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Home , Cytochrome b559, Electron acceptor, Hill reaction, Oxygen evolution, Photophosphorylation

Proc. Nat. Acad. Sci. USA Vol. 68, No. 11, pp. 2883-2892, November 1971

The Light Reactions of

DANIEL I. ARNON Department of Cell Physiology, University of California, Berkeley, Berkeley, Calif. 94720

ABSTRACT Historically, the role of light in photo- (4) and Willsttfiter (5); its last great contemporary protago- svnthesis has been ascribed either to a photolysis of nist was Otto Warburg (6). After de Saussure (7) showed that dioxide or to a photolysis of and a resultant rear- rangement of constituent atoms into of oxy- water is a reactant in photosynthesis, the C02 cleavage hy- gen and glucose (or formaldehyde). The discovery of photo- pothesis readily accounted for the deceptively simple overall demonstrated that photosynthesis in- photosynthesis equation (Eq. i): the C: 2H: 0 proportions cludes a light-induced phosphorus that in the product fitted the idea that the carbon precedes, and is independent from, a photolysis of water recombines with the or CO2. ATP formation could best be accounted for not from the photodecomposition of C02 by a photolytic disruption of the covalent bonds in C02 elements of water. or water but by the operation of a light-induced electron h 0 2 flow that results in a release offree which is trapped C02 + H20 + 02 (i) in the pyrophosphate bonds of ATP. (CH20) is now divided into (a) a non- A different hypothesis,, one that profoundly influenced re- cyclic type, in which the formation of ATP is coupled with in was forward van Niel a light-induced electron transport from water to search photosynthesis, put by (8). and a concomitant evolution of and (b) a cyclic After elucidating the nature of bacterial photosynthesis, he type which yields only ATP and produces no net change in proposed (8) that bacterial and photosynthesis are the oxidation-reduction state of any or special cases of a general process in which light energy is used acceptor. Reduced ferredoxin formed in (a) serves as an to photodecompose a donor, H2A, with the released electron donor for the reduction of NADP by an enzymic reaction that is independent of light. ATP, from both hydrogen in turn reducing C02 by dark, enzymic reactions: cyclic and noncyclic photophosphorylation, and reduced hp NADP jointly constitute the assimilatory power for the C02 + 2H2A -- (CH20) + H20 + 2A (ii) conversion of C02 to (3 moles of ATP and 2 moles of reduced NADP are required per mole of C02). The hypothesis envisaged that in plant photosynthesis Investigations, mainly with whole cells, have shown that H2A is water, whereas in green (for example) photosynthesis in green involves two photosystems, with the results that becomes the one (System II) that best uses light of "short" wavelength H2A is H2S, oxygen by- (X < 685 nm) and another (System I) that best uses product of plant photosynthesis and elemental sulfur the light of "long" wavelength (X > 685 nm). Cyclic photo- by-product of bacterial photosynthesis. phosphorylation in involves a System I In later formulations (9, 10) van Niel no longer considered photoreaction. Noncyclic photophosphorylation is widely the photodecomposition of water as being unique to plant held to involve a collaboration of two photoreactions: a short-wavelength photoreaction belonging to System II photosynthesis but postulated that "the photochemical reac- and a long-wavelength photoreaction belonging to System tion in the photosynthetic process of green bacteria, purple I. Recent findings, however, indicate that noncyclic photo- bacteria, and green plants represents, in all cases, a photo- phosphorylation may include two short-wavelength, Sys- decomposition of water" (10). According to this concept, the tem II, photoreactions that operate in series and are joined by a "dark" electron-transport chain to which is coupled distinction between plant and bacterial photosynthesis a phosphorylation site. turned on the events that followed the photodecomposition of water into H and OH. H was used for C02 reduction and OH formed a complex with an appropriate acceptor. In plant Early concepts photosynthesis, the acceptor was regenerated when the com- The first hypothesis about the role of light in photosynthesis plex was decomposed by liberating molecular oxygen. In came very appropriately from Jan Ingenhousz, who some bacterial photosynthesis, oxygen was not liberated and the years earlier had made the epochal discovery that it is "the acceptor could be regenerated only when the OH-acceptor influence of the light of the sun upon the plant" (1) that is complex was reduced by the special hydrogen donor, H2A, responsible for the "restorative" effect of vegetation on that is always required in bacterial photosynthesis. "bad" air-an observation first made in 1771 by Joseph The concept of photodecomposition of C02 or photodecom- Priestley without reference to light (2). In 1796, Ingenhousz position of water provided, each in its own historical wrote that the green plant absorbs from "carbonic acid in period, a broad, general perspective on the role of light in the the sunshine, the carbon, throwing out at that time the oxygen overall events of photosynthesis. In the last two decades, alone, and keeping the carbon to itself as nourishment" (3). however, the focus in photosynthesis research shifted toward The idea that light liberates oxygen by photodecomposing the isolation, identification, and characterization of the C02 had, with some modifications, persisted for well over a specific reactions and mechanisms by which light energy century. It seemed to have had a special attraction for some drives the photosynthetic process. This approach has led to of the most illustrious chemists in their day, e.g., von Baeyer new perspectives on the mechanisms by which light energy is 2883 Downloaded by guest on September 26, 2021 2884 Amnon Proc. Nat. Acad. Sci. USA 68 (1971)

