, some small, rapid absorbance changes around 800 nm might implicate the two molecules of B800 as the very earliest electron acceptors, perhaps in a biradical P+. B− (reviewed in Clayton 1980). At this state of knowledge, the RCs of three species of purple bacteria, the non-sulfur bacteria Rb. sphaeroides and Rhodopseudomonas viridis and the purple sulfur bacterium Chr. vinosum, show very similar properties, even though Rps. viridis does not possess BChl a but BChl b, with long-wave absorption maxima at 1020 nm (antenna) and 980 nm (special pair, P980) (see Gingras 1978; and Blankenship et al. 1995). Early research on RCs culminated in the period 1982–1987 with the crystallization and X-ray structural analysis of RCs from Rps. viridis (H. Michel 1982; J. Deisenhofer et al. 1984; H. Michel et al. 1986; J. Deisenhofer and H. Michel 1988, 1989). Similar success was then obtained with RCs from Rb. sphaeroides by G. Feher and coworkers (Allen and Feher 1984; Allen et al. 1988; and see Personal perspective by Feher 1998), by J.R. Norris and coworkers (C.H. Chang et al. 1985; P. Gast and J.R. Norris 1984), and by Arnoux et al. (1995). Although the first membrane proteins to be crystallized were rhodopsin (Michel and Oesterhelt 1980) and porin (Garavito and Rosenbush 1980), the work with RCs from Rps. viridis earned Deisenhofer and Michel (and R Huber) a Nobel Prize in 1988 because it was of great significance for the future understanding of structure and function in biological membranes. Recently the research groups of H. T. Witt and W. Saenger have crystallized and provided X-ray analysis of RC complexes in Photosystem I (Krauss et al. 1993; Jordan et al. 2001) and in Photosystem II (Zouni et al. 2001). X-ray analysis of the crystals of RCs from Rps. viridis and Rb. sphaeroides revealed their structures in exquisite detail, showing the positions of the chromophores and of bound UQ and Cyt. These definitive results are consistent with many earlier conclusions about RCs, drawn on a trail often littered with luck and faith. Acknowledgments I am indebted to Charles McDougall and Jorjan Powers for their expertise with computers. I thank Govindjee and Colin Wraight for helpful comments and for providing many references. Note 1 I introduced the term ‘reaction center’ in 1963, but some students of photosynthesis were surely aware of the concept since Emerson and Arnold’s discovery of the photosynthetic unit. References Allen JP and Feher G (1984) Crystallization of reaction center from Rhodopseudomonas sphaeroides; preliminary characterization. Proc Natl Acad Sci USA 81: 4795–4799 Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1988) Structure of the reaction center from Rhodobacter sphaeroides. In: Breton J and Vermeglio A (eds) The Photosynthetic Bacterial Reaction Center, Structure and Dynamics, pp 5–11. Plenum Press, New York Arnold W and Clayton RK (1960) The first step in photosynthesis: evidence for its electronic nature. 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Plant Physiol 35: 477–485 Emerson R, Chalmers RV and Cederstrand CN (1957) Some factors influencing the long-wave limit of photosynthesis. Proc Natl Acad Sci USA 43: 133–143 Feher G (1971) Some chemical and physical properties of a bacterial reaction center particle and its primary photochemical reactants. Photochem Photobiol 14: 373–388 Feher G (1998) Three decades of research in photosynthesis and the road leading to it. A personal account. Photosynth Res 55: 1–40 Feher G and Okamura MY (1978) Chemical composition and properties of reaction centers. In: Clayton RK and Sistrom WR (eds) The Photosynthetic Bacteria, pp 349–386. Plenum Press, New York / London French CS (1961) Light, pigments, and photosynthesis. In: McElroy WD and Glass B (eds) Light and Life, pp 447–474. The Johns Hopkins Press, Baltimore, Maryland Fromme P (1999) Biology of Photosystem I: Structural Aspects. In: Singhal GS, Renger G, Sopory SK, Irrgang K-D and Govindjee (eds) Concepts in Photobiology: Photosynthesis and Photomorphogenesis, pp 181–220. Kluwer Academic Publishers, Dordrecht, The Netherlands Garavito RM and Rosenbush JP (1980) Three-dimensional crystals of an integral membrane protein: an initial X-ray analysis. J Cell Biol 86: 327–329 Gast P and Norris JR (1984) EPR detected triplet formation in a single crystal of reaction center protein from the photosynthetic bacterium Rhodobacter sphaeroides R-26. FEBS Lett 177: 277– 280 Gingras G (1978) A comprehensive review of photochemical reaction center preparations from photosynthetic bacteria. In: Clayton RK and Sistrom WR (eds) The Photosynthetic Bacteria, pp 105–132. Plenum Press, New York / London Govindjee (1999) On the requirement of minimum number of four versus eight quanta of light for the evolution of one molecule of oxygen in photosynthesis: a historical note. Photosynth Res 59: 249–254 Govindjee (2000) Milestones in photosynthesis research. In: Yunus M, Pathre U and Mohanty P (eds) Probing Photosynthesis, pp 9–39. Taylor & Francis, London Govindjee and Rabinowitch E (1960) Two forms of chlorophyll a with distinct photochemical functions. Science 132: 355–356 71 Hill R and Bendall F (1960) Function of two cytochrome components in chloroplasts: a working hypothesis. Nature 1186: 136–137 Jordan P, Fromme P, Witt HT, Klukas O, Saenger W and Krauss N (2001) Three-dimensional structure of cyanobacterial Photosystem I at 2.5 Å resolution. Nature 411: 909–917 Ke B (2001) Photosynthesis: photobiochemistry and photobiophysics. 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