light reactions ofphotosynthesis - pnas.org · came very appropriately from jan ingenhousz, who...

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

The Light Reactions of Photosynthesis

DANIEL I. ARNON

Department of Cell Physiology, University of California, Berkeley, Berkeley, Calif. 94720

ABSTRACT Historically, the role of light in photo-svnthesis has been ascribed either to a photolysis of carbondioxide or to a photolysis of water and a resultant rear-rangement of constituent atoms into molecules of oxy-gen and glucose (or formaldehyde). The discovery ofphoto-phosphorylation demonstrated that photosynthesis in-cludes a light-induced phosphorus metabolism thatprecedes, and is independent from, a photolysis of wateror CO2. ATP formation could best be accounted for notby a photolytic disruption of the covalent bonds in C02or water but by the operation of a light-induced electronflow that results in a release of free energy which is trappedin the pyrophosphate bonds of ATP.Photophosphorylation is now divided into (a) a non-

cyclic type, in which the formation ofATP is coupled witha light-induced electron transport from water to ferredoxinand a concomitant evolution of oxygen and (b) a cyclictype which yields only ATP and produces no net change inthe oxidation-reduction state of any electron donor oracceptor. Reduced ferredoxin formed in (a) serves as anelectron donor for the reduction of NADP by an enzymicreaction that is independent of light. ATP, from bothcyclic and noncyclic photophosphorylation, and reducedNADP jointly constitute the assimilatory power for theconversion of C02 to carbohydrates (3 moles of ATP and2 moles of reduced NADP are required per mole of C02).

Investigations, mainly with whole cells, have shown thatphotosynthesis in green plants involves two photosystems,one (System II) that best uses light of "short" wavelength(X < 685 nm) and another (System I) that best useslight of "long" wavelength (X > 685 nm). Cyclic photo-phosphorylation in chloroplasts involves a System Iphotoreaction. Noncyclic photophosphorylation is widelyheld to involve a collaboration of two photoreactions:a short-wavelength photoreaction belonging to System IIand a long-wavelength photoreaction belonging to SystemI. Recent findings, however, indicate that noncyclic photo-phosphorylation may include two short-wavelength, Sys-tem II, photoreactions that operate in series and are joinedby a "dark" electron-transport chain to which is coupleda phosphorylation site.

Early concepts

The first hypothesis about the role of light in photosynthesiscame very appropriately from Jan Ingenhousz, who someyears earlier had made the epochal discovery that it is "theinfluence of the light of the sun upon the plant" (1) that isresponsible for the "restorative" effect of vegetation on"bad" air-an observation first made in 1771 by JosephPriestley without reference to light (2). In 1796, Ingenhouszwrote that the green plant absorbs from "carbonic acid inthe sunshine, the carbon, throwing out at that time the oxygenalone, and keeping the carbon to itself as nourishment" (3).The idea that light liberates oxygen by photodecomposing

C02 had, with some modifications, persisted for well over acentury. It seemed to have had a special attraction for someof the most illustrious chemists in their day, e.g., von Baeyer

2883

(4) and Willsttfiter (5); its last great contemporary protago-nist was Otto Warburg (6). After de Saussure (7) showed thatwater is a reactant in photosynthesis, the C02 cleavage hy-pothesis readily accounted for the deceptively simple overallphotosynthesis equation (Eq. i): the C: 2H: 0 proportionsin the carbohydrate product fitted the idea that the carbonfrom the photodecomposition of C02 recombines with theelements of water.

h 0 2C02 + H20 (CH20) + 02 (i)

A different hypothesis,, one that profoundly influenced re-search in photosynthesis, was put forward by van Niel (8).After elucidating the nature of bacterial photosynthesis, heproposed (8) that bacterial and plant photosynthesis arespecial cases of a general process in which light energy is usedto photodecompose a hydrogen donor, H2A, with the releasedhydrogen in turn reducing C02 by dark, enzymic reactions:

hpC02 + 2H2A -- (CH20) + H20 + 2A (ii)

The hypothesis envisaged that in plant photosynthesisH2A is water, whereas in green sulfur bacteria (for example)H2A is H2S, with the results that oxygen becomes the by-product of plant photosynthesis and elemental sulfur theby-product of bacterial photosynthesis.In later formulations (9, 10) van Niel no longer considered

the photodecomposition of water as being unique to plantphotosynthesis but postulated that "the photochemical reac-tion in the photosynthetic process of green bacteria, purplebacteria, and green plants represents, in all cases, a photo-decomposition of water" (10). According to this concept, thedistinction between plant and bacterial photosynthesisturned on the events that followed the photodecompositionof water into H and OH. H was used for C02 reduction andOH formed a complex with an appropriate acceptor. In plantphotosynthesis, the acceptor was regenerated when the com-plex was decomposed by liberating molecular oxygen. Inbacterial photosynthesis, oxygen was not liberated and theacceptor could be regenerated only when the OH-acceptorcomplex was reduced by the special hydrogen donor, H2A,that is always required in bacterial photosynthesis.The concept of photodecomposition of C02 or photodecom-

position of water provided, each in its own historicalperiod, a broad, general perspective on the role of light in theoverall events of photosynthesis. In the last two decades,however, the focus in photosynthesis research shifted towardthe isolation, identification, and characterization of thespecific reactions and mechanisms by which light energydrives the photosynthetic process. This approach has led tonew perspectives on the mechanisms by which light energy is

Proc. Nat. Acad. Sci. USA 68 (1971)

EXPERIMENTALLY IDENTIFIED FIRSTPRODUCTS OF PHOTOSYNTHESIS

I1862: STARCHWHL11883: SUCROSE, GLUCOSE CELLS

1951: NADPH21954: AT P1957: NADPH2 +ATP CHLOROPLASTS1962: Fd red1964: Fd red + AT P

FIG. 1.

used in photosynthesis and to a finding, inadmissible undereither of the two earlier hypotheses, that the photosyntheticapparatus can convert light energy into a stable form ofchemical energy, independently of the splitting of either wateror C02.