EXPERIMENTALLY IDENTIFIED FIRST results that were at best suggestive [see review (22)1. In PRODUCTS OF PHOTOSYNTHESIS short, experiments with whole cells proved, for reasons dis- I1862: STARCHWHL cussed below, incapable of yielding evidence for an indepen- 11883: SUCROSE, GLUCOSE CELLS dent light-induced phosphorus metabolism. Its occurrence in photosynthesis was discovered not in whole cells but in 1951: NADPH2 isolated chloroplasts. 1954: AT P First products of photosynthesis: 1957: NADPH2 +ATP CHLOROPLASTS experiments with isolated chloroplasts 1962: Fd red 1964: Fd red + AT P Chloroplasts were once widely believed to be the site of com- plete photosynthesis but this view was not supported by FIG. 1. critical evidence (23, 24) and was largely abandoned after Hill (25, 26) demonstrated that isolated chloroplasts could used in photosynthesis and to a finding, inadmissible under evolve oxygen but could not assimilate C02. [The failure either of the two earlier of hypotheses, that the photosynthetic isolated chloroplasts to fix C02 was also reported with the apparatus can convert light energy into a stable form of sensitive 14CO2 technique (27).] In the oxygen-producing reac- chemical energy, independently of the splitting of either water tion, which became known as the , isolated or C02. chloroplasts evolved oxygen only in the presence of artificial First products of photosynthesis: oxidants with distinctly positive oxidation-reduction poten- experiments with whole cells tials, e.g., ferric oxalate, ferricyanide, benzoquinone. In chemical terms, an insight into the role of light in photo- The Hill reaction established that the photoproduction of synthesis could come from identification of the first chemically oxygen by chloroplasts is basically independent of C02 as- defined products that are formed under the influence of light. similation. This provided strong support for the view that the In the 19th century (Fig. 1) this approach established that source of photosynthetic oxygen is water.* Left in doubt was starch is the first product of photosynthesis in chloroplasts the role of chloroplasts in the energy-storing reactions needed (11)-a conclusion that was later revised in favor of soluble for C02 assimilation. The photochemical generation by iso- carbohydrates (ref. 12). In the modern period, the powerful lated chloroplasts of a strong reductant capable of reducing new techniques of 14C (ref. 13), paper chromatography (14), C02 was deemed unlikely on experimental and theoretical and radioautography (15) aided in the identification of phos- grounds (26, 28). The first experiments with the sensitive 82p phoglyceric acid (PGA) as the first stable product of photo- technique to test the ability of isolated chloroplasts to form synthesis, formed only after a few seconds of illumination ATP, on illumination, also gave negative results (29). (16). Aside from PGA, esters of two sugars, ribulose A different perspective on the photosynthetic capacity and sedoheptulose, were soon added to the list of early prod- of isolated chloroplasts began to emerge in 1951 when three ucts of photosynthesis in green cells (17, 18). laboratories (30-32), independently and simultaneously, The discovery of PGA and other early intermediates of found that isolated chloroplasts could photoreduce NADP C02 assimilation led Calvin and his associates (19, 20) to the despite its strongly electronegative potential (Em = formulation of a photosynthetic carbon cycle (reductive -320 mV, at pH 7). This finding was followed by several pentose phosphate cycle), which was convincingly identified other developments which drastically altered the then preva- with the dark phase of photosynthesis. The chief importance lent ideas about the photosynthetic capacity of isolated of the carbon cycle to the understanding of the role of light in chloroplasts. These developments (Fig. 1) will now be dis- photosynthesis lay in revealing which of its component en- cussed in chronological order. zymic reactions require an input of energy-rich chemical In 1954, a reinvestigation of photosynthesis in isolated intermediates that must be formed by the light reactions. chloroplasts by different methods yielded evidence for a As summarized in Fig. 2, the carbon cycle shows that the con- light-dependent assimilation of C02 (ref. 33). Chloroplasts version of 1 mole of C02 to the level of hexose phosphate re- isolated from spinach leaves assimilated 14CO2 to the level quires 3 moles of ATP and 2 moles of reduced NADP. Accord- of carbohydrates, including starch, with a simultaneous ingly, the need for light energy in photosynthesis by green evolution of oxygen (34, 35). When the conversion of 14CO2 plants could now be traced to those photochemical reactions by isolated chloroplasts to sugars and starch was confirmed that generate ATP and NADPHE2. and extended in other laboratories (36-40), the capacity of The occurrence of phosphorylated compounds among the chloroplasts to carry on complete extracellular photosyn- early products of C02 assimilation suggested that light- thesis was no longer open to question. induced phosphorus assimilation may, in fact, precede carbon Because of the earlier negative results, special experimental assimilation but experimental evidence for this conclusion safeguards were deemed necessary to establish that chloro- was lacking in whole cells. When Calvin's group investigated plasts alone, without other organelles or systems and the photoassimilation of phosphorus by Scenedesmus cells with light as the only energy source, were capable of a total with the aid of carrier-free KH232PO4, they found that the synthesis of carbohydrates from C02. The chloroplasts were shortest exposure to light gave the lowest incorporation of washed and, to eliminate a possible source of chemical energy 32p into ATP and, conversely, that the highest incorporation and metabolites, their isolation was performed not as formerly ATP on of 32p into occurred short, dark exposure (21). The * Contrary to a widely held belief, this conclusion was not un- first compound to be labeled proved to be not the expected equivocally documented by experiments with 180 [(see A. H. ATP but again PGA (21). Other investigations of direct Brown and A. W. Frenkel, Annu. Rev. Plant Physiol., 4, 53 photoassimilation of phosphorus by intact cells also gave (1953)]. Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Light Reactions of Photosynthesis 2885

