Milestones in Photosynthesis Research

In: "Probing Photosynthesis: Mechanisms, Regulation And Adaptation", edited by Mohammad Yunus, Uday Pathre, and Prasanna Mohanty, published in 2000by Taylor & Francis, London, pp.9-39. A pdf file of the chapter and a correct Figure 3 is available by sending an E-mail to

Departments of Biochemistry, Plant Biology and Center of Biophysics and Computational Biology
University of Illinois at Urbana-Champaign



A tribute




Electron transport: General

Electron Transport: Acceptor Side of Photosystem II

The bicarbonate effect

Oxygen Evolution




Figure Legend


The task of writing a chapter on milestones in photosynthesis research is difficult. because there are so many milestones that I may not be able to do justice to them all. Thus, at the very outset, I beg forgiveness for incompleteness and I urge the readers that they should not consider this chapter as a record of the history of photosynthesis. Further, this chapter will not present milestones (including the ones marking one tenths of a mile) in a linear chronological manner, but according to specified topics. For an earlier thought-provoking article on the conceptual development in photosynthesis, see Myers (1974), for historical development, see Huzisige and Ke (1993), and for an introductory overview on photosynthesis, see Whitmarsh and Govindjee (1995). An overview on the milestones in the area of chlorophyll a fluorescence has been presented earlier (see Govindjee, 1995; also see Govindjee et al., 1986, and the chapter by Strasser and coauthors, this volume); thus, it will not be covered here unless I consider it pertinent to my discussion.

A tribute. This chapter is written in honor of my dear friend, a trusted colleague and a World leader in the field of Photosynthesis, Dr. Prafullachandra Vishnu Sane. I refer here only to three of his contributions. A thought-provoking new model of the distribution of the two photosystems in the thylakoid membrane system was presented elegantly by him in collaboration with Rod Park in their classic review (Park & Sane, 1971; also see Sane, 1977). His pioneering research, in collaboration with V.G. Tatake & T.S. Desai, in devising methods to record the most highly resolved thermoluminescence bands from the photosynthetic material, and for assigning these bands not only to photosystem II, but also photosystem I, have been reviewed by him in a chapter in a book (see Sane & Rutherford, 1986). I would also like to mention his discovery of heat-induced "state changes" in photosynthetic systems (see Sane et al., 1984).

The primary source of energy for nearly all life on Earth is the Sun. As early as 1845, Robert Mayer, who provided us with the Law of Conservation of Energy, had already recognized that plants convert light energy into chemical energy on a massive scale. Photosynthesis is the physico-chemical process by which oxygenic (plants, algae, cyanobacteria and prochlorophytes) and anoxygenic (photosynthetic bacteria) organisms convert light energy into redox chemical energy on a global scale. Each year 4x1018 kilojoules of free energy is stored in reduced Carbon by this process. In terms of carbon, each year about 1011 metric tons of CO2 are converted into organic matter by photosynthesis. According to Woese et al. (1990) the living organisms can be divided into three groups: archea; bacteria; and eukarya. Archea do not engage in true photosynthesis although a bacteriorhodopsin-containing organism Halobacterium salinarium (formerly H. halobium) can convert light energy into adenosine tri-phosphate (ATP). Photosynthetic bacteria and cyanobacteria, mentioned above, are clearly bacteria as the name implies, whereas plants and algae are eukarya. Oxygenic photosynthesis provides us with both food and oxygen, and anoxygenic photosynthesis only with food, needed for the survival of almost all living organisms except certain bacteria (two examples are Methanococccus janaschii and Methanobacterium autotrophicum). In addition, ancient photosynthesis is still providing us with fossil fuel; at the rate we are using it, it is not going to last forever. We need to understand the basics and the historical development of photosynthesis since it is the only process that can provide us with food, fuel and oxygen needed for the ever-increasing population, and since it is the only major process that may utilize the global increases in CO2 we are experiencing. It is now obvious that by the year 3,000, we expect the World's population to be 13 billion, but at the same time the available land for food production/person is decreasing at an alarming rate caused by our follies (increased deforestation and fossil-fuel burning) (Kendall et al., 1997; Sinha & Swaminathan, 1991; Swaminathan, 1998). Drastic measures are required to overcome this impending crisis that is already on our doorstep!

Within eight minutes of its origin, sunlight reaches the photosynthetic organisms on the Earth; almost billion chlorophyll molecules in a single chloroplast function to capture this energy within femtoseconds (10-15 s) (Note just for fun: there are as many femtoseconds in a second as there are seconds in 31 million years.) In what follows, I present a historical and a conceptual perspective on the milestones and breakthroughs in photosynthesis research dealing with the conversion of light energy into chemical energy in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine tri phosphate (ATP), which leads to the production of food from CO2 (see a general review by Whitmarsh & Govindjee, 1995). The steps for the latter were first fully deciphered by M.Calvin, A. Benson and J. Bassham at the University of California at Berkeley, for which Calvin was rewarded with a Nobel Prize in Chemistry in 1961. This aspect of photosynthesis will not be included in my chapter (see e.g., Bassham et al., 1954; Calvin, 1989 for the Calvin cycle; and Cleland et al., 1998, for the current understanding of the mechanism of the function of its key enzyme RUBISCO). Later, through the work of Kortsckak et al. (1965), Karpilov (1960) and Hatch & Slack (1966), it was discovered that in certain plants (such as sugarcane), the first product of CO2 fixation is a 4-C organic acid (e.g., oxaloacetic acid) in contrast to what Calvin and co-workers had found in the green alga Chlorella where the first stable product of CO2 fixation was a 3-C organic acid phosphoglyceric acid. This C-4 pathway is an appendage to the Calvin cycle (C-3 pathway) in plants such as sugarcane and pineapple. The discovery of the C-4 pathway has been elegantly presented by Hatch (1992). An important consideration about photosynthesis must be mentioned here: almost complete photosynthesis, i.e., CO2 fixation, occurs within the chloroplast as first convincingly shown by Arnon (1955).