First products of photosynthesis:experiments with whole cells

In chemical terms, an insight into the role of light in photo-synthesis could come from identification of the first chemicallydefined products that are formed under the influence of light.In the 19th century (Fig. 1) this approach established thatstarch is the first product of photosynthesis in chloroplasts(11)-a conclusion that was later revised in favor of solublecarbohydrates (ref. 12). In the modern period, the powerfulnew techniques of 14C (ref. 13), paper chromatography (14),and radioautography (15) aided in the identification of phos-phoglyceric acid (PGA) as the first stable product of photo-synthesis, formed only after a few seconds of illumination(16). Aside from PGA, phosphate esters of two sugars, ribuloseand sedoheptulose, were soon added to the list of early prod-ucts of photosynthesis in green cells (17, 18).The discovery of PGA and other early intermediates of

C02 assimilation led Calvin and his associates (19, 20) to theformulation of a photosynthetic carbon cycle (reductivepentose phosphate cycle), which was convincingly identifiedwith the dark phase of photosynthesis. The chief importanceof the carbon cycle to the understanding of the role of light inphotosynthesis lay in revealing which of its component en-zymic reactions require an input of energy-rich chemicalintermediates that must be formed by the light reactions.As summarized in Fig. 2, the carbon cycle shows that the con-version of 1 mole of C02 to the level of hexose phosphate re-quires 3 moles ofATP and 2 moles of reduced NADP. Accord-ingly, the need for light energy in photosynthesis by greenplants could now be traced to those photochemical reactionsthat generate ATP and NADPHE2.The occurrence of phosphorylated compounds among the

early products of C02 assimilation suggested that light-induced phosphorus assimilation may, in fact, precede carbonassimilation but experimental evidence for this conclusionwas lacking in whole cells. When Calvin's group investigatedthe photoassimilation of phosphorus by Scenedesmus cellswith the aid of carrier-free KH232PO4, they found that theshortest exposure to light gave the lowest incorporation of32p into ATP and, conversely, that the highest incorporationof 32p into ATP occurred on short, dark exposure (21). Thefirst compound to be labeled proved to be not the expectedATP but again PGA (21). Other investigations of directphotoassimilation of phosphorus by intact cells also gave

results that were at best suggestive [see review (22)1. Inshort, experiments with whole cells proved, for reasons dis-cussed below, incapable of yielding evidence for an indepen-dent light-induced phosphorus metabolism. Its occurrence inphotosynthesis was discovered not in whole cells but inisolated chloroplasts.

First products of photosynthesis:experiments with isolated chloroplastsChloroplasts were once widely believed to be the site of com-plete photosynthesis but this view was not supported bycritical evidence (23, 24) and was largely abandoned afterHill (25, 26) demonstrated that isolated chloroplasts couldevolve oxygen but could not assimilate C02. [The failure ofisolated chloroplasts to fix C02 was also reported with thesensitive 14CO2 technique (27).] In the oxygen-producing reac-tion, which became known as the Hill reaction, isolatedchloroplasts evolved oxygen only in the presence of artificialoxidants with distinctly positive oxidation-reduction poten-tials, e.g., ferric oxalate, ferricyanide, benzoquinone.The Hill reaction established that the photoproduction of

oxygen by chloroplasts is basically independent of C02 as-similation. This provided strong support for the view that thesource of photosynthetic oxygen is water.* Left in doubt wasthe role of chloroplasts in the energy-storing reactions neededfor C02 assimilation. The photochemical generation by iso-lated chloroplasts of a strong reductant capable of reducingC02 was deemed unlikely on experimental and theoreticalgrounds (26, 28). The first experiments with the sensitive 82ptechnique to test the ability of isolated chloroplasts to formATP, on illumination, also gave negative results (29).A different perspective on the photosynthetic capacity

of isolated chloroplasts began to emerge in 1951 when threelaboratories (30-32), independently and simultaneously,found that isolated chloroplasts could photoreduce NADPdespite its strongly electronegative redox potential (Em =-320 mV, at pH 7). This finding was followed by severalother developments which drastically altered the then preva-lent ideas about the photosynthetic capacity of isolatedchloroplasts. These developments (Fig. 1) will now be dis-cussed in chronological order.

In 1954, a reinvestigation of photosynthesis in isolatedchloroplasts by different methods yielded evidence for alight-dependent assimilation of C02 (ref. 33). Chloroplastsisolated from spinach leaves assimilated 14CO2 to the levelof carbohydrates, including starch, with a simultaneousevolution of oxygen (34, 35). When the conversion of 14CO2by isolated chloroplasts to sugars and starch was confirmedand extended in other laboratories (36-40), the capacity ofchloroplasts to carry on complete extracellular photosyn-thesis was no longer open to question.Because of the earlier negative results, special experimental

safeguards were deemed necessary to establish that chloro-plasts alone, without other organelles or enzyme systems andwith light as the only energy source, were capable of a totalsynthesis of carbohydrates from C02. The chloroplasts werewashed and, to eliminate a possible source of chemical energyand metabolites, their isolation was performed not as formerly* Contrary to a widely held belief, this conclusion was not un-equivocally documented by experiments with 180 [(see A. H.Brown and A. W. Frenkel, Annu. Rev. Plant Physiol., 4, 53(1953)].

2884 Amnon

Light Reactions of Photosynthesis 2885

in isotonic sugar solutions (25) but in isotonic sodium chloride(33, 35). In comparison with the parent leaves, washed salinechloroplasts gave low rates of CO2 assimilation (35)-a situa-tion similar to the first reconstruction of other cellular pro-cesses in vitro, e.g., fermentation (41, 42), protein synthesis(43), and polymerization of DNA (44). Crucial for the docu-mentation of complete photosynthesis in isolated chloroplastswere not high rates but the fact that their newly found CO2assimilation was reproducible and yielded the same inter-mediate and final products as photosynthesis by intact cells.More recently, when CO2 assimilation ceased to be a matter ofdispute and the same experimental safeguards were no longerneeded, much higher rates of CO2 assimilation by isolatedchloroplasts were obtained with modified procedures (45-48).

Since neither ATP nor reduced NADP was added to iso-lated chloroplasts that fixed C02, it was clear that theseenergy-rich compounds were being photochemically generatedfrom their respective precursors within the chloroplasts.Chloroplasts were already known to photoreduce addedNADP but nothing was known about their ability (or that ofany other photosynthetic structures) to form ATP at theexpense of light energy. A renewed attack on this problem inisolated chloroplasts was therefore undertaken.

Discovery of photosynthetic phosphorylationThe likelihood of detecting a direct role of light in ATP forma-tion was much greater in isolated chloroplasts than in intactcells. Intact cells contain only catalytic amounts of the pre-cursor adenosine phosphates (AMP, ADP) and these, becauseof permeability barriers, could not be increased by externaladditions. By contrast, in experiments with isolated chloro-plasts, it was possible to supply these normally catalyticsubstances in substrate amounts and, with the aid of labeledinorganic phosphate, determine chemically their light-in-duced conversion to ATP.

In 1954, work with the same spinach chloroplast prepara-tions that fixed CO2 led to the discovery that they were alsoable to convert light energy into chemical energy and trapit in the pyrophosphate bonds of ATP (49, 33). Severalunique features distinguished this photosynthetic phos-phorylation (photophosphorylation), as the process wasnamed, from substrate-level phosphorylation in fermentationand oxidative phosphorylation in respiration: (a) ATP forma-tion occurred in the chlorophyll-containing lamellae andwas independent of other enzyme systems or organelles(including mitochondria, which were previously considerednecessary for photosynthetic ATP formation, ref. 51); (b)no energy-rich substrate, other than absorbed photons, servedas a source of energy; (c) no oxygen was produced or con-sumed; (d) ATP formation was not accompanied by a mea-surable electron transport involving any external electrondonor or acceptor (49, 33, 50). The light-induced ATP forma-tion could be expressed by the equation:

hpn*ADP+ n -Pi n*ATP (iii)

