The Light Reactions of Photosynthesis - PNAS
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Proc. Nat. Acad. Sci. USA
Vol. 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- (4) and Willsttfiter (5); its last great contemporary protago-
svnthesis has been ascribed either to a photolysis of carbon nist was Otto Warburg (6). After de Saussure (7) showed that
dioxide or to a photolysis of water and a resultant rear-
rangement of constituent atoms into molecules 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
phosphorylation demonstrated that photosynthesis in- photosynthesis equation (Eq. i): the C: 2H: 0 proportions
cludes a light-induced phosphorus metabolism that in the carbohydrate product fitted the idea that the carbon
precedes, and is independent from, a photolysis of water from the photodecomposition of C02 recombines with the
or CO2. ATP formation could best be accounted for not
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 of free energy which is trapped C02 + H20 (CH20) + 02 (i)
in the pyrophosphate bonds of ATP.
Photophosphorylation 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 search in photosynthesis, was put forward by van Niel (8).
a light-induced electron transport from water to ferredoxin
and a concomitant evolution of oxygen 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 plant photosynthesis are
the oxidation-reduction state of any electron donor 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 hydrogen donor, H2A, with the released
electron donor for the reduction of NADP by an enzymic hydrogen in turn reducing C02 by dark, enzymic reactions:
reaction that is independent of light. ATP, from both
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 carbohydrates (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 sulfur bacteria (for example)
photosynthesis in green plants involves two photosystems, H2A is H2S, with the results that oxygen becomes the by-
one (System II) that best uses light of "short" wavelength
(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 chloroplasts 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 distinction between plant and bacterial photosynthesis
by a "dark" electron-transport chain to which is coupled
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
Downloaded by guest on March 6, 2020
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
28832884 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
1957: NADPH2 +ATP CHLOROPLASTS
First products of photosynthesis:
1962: Fd red
experiments with isolated chloroplasts
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 of
either of the two earlier 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 Hill reaction, 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, phosphate 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 redox 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 enzyme 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
of 32p into ATP occurred on 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
Downloaded by guest on March 6, 2020
photoassimilation of phosphorus by intact cells also gave (1953)].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., fermentation (41, 42), protein synthesis
(43), and polymerization of DNA (44). Crucial for the docu- RNKJILOSF TrI(c
mentation of complete photosynthesis in isolated chloroplasts DIPHOSPHATE PHOSPHATES 7 PHOSPHATE 7 STARCH
were not high rates but the fact that their newly found CO2
assimilation was reproducible and yielded the same inter- ATP J INTERMEDIATES
mediate and final products as photosynthesis by intact cells.
More recently, when CO2 assimilation ceased to be a matter of
dispute and the same experimental safeguards were no longer RIL@UOSE
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 chloroplast 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 chlorophyll-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 oxygen evolution. 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-
Downloaded by guest on March 6, 2020
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-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 molecule, 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 cytochromes) 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 iron-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
chlorophyll A! cytochrome I cytochrome 2 chloroplasts (68). In this reaction, Clostridium ferredoxin
Ihv _p replaced a native chloroplast protein (known by different
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.
Downloaded by guest on March 6, 2020
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) thatProc. 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 ferredoxins. It is noteworthy that recent evi-
added catalyst, e.g., menadione (80) or phenazine metho- dence (Shanmugam and Arnon, Biochim. Biophys. Acta, in
sulfate (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
Downloaded by guest on March 6, 2020
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 effects2888 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
80"
light-induced reduction of ferredoxin-NADP by water. A
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-
20-I 664nm 714nm 664 nm 714 nmr 664nm lr4nm
siderable agreement about some of the new facts even when
they differ about interpretations.
NONCYCLIC
PSP
CYCLIC
PSP
MODIFIED
CYCLIC PSP
Three light reactions
(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 far-red (714 nm) monochromatic light for cyclic and non-
cyclic photophosphorylation, expressed on the basis of equal wavelength photoreactions (Ila and Ilb) in the noncyclic
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 plastocyanin
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) cytochrome b559 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.
identified with System II and cyclic photophosphorylation Plastocyanin is a copper-containing protein in chloroplasts
with System I. discovered by Katoh (109) and implicated in 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 electron transport chain 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
cytochrome b599 but not that of cytochrome f. The addition
Downloaded by guest on March 6, 2020
counter to a still widely held concept (90, 94)-one that our
own laboratory embraced in 1961 (ref. 95) and abandoned in of plastocyanin restored the photooxidation of cytochromeProc. Nat. Acad. Sci. USA 68 (1971) Light Reactions of Photosynthesis 2889
-0.4 -
-0.4
NADP '
-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-,manganese, and plastoquinone (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 Ul 0 \ / 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
Downloaded by guest on March 6, 2020
ferredoxin in photoreaction Ila and in the single photoreaction wavelength, 538 nm, Knaff and Arnon, ref. 98).2890 Amnon Proc. Nat. Acad. Sci. USA 68 (1971)
241 System II I24
System I
(H20-NADP) (DPIPH2-NADP)
8
k.) ,6
S0
0)
k 4
3
v
1
w
--"" 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 (H20NADP) 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
theoretical and experimental study and led to more contro- The experimental work from the author's laboratory cited
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 bioenergetics 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-
was invoked to explain cyclic and noncyclic photophosphoryla-
port from Board of Agriculture, London, 1796).