The most visible colour on our Earth is green, a colour quite pleasing to our eyes. Nature evolved chlorophylls to capture sunlight; their structures were such that they could absorb blue and red light, and transmit green light giving leaves the colour green; nature also evolved rhodopsin in our eyes for us to see efficiently this green light. A major landmark in photosynthesis research was the elucidation of the physical and chemical properties of the chlorophyll molecule, a tetrapyrrole that has conjugated (alternating) single and double bonds giving it the spectroscopic properties of absorbing blue and red light, and a unique chemical structure allowing it to have different binding properties to different set of amino acids in various proteins to make it into light absorbing antenna, as well as reaction center chlorophylls labelled as P680 or as P700 (P for pigment trap, the numbers indicating the wavelength maxima of the first singlet excited state, in nanometers). Only P680+/P680 was destined to have the redox potential so positive (Em,7 :~ +1.1 V) that it can oxidize water to molecular oxygen (average Em,7 +0.8 V), whereas P700+/P700 (Em,7 +0.4 V) is only able to oxidize intermediates such as the hemes in cytochromes or copper in plastocyanin.

The elucidation of the chemical properties and the structure of chlorophylls was rewarded with the Nobel prizes in Chemistry to Richard Wilstätter (in 1915), to Hans Fischer (in 1930), whereas its total synthesis was recognized with a Nobel prize, also in Chemistry, to R. Woodward (in 1965). It was Duysens (1952) who had first used the term "P" for pigment representing a few reaction centre chlorophyll a or bacteriochlorophyll molecules in his Ph D thesis at the State University of Utrecht. The term P870 for bacterial reaction centre was then coined. Kok (1956, 1957), who was then at the Wageningen Agriculture University's 'ship-shaped' building, discovered P700, the reaction centre chlorophyll of what we now call Photosystem I (PSI, see a later section), whereas it was H.T. Witt and coworkers in Berlin (see Döring et al., 1969) who discovered P680, the reaction centre chlorophyll of what we now call Photosystem II, PSII. I note that my mentor and second advisor E. Rabinowitch had long suspected its presence, and Rabinowitch & Govindjee (1965) had named it as such prior to its discovery.

Although P680 and P700 are "special pairs" of chlorophyll a molecules, they are entirely different mainly because of their binding to different but specific amino acids in PSII and PSI reaction centre proteins. There are other chlorophylls too: Chl b, Chl c, and Chl d. In spite of my first advisor R. Emerson's early speculative and tentative ideas, Chl b and Chl c are only antenna pigments, i.e., they function only as light-harvesters in plants (as well as in green algae & prochlorophytes) and brown algae (as well as in diatoms & dinoflagellates), respectively. The recognition of Chl d as the major antenna pigment in Acaryochloris marina, a prochloron like prokaryote was discovered by Miyashita et al.

(1996). Whether Chl d can serve as the reaction centre Chl is under investigation in Japan. In addition, the possible role of Zinc chlorophylls versus Magnesium chlorophylls is also being actively investigated by S. Itoh and coworkers (personal communication). If confirmed, the latter two would break the stereotypic knowledge we had thus far regarding the uniqueness of Chl a as the only reaction centre chlorophyll of oxygenic photosynthesis.

The importance of the chlorophylls has been recognized by at least two books, edited by Vernon & Seely (1966) and by Scheer (1991). And finally, the recent discovery of a new type of bacteriochlorophyll, labelled as bacteriochlorophyll g (BChl g) (see Gest & Favinger, 1983; Brockman & Lipinski, 1983; Figure 1; also see Amesz, 1996) may have completed the discovery of all the various types of chlorophylls present in nature. BChl g resembles BChl b in having an ethylidene group on C8, which in the presence of oxygen and light, isomerises to give a vinyl group. Further, BChl g has a second vinyl group on C3; the product of its isomerisation is a molecule which is very similar to Chl a, the pigment of plants and green algae. Thus, heliobacteria acquire a possibly new evolutionary significance.