When photophosphorylation in chloroplasts was followedby evidence of a similar phenomenon in cell-free preparationsof such diverse types of photosynthetic organisms as photo-synthetic bacteria (52) and algae (53, 54), it became evidentthat photophosphorylation is not peculiar to plants containingchloroplasts but is a major ATP-forming process in nature

DIPHOSPHATE PHOSPHATES 7 PHOSPHATE 7 STARCH

ATP J INTERMEDIATES

RIL@UOSEMONOPHOSPHATE

FIG. 2. Schematic representation of the ATP and reducedNADP requirements {or CO2 assimilation.

that supplies ATP for the biosynthetic reactions in all typesof photosynthesis.Soon after the demonstration of photosynthetic phos-

phorylation in isolated chloroplasts, attempts were made toevaluate its physiological significance. Since, as with othercellular processes when first reproduced in vitro, the rates ofphotosynthetic phosphorylation were low, there was littleinclination at first to accord this process quantitative im-portance as a photosynthetic mechanism for converting lightinto chemical energy (55). With further improvement inexperimental methods (which included the use of brokenchloroplasts with lowered permeability barriers), rates of pho-tosynthetic phosphorylation increased 170 times (56) and more(57) over those originally described (33).The improved rates of photosynthetic phosphorylation

were equal to, or greater than, the maximum known rates ofcarbon assimilation in intact leaves. It appeared, therefore,that isolated chloroplasts retain, without substantial loss,the enzymic apparatus for photosynthetic phosphorylation-a conclusion in harmony with evidence that the phosphoryl-ating system was tightly bound in the water-insoluble lamellarportion of the chloroplasts.

Role of light in photophosphorylation

Once the main features of photophosphorylation were firmlyestablished, the next objective was to explain its mechanism,particularly its absolute dependence on illumination (33, 50).On the one hand, photophosphorylation was independent ofsuch classical manifestations of photosynthesis as oxygenevolution and CO2 assimilation; on the other hand, it seemedunlikely that light was involved in the formation ofATP itself,a reaction universally occurring in all cells independently ofphotosynthesis. Light energy, therefore, had to be used inphotophosphorylation before ATP synthesis and in a mannerunrelated to CO2 assimilation or oxygen evolution. The mostprobable mechanism for such a role seemed to be a light-induced electron flow (58, 59).

It is often difficult for the student of photosynthesis todayto realize that before the discovery of photophosphorylationthe concept of a light-induced electron transport had no sub-stantial basis in photosynthesis research. The idea that photonenergy is used in photosynthesis to transfer electrons ratherthan cumbersome atoms had a few proponents at varioustimes, for example Katz (60) and Levitt (61) but, as the litera-ture before the late 1950s shows, it did not become a viableconcept in photosynthesis-it was merely one of several specu-


RNKJILOSF TrI(c


lative ideas based on model systems. The situation changedwith the discovery of light-induced ATP formation. ATPis formed in nonphotosynthetic cells at the expense of energyreleased by electron transport. The idea that ATP may alsobe formed in photosynthesis through a special light-inducedelectron flow mechanism in chloroplasts now had a high proba-bility that could be experimentally tested.The electron flow hypothesis (58, 59) envisaged that a

chlorophyll molecule, on absorbing a quantum of light, be-comes excited and promotes an electron to an outer orbitalwith a higher energy level. This high-energy electron is thentransferred to an adjacent electron acceptor molecule, acatalyst (A) with a strongly electronegative oxidation-reduc-tion potential. The transfer of an electron from excited chloro-phyll to this first acceptor is the energy conversion step properand terminates the photochemical phase of the process. Bytransforming a flow of photons into a flow of electrons, itconstitutes a mechanism for generating a strongly electronega-tive reductant at the expense of the excitation energy ofchlorophyll.Once the strongly electronegative reductant is formed, no

further input of energy is needed. Subsequent electron trans-fers within the chloroplast liberate energy, since they constitutean electron flow from the electronegative reductant to elec-tron acceptors (thought to include chloroplast cytochromes)with more electropositive redox potentials (58, 59). Severalof the exergonic electron transfer steps, particularly thoseinvolving cytochromes, were thought to be coupled withphosphorylation. At the end of one cycle, the electron origi-nally emitted by the excited chlorophyll molecule returnsto the electron-deficient chlorophyll molecule and thequantum absorption process is repeated. A mechanism ofthis kind would account for the observed lack of any oxida-tion-reduction change in any external electron donor or ac-ceptor. Because of the envisaged cyclic pathway traversed bythe emitted electron, the process was named cyclic photo-phosphorylation (58, 59).

e

chlorophyll A ! cytochrome I cytochrome 2

Ihv _p

A cyclic electron flow that is driven by light and thatliberates chemical energy, used for the synthesis of the pyro-phosphate bonds of ATP, is unique to photosynthetic cells.The idea has been discussed elsewhere (58, 59) that cyclicphotophosphorylation may be a primitive manifestation ofphotosynthetic activity-an activity that is the commondenominator of plant and bacterial photosynthesis.Noncyclic photophosphorylationAs already alluded to, there was at first no experimentalevidence linking photophosphorylation to the photoreductionof NADP by chloroplasts. In fact, these two photochemicalactivities of chloroplasts appeared to be antagonistic (62).It was therefore wholly unexpected when a second type ofphotophosphorylation was discovered in 1957 (ref. 63) thatprovided direct experimental evidence for a coupling betweenphotoreduction of NADP and the synthesis of ATP. Here, incontrast to cyclic photophosphorylation, ATP formation was

electrons from water to NADP (or to a nonphysiologicalelectron acceptor such as ferricyanide) and a concomitantevolution of oxygen. Moreover, ATP formation in this coupledsystem greatly increased the rate of electron transfer fromwater to ferricyanide (63-65) or to NADP (66) and the rateof the concomitant oxygen evolution. It thus became apparentthat the electron transport system of chloroplasts functionsmore effectively when it is coupled, as it would be underphysiological conditions, to the synthesis of ATP. The con-

ventional Hill reaction (25, 26) now appeared to measure a

noncoupled electron transport, severed from its normallycoupled phosphorylation (63).

In extending the electron flow concept to this new reaction,it was envisaged (58, 59) that a chlorophyll molecule excitedby a captured photon transfers an electron to NADP (orto ferricyanide). It was postulated that electrons thus re-

moved from chlorophyll are replaced by electrons from water(OH-, at pH 7) with a resultant evolution of oxygen. In thismanner, light would induce an electron flow from OH- toNADP and a coupled phosphorylation. Because of the uni-directional or noncyclic nature of this electron flow, thisprocess was named noncyclic photophosphorylation (58, 59).

Role of ferredoxin

Further progress in elucidating the role of light in chloroplastreactions came from investigations of the mechanism ofNADP reduction and of the identity of the catalyst in cyclicphotophosphorylation by chloroplasts.