in isotonic sugar solutions (25) but in isotonic sodium chloride (33, 35). In comparison with the parent leaves, washed saline chloroplasts gave low rates of CO2 assimilation (35)-a situa- tion similar to the first reconstruction of other cellular pro- cesses in vitro, e.g., (41, 42), synthesis

(43), and polymerization of DNA (44). Crucial for the docu- RNKJILOSF TrI(c mentation of complete photosynthesis in isolated chloroplasts DIPHOSPHATE 7 PHOSPHATE 7 STARCH were not high rates but the fact that their newly found CO2 assimilation was reproducible and yielded the same inter- mediate and final products as photosynthesis by intact cells. ATP J INTERMEDIATES More recently, when CO2 assimilation ceased to be a matter of RIL@UOSE dispute and the same experimental safeguards were no longer MONOPHOSPHATE needed, much higher rates of CO2 assimilation by isolated chloroplasts were obtained with modified procedures (45-48). FIG. 2. Schematic representation of the ATP and reduced Since neither ATP nor reduced NADP was added to iso- NADP requirements {or CO2 assimilation. lated chloroplasts that fixed C02, it was clear that these energy-rich compounds were being photochemically generated that supplies ATP for the biosynthetic reactions in all types from their respective precursors within the chloroplasts. of photosynthesis. Chloroplasts were already known to photoreduce added Soon after the demonstration of photosynthetic phos- NADP but nothing was known about their ability (or that of phorylation in isolated chloroplasts, attempts were made to any other photosynthetic structures) to form ATP at the evaluate its physiological significance. Since, as with other expense of light energy. A renewed attack on this problem in cellular processes when first reproduced in vitro, the rates of isolated chloroplasts was therefore undertaken. photosynthetic phosphorylation were low, there was little Discovery of photosynthetic phosphorylation inclination at first to accord this process quantitative im- portance as a photosynthetic mechanism for converting light The likelihood of detecting a direct role of light in ATP forma- into chemical energy (55). With further improvement in tion was much greater in isolated chloroplasts than in intact experimental methods (which included the use of broken cells. Intact cells contain only catalytic amounts of the pre- chloroplasts with lowered permeability barriers), rates of pho- cursor adenosine phosphates (AMP, ADP) and these, because tosynthetic phosphorylation increased 170 times (56) and more of permeability barriers, could not be increased by external (57) over those originally described (33). additions. By contrast, in experiments with isolated chloro- The improved rates of photosynthetic phosphorylation plasts, it was possible to supply these normally catalytic were equal to, or greater than, the maximum known rates of substances in substrate amounts and, with the aid of labeled carbon assimilation in intact leaves. It appeared, therefore, inorganic phosphate, determine chemically their light-in- that isolated chloroplasts retain, without substantial loss, duced conversion to ATP. the enzymic apparatus for photosynthetic phosphorylation- In 1954, work with the same spinach prepara- a conclusion in harmony with evidence that the phosphoryl- tions that fixed CO2 led to the discovery that they were also ating system was tightly bound in the water-insoluble lamellar able to convert light energy into chemical energy and trap portion of the chloroplasts. it in the pyrophosphate bonds of ATP (49, 33). Several unique features distinguished this photosynthetic phos- Role of light in photophosphorylation phorylation (photophosphorylation), as the process was Once the main features of photophosphorylation were firmly named, from substrate-level phosphorylation in fermentation established, the next objective was to explain its mechanism, and oxidative phosphorylation in respiration: (a) ATP forma- particularly its absolute dependence on illumination (33, 50). tion occurred in the -containing lamellae and On the one hand, photophosphorylation was independent of was independent of other enzyme systems or organelles such classical manifestations of photosynthesis as oxygen (including mitochondria, which were previously considered evolution and CO2 assimilation; on the other hand, it seemed necessary for photosynthetic ATP formation, ref. 51); (b) unlikely that light was involved in the formation of ATP itself, no energy-rich substrate, other than absorbed photons, served a reaction universally occurring in all cells independently of as a source of energy; (c) no oxygen was produced or con- photosynthesis. Light energy, therefore, had to be used in sumed; (d) ATP formation was not accompanied by a mea- photophosphorylation before ATP synthesis and in a manner surable electron transport involving any external electron unrelated to CO2 assimilation or . The most donor or acceptor (49, 33, 50). The light-induced ATP forma- probable mechanism for such a role seemed to be a light- tion could be expressed by the equation: induced electron flow (58, 59). hp It is often difficult for the student of photosynthesis today n*ADP + n - Pi n*ATP (iii) to realize that before the discovery of photophosphorylation the concept of a light-induced electron transport had no sub- When photophosphorylation in chloroplasts was followed stantial basis in photosynthesis research. The idea that photon by evidence of a similar phenomenon in cell-free preparations energy is used in photosynthesis to transfer electrons rather of such diverse types of photosynthetic organisms as photo- than cumbersome atoms had a few proponents at various synthetic bacteria (52) and algae (53, 54), it became evident times, for example Katz (60) and Levitt (61) but, as the litera- that photophosphorylation is not peculiar to plants containing ture before the late 1950s shows, it did not become a viable chloroplasts but is a major ATP-forming process in nature concept in photosynthesis-it was merely one of several specu- Downloaded by guest on September 26, 2021 2886 Amnon Proc. Nat. Acad. Sci. USA 68 (1971)