The key character of Chl a is that its bound forms, P680* and P700*, are photoenzymes converting light into chemical redox energy within a few picoseconds, acting as the world's most efficient solar battery:

P680 + Pheophytin + photon (or exciton)-----> P680+ + Pheophytin- (1)

(uphill electron transfer overcoming about 1.7 electron volts of energy barrier by a red photon)

P700 + Ao + photon (or exciton)-----> P700+ + Ao- (2)

(uphill electron transfer overcoming about 1.1 electron volts of energy barrier by a far-red photon; Ao is a chlorophyll monomer bound at a specific site in Photosystem I)


The terms antenna and the reaction centres have already been alluded to above. The concept is simple: a large number of light harvesting molecules function to capture light energy and act as if they are "antennae"; this captured light energy, in the form of excitation energy (or excitons) is transferred to a few pigment molecules that serve as reaction centre pigment molecules where primary photochemistry takes place. By primary photochemistry, we mean conversion of light (or excitation) energy into redox chemical energy that is then stored to do useful work. The birth of this concept, i.e., of a photosynthetic unit, that includes the two components, the antenna and the reaction centre, took place in 1932 in the Kirchhoff Laboratory of Biological Sciences, at the California Institute of Technology in Pasadena, CA. There were two players: assistant Professor of Biophysics Robert Emerson (see Rabinowitch, 1961) and an undergraduate student William Arnold. Emerson & Arnold (1932a,b) discovered that under the most optimal condition of photosynthesis (single turn-over brief and saturating flashes of light, with optimal dark times between them), a maximum of only one oxygen molecule was evolved per about 2,400 chlorophyll molecules present in the green alga Chlorella (Figure 2) although the maximum quantum yield of oxygen evolution (i.e., the number of O2 evolved per quantum absorbed) must have been very high, 1/8 to 1/10 in today's numbers). Thus, the existence of a Unit (and a photoenzyme) was suggested. It was, however, Gaffron & Wohl (1936) who provided the correct and complete interpretation of the highly elegant and sophisticated experiments of Emerson & Arnold: light absorbed by most of the chlorophyll molecules in the "photosynthetic unit" is transferred to the "photoenzyme" for chemistry. The 1932 experiments were unique in another sense: they were the first experiments in Science to use repetitive flash technique increasing the Signal/Noise ratio allowing precise measurements of small quantitities of oxygen evolution by manometric methods. It is of historical interest to mention here that both the techniques of manometry and the green alga Chlorella, used in this work, were introduced to Science by Emerson's own Professor Otto Warburg, who had received the Nobel prize in Physiology & Medicine in 1931 for his studies on biochemistry. The 1932 papers of Emerson & Arnold are now classical papers and continue to be regularly cited even after 66 years of their publication. This milestone discovery was recently discussed in an elegant paper by Myers (1994), in a special issue of "Photosynthesis Research", edited by Govindjee et al. (1996), and in a recent book by Wild & Ball (1997). The Emerson-Arnold "Photosynthetic Unit" has a functional definition : about 2,400 chlorophyll molecules cooperate to evolve one oxygen molecule (and, thus, reduce one CO2 molecule). As noted above, photosynthetic unit includes antenna complexes (the light harvesters) and the reaction centres. Exciton migration occurs in femto-to- picosecond range, among the photosynthetic pigments located in the protein complexes, the antenna proteins (see Hoff & Amesz, 1995; Hoff & Deisenhofer, 1997). The first kinetic evidence for the excitation energy transfer was obtained from time-resolved ultrafast fluorescence spectroscopy experiments initiated by Brody & Rabinowitch (1957) at UIUC, Urbana, Illinois. A wonderful example of this was shown by Yamazaki et al.(1984) in red algae where one can literally watch the excitons move from phycoerythrin to phycocyanin, then to allophycocyanin, and finally to chlorophyll a in a cascade-like manner.

Major breakthroughs in the understanding of the antenna structure at the atomic level have been through the availibility of X-ray and electron diffraction crystallography. Kühlbrandt et al.(1994) have provided the atomic level structure of the largest antenna of Photosystem II, the Light-harvesting complex II b; here one can see where the Chl a and Chl b molecules are anchored. On the other hand, McDermott et al. (1995) and Walz and Ghosh (1997) have provided the atomic level structures of two light-harvesting antenna complexes of anoxygenic photosynthetic bacteria; and, Hofmann et al. (1996) have provided the atomic level structure of the peridinin-chlorophyll protein complex of the dinoflagellate Amphidinium carterae. These provide the most fascinating view of the grand design of the architecture of the photosynthetic apparatus responsible for the capture and trapping of the excitation energy. These newer structures had been preceded by the structure of the chlorobium protein complex (the so-called Fenna-Mathews-Olson protein, see Fenna & Mathews, 1976; Mathews & Fenna, 1980; Tronrud et al., 1986), of phycoerythrin (Fiener & Huber, 1993) and of phycocyanin (Schirmer et al., 1986). Thus, we are now able to ask meaningful questions about the detailed physico-chemical reactions in the antenna and finally begin to understand the molecular mechanism of excitation energy transfer in photosynthesis, an area that had been dominated in the past mainly by speculative and theoretical arguments.