Investigations of the mechanism of NADP reduction andcyclic photophosphorylation in chloroplasts led to the recogni-tion of the key role of the iron-sulfur protein, ferredoxin. Thename ferredoxin was introduced in 1962 by Mortenson et al.(67) and did not, at first, concern photosynthesis. It referredto a nonheme, iron-containing protein which they isolatedfrom Clostridium pasteurianum, an anaerobic bacteriumdevoid of chlorophyll and normally living in the soil at a

depth to which sunlight does not penetrate. A connectionbetween ferredoxin and photosynthesis was established, alsoin 1962, when C. pasteurianum ferredoxin was crystallizedand found to mediate the photoreduction ofNADP by spinachchloroplasts (68). In this reaction, Clostridium ferredoxinreplaced a native chloroplast protein (known by differentnames) that up to then was thought to be peculiar to photo-synthetic cells. Its replaceability by the bacterial ferredoxinand other considerations led to renaming the chloroplast pro-tein ferredoxin (68). A discussion of the history of ferredoxin,its occurrence and properties, is given elsewhere (69, 70).

Ferredoxin is now known to play a key role in photosyn-thesis-a role that includes (but is not limited to) a catalyticfunction in both cyclic and noncyclic photophosphorylation.Ferredoxin is an electron carrier protein whose reversibleoxidation-reduction is accompanied by characteristic changesin its absorption spectrum (Fig. 3). From the standpoint ofelectron transport, the most notable finding (68) was thestrongly electronegative oxidation-reduction potential offerredoxin, close to that of hydrogen gas (Em = -420 mV,at pH 7) and about 100 mV more electronegative than thatof NADP. Thus, ferredoxin (in its reduced state) emergedas the strongest chemically defined reductant that is photo-chemically generated by, and is isolable from, chloroplasts.The existence of stronger reductants in chloroplasts has

stoichiometrically coupled with a light-driven transfer of

2886 Amnon

been suggested on the basis of observations (71-73) that


chloroplasts photoreduce nonphysiological dyes, some ofwhich have polarographically measured oxidation-reductionpotentials that are more negative than that of ferredoxin.However, without evidence that such reductants exist inchloroplasts, these suggestions still remain speculative. Re-cent reports (74, 75) of the isolation of a "ferredoxin reducingsubstance (FRS)" from chloroplasts have not been confirmed(76).The mechanism of NADP reduction by chloroplasts was

resolved (77) into (a) a photochemical reduction of ferredoxinfollowed by two "dark" steps, (b) reoxidation of ferredoxinby ferredoxin-NADP reductase, a chlorop!ast flavoproteinenzyme, isolated in crystalline form (78), and (c) reoxidationof the reduced ferredoxin-NADP reductase by NADP.Thus, what was formerly called photoreduction of NADPturned out to be a photoreduction of ferredoxin, followed byelectron transfer to the flavin component of ferredoxin-NADP reductase and thence by hydrogen transfer to NADP(two reducing equivalents are transferred to NADP+ in theform of a hydride ion, H-).

Illuminated chloroplasts ferredoxinferredoxin-NADP reductase

H-

NADP

The role assigned to ferredoxin as the terminal electronacceptor in the photochemical events that lead to NADPreduction was further documented when the photoreductionof substrate amounts of ferredoxin was found to be accom-panied by stoichiometric oxygen evolution and ATP forma-tion (79). Earlier formulations of noncyclic photophos-phorylation (63, 64) were now further refined to show thatthe omission of NADP did not affect ATP formation. Thetrue equation for noncyclic photophosphorylation became:

4 Ferredoxin.. + 2ADP + 2Pj + 2H204 Ferredoxinred + 2ATP + 02 + 4H+ (iv)

Turning to cyclic photophosphorylation in chloroplasts,an unsolved question was its puzzling dependence on anadded catalyst, e.g., menadione (80) or phenazine metho-sulfate (81), a substance that is foreign to living cells. Nosuch additions were required for cyclic photophosphorylationin freshly prepared bacterial chromatophores (82, 83). More-over, unlike bacterial cyclic photophosphorylation, the processin chloroplasts was not sensitive to such characteristic inhibi-tors of phosphorylation as antimycin A, at low concentra-tions (84).A possible explanation of this dependence was that chloro-

plasts lose a soluble constituent like ferredoxin in the processof chloroplast isolation. Evidence was indeed obtained laterfor cyclic photophosphorylation that is dependent only oncatalytic amounts of ferredoxin and proceeds without theaddition of any other catalyst of photophosphorylation (85,86). Moreover, when catalyzed by ferredoxin, cyclic photo-phosphorylation in chloroplasts became for the first time sensi-tive to inhibition by low concentrations of antimycin A andoligomycin (69), and resembled in this respect cyclic photo-phosphorylation in bacteria (84).

Sensitivity to antimycin A and to other inhibitors providedalso a sharp distinction between ferredoxin-catalyzed cyclic

wavelength (nm)

FIG. 3. Absorption spectrum of spinach ferredoxin. Insertshows changes in absorption at 420 and 463 nm upon photoreduc-tion (Buchanan and Arnon, ref. 70).

and noncyclic photophosphorylations. Low concentrationsof antimycin A, oligomycin, and other inhibitors whichsharply inhibited cyclic photophosphorylation had no effecton noncyclic photophosphorylation (87, 69). By contrast, lowconcentrations of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea(DCMU) and o-phenanthroline, which sharply inhibited non-cyclic photophosphorylation, actually stimulated cyclicphotophosphorylation (87).There are now several other lines of evidence that point to

ferredoxin as the endogenous catalyst of cyclic photophos-phorylation in chloroplasts: (a) As expected of a true catalyst,ferredoxin stimulates cyclic photophosphorylation at lowconcentrations (1 X 10-4 M), comparable on a molar basisto those of the other known catalysts of the process; (b) whenlight intensity is restricted, ferredoxin catalyzes ATP forma-tion more effectively than any other catalyst; (c) in adequatelight, cyclic photophosphorylation by ferredoxin producesATP at a rate comparable with the maximum rates of photo-synthesis in vivo (87).

Ferredoxin emerged, therefore, as the physiological catalystof cyclic and noncyclic photophosphorylation of chloroplasts.Other substances that catalyze cyclic or noncyclic photo-phosphorylation in isolated chloroplasts appear to act assubstitutes for ferredoxins. It is noteworthy that recent evi-dence (Shanmugam and Arnon, Biochim. Biophys. Acta, inpress) suggests that cyclic photophosphorylation in bacteria]chromatophores may also be catalyzed by a ferredoxin of akind that is membrane-bound.To recapitulate, cyclic and noncyclic photophosphorylation

account for the basic feature of photosynthesis, i.e., conver-sion of light energy into chemical energy. Noncyclic photo-phosphorylation generates part of the needed ATP and allof the reductant in the form of reduced ferredoxin, which inturn serves as the electron donor for the reduction of NADPby an enzymic reaction that is independent of light. Cyclicphotophosphorylation provides the remainder of the requiredATP (literature reviewed in ref. 88). Jointly, cyclic and non-cyclic photophosphorylation generate all of the assimilatorypower, made up of ATP and reduced NADP, that is requiredfor CO2 assimilation.