lative ideas based on model systems. The situation changed electrons from water to NADP (or to a nonphysiological with the discovery of light-induced ATP formation. ATP electron acceptor such as ferricyanide) and a concomitant is formed in nonphotosynthetic cells at the expense of energy evolution of oxygen. Moreover, ATP formation in this coupled released by electron transport. The idea that ATP may also system greatly increased the rate of electron transfer from be formed in photosynthesis through a special light-induced water to ferricyanide (63-65) or to NADP (66) and the rate electron flow mechanism in chloroplasts now had a high proba- of the concomitant oxygen evolution. It thus became apparent bility that could be experimentally tested. that the electron transport system of chloroplasts functions The electron flow hypothesis (58, 59) envisaged that a more effectively when it is coupled, as it would be under chlorophyll , on absorbing a quantum of light, be- physiological conditions, to the synthesis of ATP. The con- comes excited and promotes an electron to an outer orbital ventional Hill reaction (25, 26) now appeared to measure a with a higher energy level. This high-energy electron is then noncoupled electron transport, severed from its normally transferred to an adjacent electron acceptor molecule, a coupled phosphorylation (63). catalyst (A) with a strongly electronegative oxidation-reduc- In extending the electron flow concept to this new reaction, tion potential. The transfer of an electron from excited chloro- it was envisaged (58, 59) that a chlorophyll molecule excited phyll to this first acceptor is the energy conversion step proper by a captured photon transfers an electron to NADP (or and terminates the photochemical phase of the process. By to ferricyanide). It was postulated that electrons thus re- transforming a flow of photons into a flow of electrons, it moved from chlorophyll are replaced by electrons from water constitutes a mechanism for generating a strongly electronega- (OH-, at pH 7) with a resultant evolution of oxygen. In this tive reductant at the expense of the excitation energy of manner, light would induce an electron flow from OH- to chlorophyll. NADP and a coupled phosphorylation. Because of the uni- Once the strongly electronegative reductant is formed, no directional or noncyclic nature of this electron flow, this further input of energy is needed. Subsequent electron trans- process was named noncyclic photophosphorylation (58, 59). fers within the chloroplast liberate energy, since they constitute an electron flow from the electronegative reductant to elec- Role of ferredoxin tron acceptors (thought to include chloroplast ) Further progress in elucidating the role of light in chloroplast with more electropositive redox potentials (58, 59). Several reactions came from investigations of the mechanism of of the exergonic electron transfer steps, particularly those NADP reduction and of the identity of the catalyst in cyclic involving cytochromes, were thought to be coupled with photophosphorylation by chloroplasts. phosphorylation. At the end of one cycle, the electron origi- Investigations of the mechanism of NADP reduction and nally emitted by the excited chlorophyll molecule returns cyclic photophosphorylation in chloroplasts led to the recogni- to the electron-deficient chlorophyll molecule and the tion of the key role of the -sulfur protein, ferredoxin. The quantum absorption process is repeated. A mechanism of name ferredoxin was introduced in 1962 by Mortenson et al. this kind would account for the observed lack of any oxida- (67) and did not, at first, concern photosynthesis. It referred tion-reduction change in any external electron donor or ac- to a nonheme, iron-containing protein which they isolated ceptor. Because of the envisaged cyclic pathway traversed by from Clostridium pasteurianum, an anaerobic bacterium the emitted electron, the process was named cyclic photo- devoid of chlorophyll and normally living in the soil at a phosphorylation (58, 59). depth to which sunlight does not penetrate. A connection e between ferredoxin and photosynthesis was established, also in 1962, when C. pasteurianum ferredoxin was crystallized ! and found to mediate the photoreduction of NADP by spinach I cytochrome 2 chloroplasts (68). In this reaction, Clostridium ferredoxin different Ihv _p replaced a native chloroplast protein (known by names) that up to then was thought to be peculiar to photo- A cyclic electron flow that is driven by light and that synthetic cells. Its replaceability by the bacterial ferredoxin liberates chemical energy, used for the synthesis of the pyro- and other considerations led to renaming the chloroplast pro- phosphate bonds of ATP, is unique to photosynthetic cells. tein ferredoxin (68). A discussion of the history of ferredoxin, The idea has been discussed elsewhere (58, 59) that cyclic its occurrence and properties, is given elsewhere (69, 70). photophosphorylation may be a primitive manifestation of Ferredoxin is now known to play a key role in photosyn- photosynthetic activity-an activity that is the common thesis-a role that includes (but is not limited to) a catalytic denominator of plant and bacterial photosynthesis. function in both cyclic and noncyclic photophosphorylation. Ferredoxin is an electron carrier protein whose reversible Noncyclic photophosphorylation oxidation-reduction is accompanied by characteristic changes As already alluded to, there was at first no experimental in its absorption spectrum (Fig. 3). From the standpoint of evidence linking photophosphorylation to the photoreduction electron transport, the most notable finding (68) was the of NADP by chloroplasts. In fact, these two photochemical strongly electronegative oxidation-reduction potential of activities of chloroplasts appeared to be antagonistic (62). ferredoxin, close to that of hydrogen gas (Em = -420 mV, It was therefore wholly unexpected when a second type of at pH 7) and about 100 mV more electronegative than that photophosphorylation was discovered in 1957 (ref. 63) that of NADP. Thus, ferredoxin (in its reduced state) emerged provided direct experimental evidence for a coupling between as the strongest chemically defined reductant that is photo- photoreduction of NADP and the synthesis of ATP. Here, in chemically generated by, and is isolable from, chloroplasts. contrast to cyclic photophosphorylation, ATP formation was The existence of stronger reductants in chloroplasts has stoichiometrically coupled with a light-driven transfer of been suggested on the basis of observations (71-73) that Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Light Reactions of Photosynthesis 2887