The conversion of light energy into chemical energy occurs in the World's most efficient solar battery, in the picosecond time range, in the reaction centre molecules. This conversion leads to the formation of the primary charge-separated state, P+A-, from PA, where P is the special reaction centre molecule, as already mentioned, and A is the primary electron acceptor. Examples of this reaction were already given under the section CHLOROPHYLLS. This is the only true light reaction of photosynthesis; all others can, in principle, occur in darkness. They are the only ones that are "uphill" because the free energy is positive due to the unfavourable difference in redox potentials of P/P+ and A/A-. These primary reactants and those involved in charge stabilization are located in the reaction centre complexes. The X-ray diffraction crystal structure of the reaction centre complex of the anoxygenic photosynthetic bacterium Rhodopsuedomonas viridis was the first one to be published, and was rewarded with the 1988 Nobel prize in Chemistry to H. Michel, J. Deisenhofer and R. Huber (see Deisenhofer et al., 1984,1985, 1995; Feher et al., 1989; Feher, 1998). Recently, Rhee et al. (1997) have provided a crude structure of P680-containing Photosystem II reaction centre complex at 8 Å; there are no details available. However, knowledge-based atomic level models of Photosystem II are now available (see e.g., Xiong et al., 1996, 1998; see Figure 3 and its legend ; also see a partial model by Coleman et al., 1997). Just as femtosecond-to-picosecond absorption spectroscopy have provided information on the primary charge separation in anoxygenic photosynthetic reaction centre (see e.g., Hoff & Deisenhofer, 1997; Zinth et al., 1998), information on Photosystem II are also now available (see Greenfield and Wasielewski, 1996; and Groot et al., 1997). Greenfield et al. (1997) have suggested that at ambient temperatures two time constants of charge separation can be measured:(1) approximately 8 ps, an upper limit for charge separation, due in part to equilibration of excitons among the core reaction centre chlorophylls (the "red" pool, R); and (2) approximately 50 ps, due, in part to equilibration of excitons among the accessory chlorophylls (the "blue" pool, B).

Chl a (B)* Chl a (R) P680 Pheo 50 ps >Chl a (B) Chl a (R)* P680 Pheo (3)

Chl a (B) Chl a (R)* P680 Pheo 8ps >Chl a (B) Chl a (R) P680* Pheo (4)

Chl a (B) Chl a (R) P680* Pheo 3ps >Chl a (B) Chl a (R) P680+ Pheo- (5)

We believe that the actual charge separation time is closer to 3 ps, the same as in anoxygenic photosynthetic bacteria. (In the equations shown above, * represents the molecule with an exciton or a photon.) All of the experiments on the primary photochemistry of photosystem II have been possible because of the success of Nanba & Satoh (1987) in isolating the simplest, although incomplete, reaction centre protein of photosystem II.

In contrast to PSII, a 6 Å resolution structure of P700-containing Photosystem I (PSI) reaction centre was published already five years ago by the research group of H.T. Witt of Berlin (Krauss et al., 1993); now, a 4 Å resolution structure of the same complex is available (Krauss et al., 1996). In addition to differences in the details, e.g., PSI being a Fe-S containing reaction centre, the PSI reaction centre complex contains also the core antenna pigment as well as the antenna chlorophylls. I consider this work to be one of the major milestones in the history of photosynthesis.


Electron transfers occur in pico-to-millisecond time range; they involve, in oxygenic photosynthesis, three major protein complexes: Photosystem II (water-plastoquinone oxido-reductase); Cytochrome b6/f complex (plastoquinol-plastocyanin oxido-reductase); and Photosystem I (plastocyanin-ferredoxin oxido-reductase). A fourth ATP synthase is required for ATP synthesis (see Figure 4 and its legend). An enigma, discovered by Emerson & Lewis (1943), was that the quantum yield of oxygen evolution decline suddenly beyond 680 nm, when chlorophyll a was still absorbing light; this so-called "red-drop" phenomenon led to the current concept of the two light reactions and two pigment systems when Emerson et al. (1957) discovered the Enhancement effect in photosynthesis. This concept is based on solid grounds: from the early ideas of E. Rabinowitch (1945, p.162; 1956, p. 1862) and of Hill & Bendall (1960). In the experiments on the so-called Emerson Enhancement in photosynthesis (see Figures 5 & 6 and their legends), or in the Hill Reaction, yield in far-red light (light I) by simultaneous exposure to shorter wavelength light (light II), is enhanced (Emerson et al., 1957; also see Emerson, 1958; Emerson & Rabinowitch, 1960; Govindjee & Rabinowitch, 1960; R. Govindjee et al., 1961, 1962, 1964; and a review by Myers, 1971). Further, the crucial experiments on the antagonistic effect of light I and II on the redox state of P700 (Kok, 1959; see Figure 7 and its legend), on the Chlorophyll a fluorescence yield (Govindjee et al., 1960; Butler, 1962; Duysens & Sweers, 1963), and, most importantly on the redox state of cytochrome f (Duysens et al., 1961; see Duysens, 1989) established the two light reaction scheme of photosynthesis. In fact, the existence of the series scheme of photosynthesis is best proven by light I (absorbed in photosystem I) oxidising cytochrome f and light II (absorbed in photosystem II) reducing the oxidised cytochrome f (see Figure 8 and its legend). However, without the fast absorption spectroscopic work on the new intermediate X-320 (now known to be equivalent to the so-called QA, the first bound plastoquinone molecule), the history of the discovery of the two light reactions would be incomplete (see Witt et al., 1961; Witt, 1991). Figure 9 shows the current model of the so-called Z scheme of electron transfer in oxygenic photosynthesis, whereas the legend of Fig.9 describes the current scheme. (The reader should refer to Figure 4 for the location of the components described in this figure.)