Two photosystems in plant photosynthesis

Parallel and, in the main, unrelated to investigations of cyclicand noncyclic photophosphorylation in isolated chloroplastswere investigations, chiefly at the cellular level, on the effects



PPS

80"

60

CX 40-

20-I664nm 714nm 664nm 714 nmr 664nm lr4nm

NONCYCLIC CYCLIC MODIFIED

PSP PSP CYCLIC PSP

(DPIPH7-NADP)

FIG. 4. Photophosphorylation (PSP) relative to equal lightabsorption at 664 and 714 nm. Effectiveness of red (664 nm)and far-red (714 nm) monochromatic light for cyclic and non-

cyclic photophosphorylation, expressed on the basis of equallight absorption at each wavelength (Arnon et al., ref. 102).DPIPH2, reduced 2,6-dichlorophenol indophenol.

of monochromatic light on plant photosynthesis. This workhas led to wide agreement (see reviews, 89, 90) that photo-synthesis in green plants includes two photosystems, one in-volving light reactions that proceed best in "short" wave-

length (X < 700 nm) light (System II) and another-knownas System I-that proceeds best in "long" wavelength (X >700 nm) light. It became important, therefore, to determinethe relation of cyclic and noncyclic photophosphorylation tothese two photosystems.Soon after ferredoxin-catalyzed cyclic photophosphoryla-

tion was discovered, it was found to proceed most effectivelyin long-wavelength light associated with System I. By con-

trast, noncyclic photophosphorylation (and oxygen evolu-tion) was found to proceed best in short-wavelength light andto come to an almost complete halt at wavelengths above 700nm (87). Expressed on the basis of equal absorption by chloro-plasts of light at each wavelength, the sharp decline or "reddrop" of noncyclic photophosphorylation in far-red light(714 nm) was in marked contrast to the sharp increase or

"red rise" of cyclic photophosphorylation at the longer wave-

lengths (Fig. 4). Thus, on the basis of their response tomonochromatic light, noncyclic photophosphorylation was

identified with System II and cyclic photophosphorylationwith System I.

Fig. 4 also shows that the wavelength dependence of thephosphorylation associated with electron flow from an arti-ficial electron donor (reduced 2,6-dichlorophenol indo-phenol) to NADP (91) resembles the cyclic system. Thissimilarity suggests that electron transport involved in thephotoreduction of NADP by DPIPH2 proceeds via (a portionof) System I and is not involved in the photoreduction ofNADP by water via System II (92, 93). In this view (furtherelaborated below), ferredoxin-NADP could be reduced eitherby water via System II or by an artificial electron donor viaSystem I. System I identified with cyclic, and System II

identified with noncyclic, electron transport could thus beregarded as parallel processes in chloroplasts, each basicallycapable of proceeding independently of the other (92, 93).The idea that Systems I and II operate in parallel runs

counter to a still widely held concept (90, 94)-one that our

own laboratory embraced in 1961 (ref. 95) and abandoned in

1965 (ref. 92)-that the two photosystems must operate inseries and, through such collaboration, bring about thelight-induced reduction of ferredoxin-NADP by water. Adetailed discussion of the relative merits of each concept isnot possible within the space allotted to this survey. Theaim here will be to cover in broad outline some recent findingspertaining 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 whenthey differ about interpretations.

Three light reactions

Until recently there was general agreement that System IIincluded only one short-wavelength light reaction. But recentexperiments in our laboratory on electron transport in isolatedchloroplasts have yielded evidence (96-104) for two short-wavelength photoreactions (Ila and Ilb) in the noncyclicelectron transport from water to ferredoxin-NADP. Thesetwo photoreactions of System II are linked in series; togetherwith the parallel single photoreaction of System I they form,in our view, the three light reactions of plant photosynthesis(Fig. 5).According to this concept, in System 11 (Fig. 5, left)

photoreaction Ilb oxidizes water and reduces Component550 (C550), while photoreaction Ila oxidizes plastocyanin(PC) and reduces ferredoxin. These two light reactions arejoined by a "dark" electron transfer chain which includes(but is not limited to) cytochrome b559 and is coupled to thenoncyclic phosphorylation site.Component 550 is a newly discovered photoreactive chloro-

plast component, distinct from cytochromes, which undergoesphotoreduction by electrons from water (or a substituteelectron donor) only when chloroplasts are illuminated bySystem II (short-wavelength) light (96-98). The photoreduc-tion of Component 550 is measured by a decrease in absor-bance at 550 nm (546 nm at 770K) but its chemical nature isotherwise still unknown. The existence of Component 550has now been confirmed by Erixon and Butler (105, 106),Boardman et al. (107), and Bendall and Sofrova (108). Erixonand Butler (106) have also added the significant observationthat Component 550 acts as the previously postulatedquencher (Q) of fluorescence of System II.

Plastocyanin is a copper-containing protein in chloroplastsdiscovered by Katoh (109) and implicated in noncyclicelectron transport from water to NADP (110-113). Cyto-chrome b559 is one of the three cytochromes native to chloro-plasts, the other two being cytochromes f and bN. Cytochromeb559 has been associated with chloroplast fragments enrichedin System II (refs. 114-116). Some investigators (117-119)have proposed that it serves as an electron donor to cyto-chrome f in an electron transport chain that joins SystemsII and I. In this formulation, plastocyanin is a requlirementfor the photooxidation by System I of both cytochromesb559 andf:H20-*- hvip,, Q cyt. b559 - cyt.f->PC

hvi Fd-NADP

Our recent experiments show that removal of plastocyaninfrom chloroplasts abolished their capacity to photooxidizecytochrome b599 but not that of cytochrome f. The additionof plastocyanin restored the photooxidation of cytochrome

2888 Amnon


NADP '

0

en

0

-0

w

+0.2

+0.8L

-0.4

-0.2

0

0

+0.2

w

+0.4

+0.6

+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-,manganese, and plastoquinone (PQ) (Knaff and Arnon, ref. 96).

b559 (ref. 100). In contrast, the removal of plastocyanin hadno effect on the photoreduction of cytochrome b559 by Com-ponent 550, nor on the photoreduction of Component 550itself by photoreaction IIb (97). These findings, buttressedby the observation (99, 96, 100) that cytochrome b5i9, unlikecytochrome f, is photooxidized effectively only by System II(short-wavelength) light, account for the proposed sequenceof electron carriers in System II (Fig. 5).Cytochromes b6 and f have previously been assigned to

cyclic photophosphorylation (92, 93) and are now also con-

sidered components of System I. The present concept placescytochrome f and P700 in System I and, in contrast to theolder hypothesis, predicts that neither would be required forthe photoreduction of ferredoxin-NADP by water via SystemII. [P700 represents a small component part of the totalchlorophyll a and has in situ an absorption peak at 700 nm;P700 is considered to act as the terminal trap for the lightenergy absorbed by the "bulk" chlorophyll of System I andto participate directly in photochemical reactions (120). ]Experimental verification of this prediction was sought

from experiments with chloroplast fragments (101, 104). Theconcept of three photoreactions arranged in two parallelphotosystems was greatly strengthened when chloroplastswere subdivided into separate fragments with propertiesand activities corresponding to those ascribed here to SystemII 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 ofSystem I activity and free of functional P700 and cytochromef. The isolation of such chloroplast fragments of System II isconceivable according to the new hypothesis but theoreticallyimpossible according to the old hypothesis.