chloroplasts photoreduce nonphysiological dyes, some of which have polarographically measured oxidation-reduction potentials that are more negative than that of ferredoxin. However, without evidence that such reductants exist in chloroplasts, these suggestions still remain speculative. Re- cent reports (74, 75) of the isolation of a "ferredoxin reducing substance (FRS)" from chloroplasts have not been confirmed (76). The mechanism of NADP reduction by chloroplasts was resolved (77) into (a) a photochemical reduction of ferredoxin followed by two "dark" steps, (b) reoxidation of ferredoxin by ferredoxin-NADP reductase, a chlorop!ast flavoprotein enzyme, isolated in crystalline form (78), and (c) reoxidation wavelength (nm) of the reduced ferredoxin-NADP reductase by NADP. FIG. 3. Absorption spectrum of spinach ferredoxin. Insert Thus, what was formerly called photoreduction of NADP shows changes in absorption at 420 and 463 nm upon photoreduc- turned out to be a photoreduction of ferredoxin, followed by tion (Buchanan and Arnon, ref. 70). electron transfer to the flavin component of ferredoxin- NADP reductase and thence by hydrogen transfer to NADP and noncyclic photophosphorylations. Low concentrations (two reducing equivalents are transferred to NADP+ in the of antimycin A, oligomycin, and other inhibitors which form of a hydride ion, H-). sharply inhibited cyclic photophosphorylation had no effect on noncyclic photophosphorylation (87, 69). By contrast, low Illuminated chloroplasts ferredoxin concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea ferredoxin-NADP reductase (DCMU) and o-phenanthroline, which sharply inhibited non- H- cyclic photophosphorylation, actually stimulated cyclic photophosphorylation (87). NADP There are now several other lines of evidence that point to The role assigned to ferredoxin as the terminal electron ferredoxin as the endogenous catalyst of cyclic photophos- acceptor in the photochemical events that lead to NADP phorylation in chloroplasts: (a) As expected of a true catalyst, reduction was further documented when the photoreduction ferredoxin stimulates cyclic photophosphorylation at low of substrate amounts of ferredoxin was found to be accom- concentrations (1 X 10-4 M), comparable on a molar basis panied by stoichiometric oxygen evolution and ATP forma- to those of the other known catalysts of the process; (b) when tion (79). Earlier formulations of noncyclic photophos- light intensity is restricted, ferredoxin catalyzes ATP forma- phorylation (63, 64) were now further refined to show that tion more effectively than any other catalyst; (c) in adequate the omission of NADP did not affect ATP formation. The light, cyclic photophosphorylation by ferredoxin produces true equation for noncyclic photophosphorylation became: ATP at a rate comparable with the maximum rates of photo- synthesis in vivo (87). 4 Ferredoxin.. + 2ADP + 2Pj + 2H20 Ferredoxin emerged, therefore, as the physiological catalyst 4 Ferredoxinred + 2ATP + 02 + 4H+ (iv) of cyclic and noncyclic photophosphorylation of chloroplasts. Other substances that catalyze cyclic or noncyclic photo- Turning to cyclic photophosphorylation in chloroplasts, phosphorylation in isolated chloroplasts appear to act as an unsolved question was its puzzling dependence on an substitutes for . It is noteworthy that recent evi- added catalyst, e.g., menadione (80) or phenazine metho- dence (Shanmugam and Arnon, Biochim. Biophys. Acta, in (81), a substance that is foreign to living cells. No press) suggests that cyclic photophosphorylation in bacteria] such additions were required for cyclic photophosphorylation chromatophores may also be catalyzed by a ferredoxin of a in freshly prepared bacterial chromatophores (82, 83). More- kind that is membrane-bound. over, unlike bacterial cyclic photophosphorylation, the process To recapitulate, cyclic and noncyclic photophosphorylation in chloroplasts was not sensitive to such characteristic inhibi- account for the basic feature of photosynthesis, i.e., conver- tors of phosphorylation as antimycin A, at low concentra- sion of light energy into chemical energy. Noncyclic photo- tions (84). phosphorylation generates part of the needed ATP and all A possible explanation of this dependence was that chloro- of the reductant in the form of reduced ferredoxin, which in plasts lose a soluble constituent like ferredoxin in the process turn serves as the electron donor for the reduction of NADP of chloroplast isolation. Evidence was indeed obtained later by an enzymic reaction that is independent of light. Cyclic for cyclic photophosphorylation that is dependent only on photophosphorylation provides the remainder of the required catalytic amounts of ferredoxin and proceeds without the ATP (literature reviewed in ref. 88). Jointly, cyclic and non- addition of any other catalyst of photophosphorylation (85, cyclic photophosphorylation generate all of the assimilatory 86). Moreover, when catalyzed by ferredoxin, cyclic photo- power, made up of ATP and reduced NADP, that is required phosphorylation in chloroplasts became for the first time sensi- for CO2 assimilation. tive to inhibition by low concentrations of antimycin A and oligomycin (69), and resembled in this respect cyclic photo- Two photosystems in plant photosynthesis phosphorylation in bacteria (84). Parallel and, in the main, unrelated to investigations of cyclic Sensitivity to antimycin A and to other inhibitors provided and noncyclic photophosphorylation in isolated chloroplasts also a sharp distinction between ferredoxin-catalyzed cyclic were investigations, chiefly at the cellular level, on the effects Downloaded by guest on September 26, 2021 2888 Amnon Proc. Nat. Acad. Sci. USA 68 (1971)