Electron transport: General

The mechanism by which electrons are transferred from one molecule to another has now been explained by R. Marcus (1993) who was awarded the 1992 Nobel prize in Chemistry for it.. A brief discussion follows. When a system such as e.g., DA is converted into D+A-, i.e., an electron is transferred from D to A, the energy curves of the two states DA and D+A- are shifted with respect to each other on axis of the reaction path (see Figure 10). Marcus defined a new term called the reorganizational energy (l ) by the length of the vertical line from the energy curve of the DA state to where the line hits the D+A- curve. The rate constant of the reaction is given by:

k= A exp ( -D G*/kBT) (6),

where A depends on the nature of the electron transfer reaction, kB is the Boltzmann constant, T is the absolute temperature in Kelvin, and D G* is the activation free energy for the reaction. And,

D G* = l /4 (1+ D Go/l )2 (7),

where D Go is the standard free energy for the reaction, and l is the reorganizational energy, as mentioned above. A major prediction of the Marcus equation (7) is that as D Go is varied from zero to some negative value, D G* decreases and becomes zero at D Go = -l , then increases when D Go is made still more negative , i.e., -D Go > l . The initial decrease of D G* with increasingly negative D Go was as expected, but what Marcus equation explained was the rate of electron flow in what he had called the "inverted region": increases in D G* when D Go was made increasingly negative. This prediction was made in 1960, but was confirmed experimentally only in 1984 by G.L. Closs and coworkers. Marcus (1993) has extended his theory to explain the characterestics of electron transfer in the bacterial reaction center. It is interesting to point out that electron transfers during the primary charge separation, discussed earlier, do not slow down at cryogenic temperatures; in fact, they become temperature independent. Such a phenomenon had been discovered by DeVault & Chance (1966) (also see DeVault, 1989): it was suggested that electrons can "tunnel" through barriers as if they squeeze through with certain probability. Further, if the energetics and other entropic parameters are constant, electron transfer rates can be predicted from the distances of the acceptor from the donor molecules. Moser et al. (1992) showed that if the acceptor is 1.4 Å further away, the transfer rate decreases by an order of magnitude. This, known as the "Dutton ruler", does not explain why electrons flow only on one side of the bacterial reaction centre molecule, not the other. Future prospects of understanding the mechanism of electron transfer are increasing as we begin to have atomic structures of the various intermediates. For example, we now have access to atomic level structures of the hydrophilic parts of cytochrome f (Martinez et al., 1994); of plastocyanin (Redinbo et al., 1993); and of the Cyt b/c complex of beef heart mitochondria (Xia et al., 1997) that may have similarities to Cyt b6/f complex of plants. Zhang et al. (1998) have described a model of electron transfer in which movement of domains occurs in cytochrome b/c1 complex; I expect it to have application to electron transfer in cytb6/f complex. However, in spite of the available structure of the reaction centre of anoxygenic photosynthetic bacteria, which have tremendous similarities in their two halves, we still do not fully understand why the electrons flow mostly on one, rather than the other side of the molecule. Here, a combination of molecular biology and physics & physical chemistry have begun to play important roles in the understanding of this process. We are still waiting for the final understanding.

Electron Transport: Acceptor Side of Photosystem II

A major advancement took place in the understanding of the functioning of the electron transfer pathway when Velthuys & Amesz (1974) and Bouges-Bocquet (1973) showed that there is a two electron gate on the acceptor side of PSII (see Figure 11 and its legend ); a similar gate was discovered later in anoxygenic photosynthetic bacteria, independently by Vermeglio (1977) and by Wraight (1977). Electrons from the reaction centers are transferred first to a bound quinone molecule QA which is, surprisingly, a one-electron acceptor. The reduced QA transfers its electron to a loosely bound quinone QB; the reduced QB is tightly bound and has a long lifetime; it waits for a second electron until it can be doubly reduced to QB2-. The latter then becomes a quinol (QBH2) after grabbing two protons coming from the side of the membrane it is located on. The electron flow at the QAQB site can be written as (see a review by Crofts & Wraight, 1983):

QAQB+ light-------> QA -QB-----> QAQB- (8)

QAQB- + light ------> QA QB2- (9)

QA QB2-+ 2H+------> QA QBH2 (10)

QA QBH2 + PQ-------> QAQB + PQH2. (11)

Although it is not certain, especially in plants, whether the protonation steps occur before the electron flow or vice versa, the above steps are referred to as the "two electron gate".