Photoreduction of primary electron acceptors at 770K

The new scheme for electron transport envisages two primaryelectron acceptors: Component 550 in photoreaction lIb andferredoxin in photoreaction Ila and in the single photoreaction

of System I. A primary electron acceptor would be expected toundergo reduction by an electron transferred solely as a resultof photon capture at temperatures low enough to inhibitchemical reactions. This expectation was fulfilled for photo-reaction IIb when the photoreduction of Component 550 was

indeed found to occur at liquid-nitrogen temperature (Fig. 6).Evidence for the photoreduction of ferredoxin at 770K was

sought by electron paramagnetic resonance spectroscopy-atechnique that, unlike absorption spectrophotometry, wouldpermit detection of ferredoxin reduction without the inter-ference of chlorophyll or chloroplast cytochromes. When wholespinach chloroplasts were illuminated at 770K from 10 to 50

s ASPINA H

-2- ~~548-

540 55056

Ul 0 \ / M

K 546 LETTUCE546

540 550 560

wave/ength (nm)FIG. 6. Light-induced absorbance changes of Component 550

in the region 540-560 nm at - 1890C (5 mM FeCy; referencewavelength, 538 nm, Knaff and Arnon, ref. 98).


-0.4 -

-0.2

+0.6


241 System II(H20-NADP)

k.)S00)k

FIG. 7. Low-temperature light-induced EPR signals in spinachchloroplasts. Illumination: 770K; EPR: 250K (Malkin andBearden, ref. 121).

min and examined by EPR spectroscopy at 250K, light-in-duced changes in the EPR spectrum were observed (121).Fig. 7 shows the EPR spectrum of chloroplasts in the darkand when illuminated for 20 min at 770K. Illumination in-duced increased EPR absorption at g-values of 1.86, 1.94, and2.05. No other light-induced absorptions were detected whenthe scanning range was widened to include g-values from 1.5to 6. The large (off-scale) signal-in the g = 2.00 region, whichoccurred in both the light and dark samples, was due to freeradicals (not associated with ferredoxin) previously observedin photosynthetic systems by the EPR technique (122).

It appears that the light-induced EPR spectrum of chloro-plasts was produced by a bound type of plant ferredoxin differ-ent from the soluble ferredoxin contained in whole chloroplasts.Illumination at 770K of broken chloroplasts, prepared byosmotically disrupting whole chloroplasts and washing to re-

move any remaining soluble ferredoxin, gave anEPR spectrumsimilar to that observed with whole chloroplasts (121). Theabsence of soluble ferredoxin in this chloroplast preparationwas evidenced by its inability (without added ferredoxin) toreduce NADP photochemically with either water or reduceddye as the hydrogen donor.

Quantum efficiency of photosynthesisFew questions in photosynthesis have received more intensivetheoretical and experimental study and led to more contro-versy than the efficiency of the quantum conversion process.The extensive literature on this subject, which is beyond thescope of this article, concerns quantum efficiency of completephotosynthesis in whole cells, as measured by oxygen evolu-tion or CO2 fixation. A different approach to the bioenergeticsof photosynthesis emerged from the electron flow theory thatwas invoked to explain cyclic and noncyclic photophosphoryla-tion. Transfer of one electron from excited chlorophyll to a

primary electron acceptor, Component 550 or ferredoxin, isdepicted as a one-quantum photochemical act. Since twophotoacts are involved in transfer of an electron from waterto ferredoxin-NADP (Fig. 5), the theoretical quantum re-

quirement for System II becomes two quanta per electron or

eight quanta per molecule of 02 (Eq. iv). Likewise, with only

System I(DPIPH2-NADP)

w v --""1 19~~~

I24

8

,6

4

3

2

550 600 650 700 730 550 600 650 700 730wavelength (nm)

FIG. 8. Quantum requirements of light-induced electrontransport by System II (H20 NADP) and System I (DPIPH2- NADP) in monochromatic light of different wavelengths.(Data of B. D. McSwain.)

one photoact in System I, its light-induced electron transportwould have a theoretical quantum requirement of one quan-tum per electron.

Fig. 8 shows experimental measurements close to two quantaper electron for System II and one quantum per electron forSystem I. The quantum requirement measurements in Fig. 8demonstrate again the contrasting responses of Systems II andI to monochromatic light: increasing quantum efficiency forSystem II and decreasing quantum efficiency for System I atthe shorter wavelengths and a reverse situation at the longerwavelengths. Similar results, despite many variations in condi-tions and experimental design, have been obtained by otherinvestigators (123-129).

Since the assimilation of one molecule of CO2 to the level ofhexose via the reductive pentose phosphate cycle requires twomolecules of NADPH2 and three molecules of ATP (Fig. 2), itbecomes possible to arrive at the maximum theoretical quan-tum efficiency of photosynthesis from measurements of therespective quantum efficiencies for cyclic and noncyclic elec-tron transport. A requirement of two quanta per electron forSystem II means that eight quanta would be required toproduce two NADPH2 and two ATP (accompanied by evolu-tion of one 02) via noncyclic photophosphorylation. Two addi-tional quanta assigned to generate one ATP via cyclic photo-phosphorylation would give a total requirement of about tenquanta per molecule of CO2 assimilated or 02 liberated-avalue in good agreement with measurements of the overallquantum efficiency of photosynthesis in intact cells.

The experimental work from the author's laboratory citedherein was supported, in part, by National Science FoundationGrant GB-23796.

1. Ingenhousz, J., Experiments Upon Vegetables (Elmsly andPayne, London, 1779).

2. Priestley, J., Phil. Trans. Roy. Soc. London, 62, 147 (1772).3. Ingenhousz, J., Essay on the Food of Plants and the Reno-

vation of Soils (Appendix to 15th Chapter of General Re-port from Board of Agriculture, London, 1796).

4. von Baeyer, A., Ber. Deut. Chem. Ges., 3, 63 (1864).5. WillsttAter, R., and A. Stoll, Untersuchungen Ober Die

Assimilation der Kohlensdure (Springer, Berlin, 1918).6. Warburg, 0., G. Krippahl, and A. Lehmann, Amer. J. Bot.,

56, 961 (1969).7. de Saussure, T., Recherches Chimiques Sur La Vegetation

(V. Nyon, Paris, 1804).8. van Niel, C. B., Arch. Mikrobiol. Z., 3, 1 (1931).9. van Niel, C. B., Advan. Enzymol., 1, 263 (1941).

3200 3300 34Magnetic field (gauss)

2890 Amnon


10. van Niel, C. B., Photosynthesis in Plants, ed. J. Franck andW. E. Loomis (Iowa State College Press, Ames, 1949).