1965 (ref. 92)-that the two photosystems must operate in PPS series and, through such collaboration, bring about the light-induced reduction of ferredoxin-NADP by water. A 80" detailed discussion of the relative merits of each concept is 60 not possible within the space allotted to this survey. The aim here will be to cover in broad outline some recent findings CX 40- pertaining to electron transport in chloroplasts and the in- terpretation our laboratory placed on them. It may be perti- nent to note that different laboratories are already in con- siderable agreement about some of the new facts even when they differ about interpretations. 20-I664nm 714nm 664 nm 664nm 714 nmr lr4nm NONCYCLIC CYCLIC MODIFIED Three light reactions PSP PSP CYCLIC PSP (DPIPH7-NADP) Until recently there was general agreement that System II included only one short-wavelength light reaction. But recent FIG. 4. Photophosphorylation (PSP) relative to equal light experiments in our laboratory on electron transport in isolated absorption at 664 and 714 nm. Effectiveness of red (664 nm) chloroplasts have yielded evidence (96-104) for two short- and non- far-red (714 nm) monochromatic light for cyclic and wavelength photoreactions (Ila and Ilb) in the noncyclic cyclic photophosphorylation, expressed on the basis of equal light absorption at each wavelength (Arnon et al., ref. 102). electron transport from water to ferredoxin-NADP. These DPIPH2, reduced 2,6-dichlorophenol indophenol. two photoreactions of System II are linked in series; together with the parallel single photoreaction of System I they form, of monochromatic light on plant photosynthesis. This work in our view, the three light reactions of plant photosynthesis has led to wide agreement (see reviews, 89, 90) that photo- (Fig. 5). synthesis in green plants includes two photosystems, one in- According to this concept, in System 11 (Fig. 5, left) volving light reactions that proceed best in "short" wave- photoreaction Ilb oxidizes water and reduces Component length (X < 700 nm) light (System II) and another-known 550 (C550), while photoreaction Ila oxidizes as System I-that proceeds best in "long" wavelength (X > (PC) and reduces ferredoxin. These two light reactions are 700 nm) light. It became important, therefore, to determine joined by a "dark" electron transfer chain which includes the relation of cyclic and noncyclic photophosphorylation to (but is not limited to) and is coupled to the these two photosystems. noncyclic phosphorylation site. Soon after ferredoxin-catalyzed cyclic photophosphoryla- Component 550 is a newly discovered photoreactive chloro- tion was discovered, it was found to proceed most effectively plast component, distinct from cytochromes, which undergoes in long-wavelength light associated with System I. By con- photoreduction by electrons from water (or a substitute trast, noncyclic photophosphorylation (and oxygen evolu- electron donor) only when chloroplasts are illuminated by tion) was found to proceed best in short-wavelength light and System II (short-wavelength) light (96-98). The photoreduc- to come to an almost complete halt at wavelengths above 700 tion of Component 550 is measured by a decrease in absor- nm (87). Expressed on the basis of equal absorption by chloro- bance at 550 nm (546 nm at 770K) but its chemical nature is plasts of light at each wavelength, the sharp decline or "red otherwise still unknown. The existence of Component 550 drop" of noncyclic photophosphorylation in far-red light has now been confirmed by Erixon and Butler (105, 106), (714 nm) was in marked contrast to the sharp increase or Boardman et al. (107), and Bendall and Sofrova (108). Erixon "red rise" of cyclic photophosphorylation at the longer wave- and Butler (106) have also added the significant observation lengths (Fig. 4). Thus, on the basis of their response to that Component 550 acts as the previously postulated monochromatic light, noncyclic photophosphorylation was quencher (Q) of fluorescence of System II. a identified with System II and cyclic photophosphorylation Plastocyanin is copper-containing protein in chloroplasts Katoh and in with System I. discovered by (109) implicated noncyclic Fig. 4 also shows that the wavelength dependence of the electron transport from water to NADP (110-113). Cyto- phosphorylation associated with electron flow from an arti- chrome b559 is one of the three cytochromes native to chloro- ficial electron donor (reduced 2,6-dichlorophenol indo- plasts, the other two being cytochromes f and bN. Cytochrome phenol) to NADP (91) resembles the cyclic system. This b559 has been associated with chloroplast fragments enriched similarity suggests that electron transport involved in the in System II (refs. 114-116). Some investigators (117-119) photoreduction of NADP by DPIPH2 proceeds via (a portion have proposed that it serves as an electron donor to cyto- of) System I and is not involved in the photoreduction of chrome f in an that joins Systems NADP by water via System II (92, 93). In this view (further II and I. In this formulation, plastocyanin is a requlirement elaborated below), ferredoxin-NADP could be reduced either for the photooxidation by System I of both cytochromes by water via System II or by an artificial electron donor via b559 andf: System I. System I identified with cyclic, and System II H20-*- hvip,, Q cyt. b559 - cyt.f->PC identified with noncyclic, electron transport could thus be hvi Fd-NADP regarded as parallel processes in chloroplasts, each basically capable of proceeding independently of the other (92, 93). Our recent experiments show that removal of plastocyanin The idea that Systems I and II operate in parallel runs from chloroplasts abolished their capacity to photooxidize counter to a still widely held concept (90, 94)-one that our cytochrome b599 but not that of cytochrome f. The addition own laboratory embraced in 1961 (ref. 95) and abandoned in of plastocyanin restored the photooxidation of cytochrome Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Light Reactions of Photosynthesis 2889