The bicarbonate effect. I have had a personal interest in this two electron "gate". Over the past twenty-five years or more, we have been trying to understand the role of bicarbonate in the reactions on the two photosystems, a phenomenon discovered by Warburg & Krippahl (1960). Warburg (1964) took this phenomenon to suggest that oxygen in photosynthesis originated in CO2, not in water, contradicting the existing idea that oxygen originated in water. He argued that (1) the 18O experiments of Ruben et al. (1941) could not be trusted because of equilibration with both water and CO2; (2) the hypothesis of C.B. van Niel was only from comparative biochemistry between oxygenic and anoxygenic organisms, and thus had no definitive value; and (3) the requirement of bicarbonate in the Hill reaction showed that it cannot be taken to prove that CO2 was not the source of oxygen in photosynthesis. I note here parenthetically that the discovery of the Hill reaction (see Hill & Scarisbrick, 1940), i.e., reduction of an artificially added electron acceptor and evolution of oxygen by chloroplasts, was itself a major finding that allowed tremendous number of biochemical studies that ensued from that time; it opened the field to look under the "hood" of the car, so to say. These were the arguments that fascinated me. I presented them to a photosynthesis course I was teaching in the early seventies; it was attended, among others, by Prasanna Mohanty and Alan Stemler, my PhD students. Alan jumped at the idea of working on the exciting project of attempting to understand the role of bicarbonate in the Hill reaction. Our first paper (Stemler & Govindjee, 1973) suggested that bicarbonate may function on the water side of photosynthesis. However, very soon we discovered a major effect on the acceptor side of photosystem II. We located a role on the electron acceptor side, specifically at the two-electron gate (Wydrzynski & Govindjee, 1975; Govindjee et al., 1976; Jursinic et al., 1976; Khanna et al., 1977, 1981; Blubaugh & Govindjee, 1988; Eaton-Rye & Govindjee, 1988; Xu et al., 1990; Govindjee, 1993, and, Govindjee & van Rensen, 1993). Bicarbonate seems to be involved not so much in the electron flow per se, but in the protonation steps (also see van Rensen et al., 1988). Interestingly, this effect is absent in anoxygenic photosynthetic bacteria (see e.g., Shopes et al.,.1988; Wang et al., 1992). Differences between photosynthetic bacteria and photosystem II are, we believe, due to a preponderance of positively charged amino acids in the QB binding region of PSII over that in photosynthetic bacteria (Xiong et al., 1996; 1998a). This may be responsible for different protonation channels in anoxygenic photosynthetic bacteria and in PSII. This bicarbonate site seems close to (Khanna et al., 1981) , but not identical to that where certain herbicides bind and act (Velthuys,1981; Wraight ,1981). Terbutryn inhibits and kills plants by displacing QB, and bicarbonate seems to be involved in the protonation of the reduced form of QB. One of the binding sites of bicarbonate has been shown to be on the non-heme iron ( see e.g., Diner et al., 1991) and the other near an arginine residue, D1-R-257 (Xiong et al., 1998b). It is not yet clear how this acceptor side binding (see Figure 12) would provide another major bicarbonate action on the oxygen evolving complex expounded by O. Warburg, H. Metzner and A. Stemler (see Stemler, 1982), and now being investigated in great detail by Klimov & coworkers (Allakhverdiev et al., 1997; Hulsebosch et al., 1998; Klimov et al., 1997; Yruela et al., 1998). It calls for further research.

Oxygen Evolution

The answer to the question of the minimum number of quanta needed to evolve one oxygen molecule was solved in favour of 8-10 (Emerson & Lewis, 1943; Emerson, 1958; R. Govindjee et al., 1968) rather than of 3 to 4 (Warburg & Negelein, 1923). This is in agreement with the scheme that two light reactions are needed for oxygen evolution and NADP+ reduction. A major breakthrough in the oxygen evolution steps was the discovery by Joliot et al. (1969) that oxygen/flash as a function of flash number oscillates with a period of 4 indicating accumulation of 4 positive charges on some intermediate before water is oxidized to molecular oxygen (Figure 13). A theory evolved by Kok et al.(1970) became the major framework (for alternate models, see Mar & Govindjee, 1972), where the system started mostly in the S1 state in darkness, and oxygen release took place when S4 was converted to S0 (S representing the redox state of the tetranuclear Mn complex):

` S1 + light flash #1; or #5---> S2 (12)

S2 + light flash # 2; or #6---->S3 (13)

S3+ light flash # 3; or # 7----> S4 (14)

S4 + 2H2O-----> S0+ O2 + 4 H+ (15)

S0 + light flash # 4; or # 8---> S1. (16)

The elegant measurements of Dismukes & Siderer (1980) on multiline EPR (electron paramagtnetic resonance) signals of Mn, and those of M. Klein and coworkers (see Yachandra et al., 1996; Roelofs et al., 1996) on EXAFS (Extended X-ray Absorption Fine Structure) and XAS ( X-ray Absorption Spectroscopy) led to the identification of the S-state intermediates with mixed valence Mn complex (a tetranuclear Mn/PSII). The S0 is suggested to be Mn (II), Mn (III), Mn (IV), Mn (IV) (alternatively, Mn (III), Mn (III), Mn (III), Mn (IV)); S1 to be Mn (III), Mn (III), Mn (IV), Mn (IV); and both S2 & S3 as Mn (III), Mn (IV), Mn (IV), Mn (IV), Mn (IV). The mechanism of water oxidation to dioxygen is not yet known; however, there are theories as to how this occurs. I refer the readers to read Renger (1997) and Babcock & co-workers (Hoganson& Babcock,1997; Tommos & Babcock, 1998) to obtain a glimpse of the current and two different thoughts on the mechanism of oxygen evolution. The recent finding of Messinger et al. (1997a) that S0 is also paramagnetic will further enhance our ability to understand the transitions of the S states, presented above. In addition, the possibility of showing that the S states can be reduced to S(-3) state lends credence to the current picture of the valence state of the Mn in the water oxidase (see Messinger et al., 1997b). On the personal side, I want to mention that Coleman & Govindjee (1987) were the first to suggest that the Mn atoms of the Mn cluster were bound to the reaction centre proteins D1 & D2; and, Kambara et al. (1985) and Padhye et al. (1986) were the first to suggest that an organic radical, specifically a histidine, may be the charge accumulator in addition to Mn during S2 to S3 transition. The role of histidine is being actively investigated in several laboratories; first, it seemed evidence was obtained, then, it was challenged; now, I am waiting for the final judgement before citing any paper on this topic. Following the early pioneering work of W.F.J. Vermaas and of B. Diner and coworkers, Bowlby et al. (1996) have reported that Glutamic-65 and Histidine 337 on D1 protein may be ligands to Mn; and, Aspartic 103 and Glutamic 104, also on D1, may be ligands to Calcium. On the other hand, T. Wydrzynski and coworkers (Messinger et al., 1995) are providing new information, using elegant mass spectroscopic methods, on the exchangeable water molecules at the site of water oxidation. Evidence for the proximity of a Calcium to the Mn cluster, discussed above, has now been implied from X-ray absorption spectroscopy (Latimer et al., 1995). Oxygen, the by-product of water oxidation, is released through the operation of the "oxygen clock" that involves 4 Mn atoms/PSII (see a detailed earlier review by Debus, 1992).