11. Sachs, J., Jahrb. Wise Bot., 3, 184 (1863).12. Boehm, J., Bot. Zeitg., 41, 33, 49 (1883).13. Ruben, S., and M. Kamen, Phy8. Rev., 59, 49 (1941).14. Martin, A. J. P., and R. L. M. Synge, Les Prix Nobel en

1952 (Norstedt, Stockholm, 1952).15. Fink, R. M., and K. Fink, Science, 107, 253 (1948).16. Benson, A. A., and M. Calvin, Annu. Rev. Plant Physiol.,

1, 25 (1950).17. Benson, A. A., J. Amer. Chem. Soc., 73, 2971 (1951).18. Benson, A. A., J. A. Bassham, M. Calvin, A. G. Hall,

H. E. Hirsch, S., Kawaguchi, V. Lynch, and N. E. Tolbert,J. Biol. Chem., 196, 703 (1952).

19. Bassham, J. A., A. A. Benson, L. D. Kay, A. Z. Harris,A. T. Wilson, and M. Calvin, J. Amer. Chem. Soc., 76, 1760(1954).

20. Bassham, J. M., and M. Calvin, The Path of Carbon inPhotosynthesis (Prentice-Hall, Engelwood Cliffs, N.J.,1957).

21.- Goodman, M., D. F. Bradley, and M. Calvin, J. Amer.Chem. Soc., 75, 1962 (1953).

22. Arnon, D. I., Annu. Rev. Plant Physiol., 7, 325 (1956).23. Sachs, J., Lectures on the Physiology of Plants (Clarendon

Press, Oxford, 1887).24. Pfeffer, W., Physiology of Plants (Clarendon Press, Ox-

ford, 1900).25. Hill, R., Proc. Roy. Soc. Ser. B, 127, 192 (1939).26. Hill, R., Symp. Soc. Exp. Biol., 5, 223 (1951).27. Brown, A. H., and J. Franck, Arch. Biochem., 16, 55 (1948).28. Wessels, J. S. C., and E. Havinga, Rec. Trav. Chim. Pays-

Bas, 71,7 (1952).29. Aronoff, S., and M. Calvin, Plant Physiol., 23, 351 (1948).30. Vishniac, W., and S. Ochoa, Nature, 167, 768 (1951).31. Tolmach, L. J., Nature, 167, 946 (1951).32. Arnon, D. I., Nature, 167, 1008 (1951).33. Arnon. D. I., M. B. Allen, and F. R. Whatley, Nature, 174,

394 (1954).34. Arnon, D. I., Paper presented at the Cell Symposium,

Amer. Assoc. Advan. Sci., Berkeley Meeting, 1954;Science, 122, 9 (1955).

35. Allen, M. B., D. I. Arnon, J. B. Capindale, F. R. Whatley,and L. J. Durham, J. Amer. Chem. Soc., 77, 4149 (1955).

36. Gibbs, M., and M. A. Cynkin, Nature, 182, 1241 (1958).37. Gibbs, M., and N. Calo, Plant Physiol., 34, 318 (1959).38. Tolbert, N. E., in Brookhaven Symp. Biol., No. 11 (1958),

p. 271.39. Smillie, R. M., and R. C. Fuller, Plant Physiol., 34, 651

(1959).40. Smillie, R. M., and G. Krotkov, Can. J. Bot., 37, 1217

(1959).41. Buchner, E., Ber. Deut. Chem. Ges., 30, 117, 1110 (1897).42. Rubner, M., in Die Ernahrungsphysiologie der Hefezelle bei

Alkoholischer Gdirung (Veit, Leipzig, 1913), p. 59.43. Zamecnik, P. C., and E. B. Keller, J. Biol. Chem., 209, 337

(1954).44. Kornberg, A., Les Prix Nobel en 1959 (Norstedt, Stock-

holm, 1960).45. Walker, D. A., Plant Physiol., 40, 1157 (1965).46. Walker, D. A., Biochem. J., 92, 22C (1964).47. Jensen, R. G., and J. A. Bassham, Proc. Nat. Acad. Sci.

USA, 56, 1096 (1966).48. Kalberer, P. P., B. B. Buchanan, and D. I. Arnon, Proc.

Nat. Acad. Sci. USA, 57, 1542 (1967).49. Arnon, D. I., M. B. Allen, and F. R. Whatley, in Rapports

et Communications Parvenus Avant le Congres Aux Sections11 et 12 de 8e Congres International de Botanique, Paris,July 1964, pp. 1-2.

50. Arnon, D. I., F. R. Whatley, and M. B. Allen, J. Amer.Chem. Soc., 76, 6324 (1954).

51. Vishniac, W., and S. Ochoa, J. Biol. Chem., 198, 501(1952).

52. Frenkel, A., J. Amer. Chem. Soc., 76, 5568 (1954).53. Thomas, J. B., and A. J. M. Haans, Biochim. Biophys.

Acta, 18, 286 (1955).54. Petrack, B., and F. Lipmann, in Light and Life, ed. W. D.

McElroy and B. Glass (Johns Hopkins Univ. Press,Baltimore, 1961), p. 621.

55. Rabinowitch, E., in Research in Photosynthesis, ed. H.Gaffron (Interscience, New York, 1957), p. 345.

56. Allen, M. B., F. R. Whatley, and D. I. Arnon, Biochim.Biophys. Acta, 27, 16 (1958).

57. Jagendorf, A. T., and M. Avron, J. Biol. Chem., 231, 277(1958).

58. Arnon, D. I., Nature, 184, 10 (1959).59. Arnon, D. I., in Light and Life, ed. W. D. McElroy and

B. Glass (Johns Hopkins Univ. Press, Baltimore, 1961),pp. 489-566.

60. Katz, D., in Photosynthesis in Plants, ed. J. Franck andW. E. Loomis (Iowa State College Press, Ames, 1949),p. 287.

61. Levitt, L. S., Science, 118, 696 (1953); 120, 33 (1954).62. Arnon, D. I., M. B. Allen, and F. R. Whatley, Biochim.

Biophys. Acta, 20,449 (1956).63. Arnon, D. I., F. R. Whatley, and M. B. Allen, Paper pre-

sented at Amer. Chem. Soc. Meeting, New York, 1957;Science, 127, 1026 (1958).

64. Arnon, D. I., F. R. Whatley, and M. R. Allen, Biochim.Biophys. Acta, 32, 47 (1959).

65. Avron, M., D. W. Krogmann, and A. T. Jagendorf,Biochim. Biophys. Acta, 30, 144 (1958).

66. Davenport, H. E., Biochem. J., 77, 471 (1960).67. Mortenson, L. E., R. C. Valentine, and J. E. Carnahan,

Biochem. Biophys. Res. Commun., 7, 448 (1962).68. Tagawa, K., and D. I. Arnon, Nature, 195, 537 (1962).69. Arnon, D. I., Naturwiseenschaften, 56, 295 (1969).70. Buchanan, B. B., and D. I. Arnon, Advan. Enzymol., 33,

120 (1970).71. Zweig, G., and M. Avron, Biochem. Biophys. Res. Commun.,

19, 397 (1965).72. Kok, B., H. J. Rurainski, and 0. V. H. Owens, Biochim.