-0.4 - NADP ' -0.4

-0.2 -0.2

0 0 en

0 0 +0.2 -0 +0.2 w w

+0.4

+0.6 +0.6

+0.8L +0.8

fib

SYSTEMII SYSTEM I (noncycl c) (cyclic)

FIG 5. Scheme for three light reactions in plant photosynthesis. Explanation in text; fp stands for ferredoxin-NADP reductase. Discussed elsewhere (93) are the roles of Cl-,, and (PQ) (Knaff and Arnon, ref. 96).

b559 (ref. 100). In contrast, the removal of plastocyanin had of System I. A primary electron acceptor would be expected to no effect on the photoreduction of cytochrome b559 by Com- undergo reduction by an electron transferred solely as a result ponent 550, nor on the photoreduction of Component 550 of photon capture at temperatures low enough to inhibit itself by photoreaction IIb (97). These findings, buttressed chemical reactions. This expectation was fulfilled for photo- by the observation (99, 96, 100) that cytochrome b5i9, unlike reaction IIb when the photoreduction of Component 550 was cytochrome f, is photooxidized effectively only by System II indeed found to occur at liquid-nitrogen temperature (Fig. 6). (short-wavelength) light, account for the proposed sequence Evidence for the photoreduction of ferredoxin at 770K was of electron carriers in System II (Fig. 5). sought by electron paramagnetic resonance spectroscopy-a Cytochromes b6 and f have previously been assigned to technique that, unlike absorption spectrophotometry, would cyclic photophosphorylation (92, 93) and are now also con- permit detection of ferredoxin reduction without the inter- sidered components of System I. The present concept places ference of chlorophyll or chloroplast cytochromes. When whole cytochrome f and P700 in System I and, in contrast to the spinach chloroplasts were illuminated at 770K from 10 to 50 older hypothesis, predicts that neither would be required for the photoreduction of ferredoxin-NADP by water via System II. [P700 represents a small component part of the total chlorophyll a and has in situ an absorption peak at 700 nm; P700 is considered to act as the terminal trap for the light s ASPINA H energy absorbed by the "bulk" chlorophyll of System I and to participate directly in photochemical reactions (120). ] -2- ~~548- Experimental verification of this prediction was sought 540 55056 from experiments with chloroplast fragments (101, 104). The concept of three photoreactions arranged in two parallel

photosystems was greatly strengthened when chloroplasts 0 Ul \ / M were subdivided into separate fragments with properties and activities corresponding to those ascribed here to System II and System I, respectively (101, 104). An especially perti- nent demonstration was the photoreduction of ferredoxin- NADP by water by the use of chloroplast fragments devoid of System I activity and free of functional P700 and cytochrome f. The isolation of such chloroplast fragments of System II is conceivable according to the new hypothesis but theoretically K 546 LETTUCE impossible according to the old hypothesis. 546 540 550 560 Photoreduction of primary electron acceptors at 770K wave/ength (nm) The new scheme for electron transport envisages two primary FIG. 6. Light-induced absorbance changes of Component 550 electron acceptors: Component 550 in photoreaction lIb and in the region 540-560 nm at - 1890C (5 mM FeCy; reference ferredoxin in photoreaction Ila and in the single photoreaction wavelength, 538 nm, Knaff and Arnon, ref. 98). Downloaded by guest on September 26, 2021 2890 Amnon Proc. Nat. Acad. Sci. USA 68 (1971)