A major tool to study the S-states was shown to be thermoluminescence, a phenomenon discovered by W. Arnold ( see reviews by Sane and Rutherford, 1986; Vass & Govindjee, 1996). Thermoluminescence (TL) is light emission, that originates mainly from PSII. If one shines light on a photosynthetic material, and freezes to 77K, all the separated charges are frozen, and heating the sample in darkness leads to thermoluminescence bands originating from the back reactions (i.e., recombination of charges) of say, e.g., S2 with QB- or S2 with QA-, etc. P.V. Sane, in collaboration with V.G. Tatake and T.S. Desai, was responsible for placing India on the World map of pioneering research in this area of thermoluminescence. The design of the instrument at the Bhabha Atomic Research Centre (BARC), Mumbai, India, where this work was done, provided very high Signal/Noise ratio. Several (6 or 7) TL bands were fully separated from each other and these bands, through separation of pigment systems I and II and through the ingenious use of inhibitors and electron carriers, were related to specific reactions in PSII and even PSI (see Sane & Rutherford, 1986; and Vass & Govindjee, 1996, for reviews). It was because calculations, by Tatake et al. (1981), of the activation energies of the various TL bands did not match the Randall-Wilkins theory that I approached Don DeVault for help; this led to the formulation by DeVault et al. (1983) of the most appropriate theory for TL in plants. Without the leadership provided by P.V. Sane, researches on TL at BARC would have been in directions different from those on the photosynthetic systems. Relationship of the S-states to the TL was, however, provided by Inoue &Shibata (1978) when they discovered the period 4 oscillation in TL and related it to oxygen evolution steps (see reviews by Vass & Inoue, 1992; and Inoue, 1996). I refer the readers to the cited reviews to discover for themselves the discoveries of A.W. Rutherford, G. Renger, and the research group in Szeged, Hungary without whose work, TL will not be where it is today. Currently, the TL method is being used to monitor and characterise site-directed mutants both on the acceptor (see e.g., Mäenepää et al., 1995) as well as the donor side of PSII (see e.g., Kramer et al., 1994).


ATP is the energy currency of life. Production of glucose (food) from CO2 requires both NADPH and ATP. Below, we will discuss the breakthroughs that led to the understanding of ATP synthesis: chemiosmotic hypothesis; and the alternate binding site hypothesis.

After indications in several laboratories that algal cells produce ATP in light, Arnon et al. (1954) and Frenkel (1954) discovered photophosphorylation in chloroplasts of plants, and chromatophores of anoxygenic photosynthetic bacteria, respectively. This was a major breakthrough and it took many years to recognize that only in some anoxygenic photosynthetic bacteria, almost all light energy is first converted into ATP energy and then this energy is used for the reversed electron flow to produce the reducing power in the form of NADH. In oxygenic organisms, however, this is not the case as correctly expressed by Rabinowitch (1956). As we know today, ATP synthesis follows electron transport steps that first store energy temporarily by creating a proton motive force; this energy would be otherwise lost if not used for ATP formation (see the scheme of Hill & Bendall, 1960). P. Mitchell (1961), who received the Nobel prize in Chemistry in 1978, provided the theory that a proton motive force ( that is a sum of a pH gradient and a membrane potential) is the energy source of ATP synthesis. Membranes are normally impermeable to protons; and, protons are transferred from one side of the membrane to the other by virtue of the alternate electron and hydrogen atom transfers due to specific location of the electron and hydrogen atom carriers and the energetics of the light-induced electron transfers in photosynthesis. An early breakthrough was the independent experiment of Shen & Shen (1962) and of Hind & Jagendorf (1963) showing that light forms some entity (XE) that can be used later in darkness to produce ATP. Hind & Jagendorf showed that XE is a pH gradient that drives ATP synthesis. Jagendorf & Uribe (1966) showed that ATP can be synthesized from pH gradient created by acid-base transition in total darkness. These experiments, along with those of the others, provided major evidence for the chemiosmotic hypothesis of Mitchell. In photosystem II, water oxidation complex that liberates protons is on the inner side (the lumen) of the thylakoid membranes. Further, plastoquinone is reduced to plastoquinol on the outer side (stroma side) of the thylakoid membrane; the plastoquinol (a hydrogen atom carrier) moves towards Rieske Iron sulfur and cytochrome f (both are electron, not hydrogen atom carriers) that are located towards the lumen side. Here, plastoquinol delivers its electrons to the Rieske Iron sulfur centre and the cytochrome f, leaving the protons to be released into the lumen. This arrangement, thus, allows natural proton translocation from the stroma to the lumen side as the electron transport takes place in PSII. This adds to the pH gradient to be used for ATP synthesis. The equivalence of pH gradient and membrane potential in synthesizing ATP was shown by the elegant experiments of Gräber et al. (1984) and Hangarter & Good (1982) when they varied one keeping the other constant and showing that it was the sum of the two that was important for initiating ATP synthesis.