Biophys. Acta. 109, 347 (1965).73. Black, C. C., Biochim. Biophys. Acta, 120, 332 (1966).74. Yocum, C. F., and A. San Pietro, Biochem. Biophys. Re8.

Commun., 36, 614 (1969).75. Yocum, C. F., and A. San Pietro, Arch. Biochem. Biophye.,

140, 152 (1970).76. Tsujimoto, H. Y., and R. K. Chain, Plant Physiol., 47S,

33 (1971).77. Shin, M., and D. I. Arnon, J. Biol. Chem., 240, 1405

(1965).78. Shin, M., K. Tagawa, and D. I. Arnon, Biochem. Z., 338,84

(1963).79. Arnon, D. J., H. Y. Tsujimoto, and B. D. McSwain,

Proc. Nat. Acad. Sci. USA, 51, 1274 (1964).80. Arnon, D. I., F. R. Whatley, and M. B. Allen, Biochim.

Biophys. Acta, 16, 607 (1955).81. Jagendorf, A. T., and M. Avron, J. Biol. Chem., 231, 277

(1958).82. Newton, J. W., and M. Kamen, Biochim. Biophys. Acta, 25,

462 (1957).83. Anderson, I. C., and R. C. Fuller, Arch. Biochem. Biophy8.,

76, 168 (1958).84. Baltscheffsky, H., Sv. Kem. Tidekr., 72, 310 (1960).85. Tagawa, K., H. Y. Tsujimoto, and D. I. Arnon, Nature,

199, 1247 (1963).86. Tagawa, K., H. Y. Tsujimoto, and D. I. Arnon, Proc.

Nat. Acad. Sci. USA, 49, 567 (1963).87. Anon, D. I., H. Y. Tsujimoto, and B. D. McSwain,

Nature, 214, 562 (1967).88. Schurmann, P., B. B. Buchanan, and D. I. Anon, Proc.

2nd Intern. Cong. on Photosynthesis Res., Stresa, Italy, 1971,in press.

89. Smith, J. H. C., and C. S. French, Annu. Rev. Plant Physiol.,14, 181 (1963).

90. Boardman, N. K., Annu. Rev. Plant Physiol., 21, 115(1970).

91. Vernon, L. P., and W. S. Zaugg, J. Biol. Chem., 235, 2728(1960).

92. Arnon, D. I., H. Y. Tsujimoto, and B. D. McSwain,Nature, 207, 1367 (1965).

93. Arnon, D. I., Physiol. Rev., 47, 317 (1967).



94. Levine, R. P., Annu. Rev. Plant Physiol., 20, 523 (1969).95. Losada, M., F. R. Whatley, and D. I. Arnon, Nature, 190,

606 (1961).96. Knaff, D. B., and D. I. Arnon, Proc. Nat. Acad. Sci. USA,

64, 715 (1969).97. Knaff, D. B., and D. I. Arnon, Biochim. Biophys. Acta, 226,

400 (1971).98. Knaff, D. B., and D. I. Arnon, Proc. Nat. Acad. Sci. USA,

63,963 (1969).99. Knaff, D. B., and D. I. Arnon, Proc. Nat. Acad. Sci. USA,

63, 956 (1969).100. Knaff, D. B., and D. I. Arnon, Biochim. Biophys. Acta, 223,

201 (1970).101. Arnon, D. I., R. K. Chain, B. D. McSwain, H. Y. Tsu-

jimoto, and D. B. Knaff, Proc. Nat. Acad. Sci. USA, 67,1404 (1970).

102. Arnon, D. I., D. B. Knaff, B. D. McSwain, R. K. Chain,and H. Y. Tsujimoto, Photochem. Photobiol., 14, 397(1971)

103. Arnon, D. I., D. B. Knaff, B. D. McSwain, H. Y. Tsuji-moto, R. K. Chain, R. Malkin, and A. J. Bearden, in EnergyTransduction in Respiration and Photosynthesis, ed. E.Quagliariello, S. Papa, and E. C. Slater (Adriatica Editrice,Bari, Italy, in press).

104. Malkin, R., Biochim. Biophys. Acta, in press.105. Erixon, K., and W. L. Butler, Photochem. Photobiol., 14,

427 (1971).106. Erixon, K., and W. L. Butler, Biochim. Biophys. Acta, 234,

381 (1971).107. Boardman, N. K., J. M. Anderson, and R. G. Hiller,

Biochim. Biophys. Acta, 234, 126 (1971).108. Bendall, D. S., and D. S. Sofrova, Biochim. Biophys. Acta,

234, 371 (1971).109. Katoh, S., Nature, 186, 533 (1960).110. Katoh, S., and A. Takamiya, Biochim. Biophys. Acta, 99,

156 (1965).111. Katoh, S., and A. San Pietro, in The Biochemistry of Copper,

112.

113.

114.

115.

116.

117.

118.

119.

120.121.

122.123.

124.

125.126.

127.128.129.

ed. J. Peisach, P. Aisen, and W. E. Blumberg (AcademicPress, New York, 1966), p. 407.Gorman, D. S., and R. P. Levine, Plant Physiol., 41, 1648(1966).Elstner, E., E. Pistorius, P. Boger, and A. Trebst, Planta,79, 146 (1968).Boardman, N. K., and J. Anderson, Biochim. Biophys.Acta, 143, 187 (1967).Anderson, J. M., and N. K. Boardman, Biochim. Biophys.Acta, 112, 403 (1966).Arnon, D. I., H. Y. Tsujimoto, B. D. McSwain, and R. K.Chain, in Comparative Biochemistry and Biophysics ofPhotosynthesis, ed. K. Shibata, A. Takamiya, A. T.Jagendorf, and R. C. Fuller (University Park Press, StateCollege, Pa., 1968), p. 113.Cramer, W. A., and W. L. Butler, Biochim. Biophys. Acta,143, 332 (1967).Fan, H. N., and W. A. Cramer, Biochim. Biophys. Acta,216, 200 (1970).Bendall, D. S., and R. Hill, Annu. Rev. Plant Physiol., 19,167 (1968).Kok, B., Biochim. Biophys. Acta, 48, 527 (1961).Malkin, R., and A. J. Bearden, Proc. Nat. Acad. Sci. USA,68, 16 (1971).Weaver, E. C., Annu. Rev. Plant Physiol., 10, 283 (1968).Yin, H. C., Y. K. Shen, G. M. Shen, S. Y. Yang, and K. S.Chiu, Sci. Sinica, 10, 976 (1961).Hoch, G., and I. Martin, Arch. Biochem. Biophys., 102, 430(1963).Sauer, K., and R. Park, Biochemistry, 4, 2791 (1965).Sauer, K., and J. Biggins, Biochim. Biophys. Acta, 102, 55(1965).Schwartz, M., Biochim. Biophys. Acta, 131, 559 (1967).Schwartz, M., Biochim. Biophys. Acta, 102, 361 (1965).Avron, M., and G. Ben-Hayyim, in Progress in Photo-synthesis Research, ed. H. Metzner (Laupp, Tubingen,1969), Vol. 3, p. 1185.

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