241 System II System I I24 (H20-NADP) (DPIPH2-NADP) 8

k.) ,6 S0 0) k 4 3 w v 1 --"" 19~~~ 2

550 600 650 700 730 550 600 650 700 730 wavelength (nm) FIG. 8. Quantum requirements of light-induced electron transport by System II (H20 NADP) and System I (DPIPH2 - NADP) in monochromatic light of different wavelengths. 3200 3300 34 (Data of B. D. McSwain.) Magnetic field (gauss) FIG. 7. Low-temperature light-induced EPR signals in spinach one photoact in System I, its light-induced electron transport chloroplasts. Illumination: 770K; EPR: 250K (Malkin and would have a theoretical quantum requirement of one quan- Bearden, ref. 121). tum per electron. Fig. 8 shows experimental measurements close to two quanta min and examined by EPR spectroscopy at 250K, light-in- per electron for System II and one quantum per electron for duced changes in the EPR spectrum were observed (121). System I. The quantum requirement measurements in Fig. 8 Fig. 7 shows the EPR spectrum of chloroplasts in the dark demonstrate again the contrasting responses of Systems II and and when illuminated for 20 min at 770K. Illumination in- I to monochromatic light: increasing quantum efficiency for duced increased EPR absorption at g-values of 1.86, 1.94, and System II and decreasing quantum efficiency for System I at 2.05. No other light-induced absorptions were detected when the shorter wavelengths and a reverse situation at the longer the scanning range was widened to include g-values from 1.5 wavelengths. Similar results, despite many variations in condi- to 6. The large (off-scale) signal-in the g = 2.00 region, which tions and experimental design, have been obtained by other occurred in both the light and dark samples, was due to free investigators (123-129). radicals (not associated with ferredoxin) previously observed Since the assimilation of one molecule of CO2 to the level of in photosynthetic systems by the EPR technique (122). hexose via the reductive pentose phosphate cycle requires two It appears that the light-induced EPR spectrum of chloro- molecules of NADPH2 and three molecules of ATP (Fig. 2), it plasts was produced by a bound type of plant ferredoxin differ- becomes possible to arrive at the maximum theoretical quan- ent from the soluble ferredoxin contained in whole chloroplasts. tum efficiency of photosynthesis from measurements of the Illumination at 770K of broken chloroplasts, prepared by respective quantum efficiencies for cyclic and noncyclic elec- osmotically disrupting whole chloroplasts and washing to re- tron transport. A requirement of two quanta per electron for move any remaining soluble ferredoxin, gave an EPR spectrum System II means that eight quanta would be required to similar to that observed with whole chloroplasts (121). The produce two NADPH2 and two ATP (accompanied by evolu- absence of soluble ferredoxin in this chloroplast preparation tion of one 02) via noncyclic photophosphorylation. Two addi- was evidenced by its inability (without added ferredoxin) to tional quanta assigned to generate one ATP via cyclic photo- reduce NADP photochemically with either water or reduced phosphorylation would give a total requirement of about ten dye as the hydrogen donor. quanta per molecule of CO2 assimilated or 02 liberated-a value in good agreement with measurements of the overall Quantum efficiency of photosynthesis quantum efficiency of photosynthesis in intact cells. Few questions in photosynthesis have received more intensive The experimental work from the author's laboratory cited theoretical and experimental study and led to more contro- herein was supported, in part, by National Science Foundation versy than the efficiency of the quantum conversion process. Grant GB-23796. The extensive literature on this subject, which is beyond the 1. Ingenhousz, J., Experiments Upon Vegetables (Elmsly and scope of this article, concerns quantum efficiency of complete Payne, London, 1779). photosynthesis in whole cells, as measured by oxygen evolu- 2. Priestley, J., Phil. Trans. Roy. Soc. London, 62, 147 (1772). tion or CO2 fixation. A different approach to the 3. Ingenhousz, J., Essay on the Food of Plants and the Reno- of photosynthesis emerged from the electron flow theory that vation of Soils (Appendix to 15th Chapter of General Re- port from Board of was invoked to explain cyclic and Agriculture, London, 1796). noncyclic photophosphoryla- 4. von Baeyer, A., Ber. Deut. Chem. Ges., 3, 63 (1864). tion. Transfer of one electron from excited chlorophyll to a 5. WillsttAter, R., and A. Stoll, Untersuchungen Ober Die primary electron acceptor, Component 550 or ferredoxin, is Assimilation der Kohlensdure (Springer, Berlin, 1918). depicted as a one-quantum photochemical act. Since two 6. Warburg, 0., G. Krippahl, and A. Lehmann, Amer. J. Bot., photoacts are involved in transfer of an electron from water 56, 961 (1969). 7. de Saussure, T., Recherches Chimiques Sur La Vegetation to ferredoxin-NADP (Fig. 5), the theoretical quantum re- (V. Nyon, Paris, 1804). quirement for System II becomes two quanta per electron or 8. van Niel, C. B., Arch. Mikrobiol. Z., 3, 1 (1931). eight quanta per molecule of 02 (Eq. iv). Likewise, with only 9. van Niel, C. B., Advan. Enzymol., 1, 263 (1941). Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Light Reactions of Photosynthesis 2891

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