The understanding of the mechanism by which ATP synthesis takes place at the ATP synthase using the proton motive force has been influenced by the following three breakthroughs. (1) The binding change hypothesis of P. Boyer and co-workers (see Boyer et al., 1973; and a review by Boyer, 1997; also see Figure 14 and its legend) that suggests that at one time (a) ADP and Pi (inorganic phosphate) bind weakly at one site of the alpha-beta pair of the F1 part of the ATP synthase enzyme; (b) bound ATP is formed from bound ADP and Pi, without the use of energy at a second alpha-beta pair; and (c) the pH gradient energy is converted into rotational energy, mainly of the gamma subunit that extends upto the third alpha-beta pair, that is used to flip off the ATP free from the third alpha-beta binding site. These three sites alternate in time. (2) The rotation feature of the Boyer model has been elegantly demonstrated directly by fluorescence microscopy and by photoselection and other experiments (see Capaldi, R.A. 1994; Duncan et al., 1995. Sabbert et al., 1996, 1997; Junge et al., 1997) (3) Atomic resolution structure (Abrahams et al., 1994) of beef-heart mitochondrial F1 showed that the gamma subunit indeed looks through the alpha-beta pairs; and in agreement with the Boyer model, the structure shows one alpha-beta pair site empty (as if ATP was released); another with bound ADP and Pi; and the third with an equivalent of bound ATP. I consider it highly likely that the plant, algal, and cyanobacterial ATP synthase will be basically similar to that of the cow.


In my opinion, the mechanism of photosynthesis has been probed by several means. Its face has changed by experiments of many investigators. I restrict my personal list to deal mainly with oxygen evolution. It includes, among others, the experiments of my first mentor Robert Emerson (Emerson & Arnold,1932). U sing the repetitive flash method and Warburg’s manometry, they led to the concept of "Photosynthetic Unit" where excitation energy, absorbed by hundreds of antenna pigment molecules, is transferred to the reaction center chlorophyll molecules, the "photoenzyme" or the "energy trap" for chemistry. Next, was the discovery of the "Red Drop" (Emerson & Lewis, 1943) in the quantum yield action spectrum of oxygen evolution in the green alga Chlorella pyrenoidosa, and the enhancement effect of certain wavelengths of light on the yield of oxygen evolution in the "red drop" region (Emerson et al., 1957), discovered by the use of an excellent monochromator and state-of-the art manometry; it lead to the concept of two-light reaction and two-pigment system mechanism of electron transport. It became well-known due to the working hypothesis of Hill & Bendall (1960) and became a scientific fact by the experiments of Duysens et al. (1961) on the antagonistic effect of light absorbed in pigment system I and II on the redox state of cytochrome f. The important concept that there is an "oxygen clock", where four positive charges must accumulate before water can be oxidized to oxygen was enshrined before us by (a) the experiments of Joliot et al. (1969) on the periodicity of four in the plots of the amount of oxygen evolved per flash as a function of flash number, and (b) the so-called "S-states" model of charge accumulation enunciated by Kok et al. (1970). Such a periodicity of four, reflecting indirectly the S-states, has also been observed in chlorophyll a fluorescence (Delosme, 1971; Joliot & Joliot, 1971), and recently, Shinkarev et al. (1997) have even managed to obtain the kinetics of the last step of oxygen evolution from analysis of the data on the decay of chlorophyll a fluorescence in single flashes of light. Thermoluminescence, discovered by W. Arnold & H. Sherwood (see Arnold, 1991), that has been exploited both by P.V. Sane (Sane & Rutherford, 1986) as well as myself (DeVault et al., 1983; Vass & Govindjee, 1996), has uniquely probed the characteresteristics of the S-states of oxygen evolution (Inoue & Shibata, 1978; Inoue, 1996). The most elegant probes for showing that Kok’s S-states are Manganese are the low temperature EPR (Dismukes& Siderer, 1980); and the Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy (M. Klein, K. Sauer & co-workers; see Yachandra et al., 1996). At the end, I do want to emphasise two elegant conceptual work on another area, that of phosphorylation: (a) the chemiosmotic theory of P. Mitchell in which proton motive force across a membrane provide energy for ATP synthesis (Mitchell, 1961) and (b) the elegant theory of Paul Boyer as to how this comes about: by conversion of electrochemical energy to conformational energy (see Boyer, 1977). Finally, I wish to mention that mimicking photosynthesis in vitro has been a dream of many, and the recent success of Steinberg-Yfrach et al. (1998) in making "lots of ATP" in artificial liposome membranes, energised by a synthetic system (carotene-porphyrin-quinone, is highly commendable. It would have made my second mentor Eugene Rabinowitch quite happy.


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