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).
to ferredoxin-NADP (Fig. 5), the theoretical quantum re- 7. de Saussure, T., Recherches Chimiques Sur La Vegetation
(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).
Downloaded by guest on March 6, 2020
eight quanta per molecule of 02 (Eq. iv). Likewise, with only 9. van Niel, C. B., Advan. Enzymol., 1, 263 (1941).Proc. Nat. Acad. Sci. USA 68 (1971) Light Reactions of Photosynthesis 2891
10. van Niel, C. B., Photosynthesis in Plants, ed. J. Franck and McElroy and B. Glass (Johns Hopkins Univ. Press,
W. E. Loomis (Iowa State College Press, Ames, 1949). Baltimore, 1961), p. 621.
11. Sachs, J., Jahrb. Wise Bot., 3, 184 (1863). 55. Rabinowitch, E., in Research in Photosynthesis, ed. H.
12. Boehm, J., Bot. Zeitg., 41, 33, 49 (1883). Gaffron (Interscience, New York, 1957), p. 345.
13. Ruben, S., and M. Kamen, Phy8. Rev., 59, 49 (1941). 56. Allen, M. B., F. R. Whatley, and D. I. Arnon, Biochim.
14. Martin, A. J. P., and R. L. M. Synge, Les Prix Nobel en Biophys. Acta, 27, 16 (1958).
1952 (Norstedt, Stockholm, 1952). 57. Jagendorf, A. T., and M. Avron, J. Biol. Chem., 231, 277
15. Fink, R. M., and K. Fink, Science, 107, 253 (1948). (1958).
16. Benson, A. A., and M. Calvin, Annu. Rev. Plant Physiol., 58. Arnon, D. I., Nature, 184, 10 (1959).
1, 25 (1950). 59. Arnon, D. I., in Light and Life, ed. W. D. McElroy and
17. Benson, A. A., J. Amer. Chem. Soc., 73, 2971 (1951). B. Glass (Johns Hopkins Univ. Press, Baltimore, 1961),
18. Benson, A. A., J. A. Bassham, M. Calvin, A. G. Hall, pp. 489-566.
H. E. Hirsch, S., Kawaguchi, V. Lynch, and N. E. Tolbert, 60. Katz, D., in Photosynthesis in Plants, ed. J. Franck and
J. Biol. Chem., 196, 703 (1952). W. E. Loomis (Iowa State College Press, Ames, 1949),
19. Bassham, J. A., A. A. Benson, L. D. Kay, A. Z. Harris, p. 287.
A. T. Wilson, and M. Calvin, J. Amer. Chem. Soc., 76, 1760 61. Levitt, L. S., Science, 118, 696 (1953); 120, 33 (1954).
(1954). 62. Arnon, D. I., M. B. Allen, and F. R. Whatley, Biochim.
20. Bassham, J. M., and M. Calvin, The Path of Carbon in Biophys. Acta, 20,449 (1956).
Photosynthesis (Prentice-Hall, Engelwood Cliffs, N.J., 63. Arnon, D. I., F. R. Whatley, and M. B. Allen, Paper pre-
1957). sented at Amer. Chem. Soc. Meeting, New York, 1957;
21.- Goodman, M., D. F. Bradley, and M. Calvin, J. Amer. Science, 127, 1026 (1958).
Chem. Soc., 75, 1962 (1953). 64. Arnon, D. I., F. R. Whatley, and M. R. Allen, Biochim.
22. Arnon, D. I., Annu. Rev. Plant Physiol., 7, 325 (1956). Biophys. Acta, 32, 47 (1959).
23. Sachs, J., Lectures on the Physiology of Plants (Clarendon 65. Avron, M., D. W. Krogmann, and A. T. Jagendorf,
Press, Oxford, 1887). Biochim. Biophys. Acta, 30, 144 (1958).
24. Pfeffer, W., Physiology of Plants (Clarendon Press, Ox- 66. Davenport, H. E., Biochem. J., 77, 471 (1960).
ford, 1900). 67. Mortenson, L. E., R. C. Valentine, and J. E. Carnahan,
25. Hill, R., Proc. Roy. Soc. Ser. B, 127, 192 (1939). Biochem. Biophys. Res. Commun., 7, 448 (1962).
26. Hill, R., Symp. Soc. Exp. Biol., 5, 223 (1951). 68. Tagawa, K., and D. I. Arnon, Nature, 195, 537 (1962).
27. Brown, A. H., and J. Franck, Arch. Biochem., 16, 55 (1948). 69. Arnon, D. I., Naturwiseenschaften, 56, 295 (1969).
28. Wessels, J. S. C., and E. Havinga, Rec. Trav. Chim. Pays- 70. Buchanan, B. B., and D. I. Arnon, Advan. Enzymol., 33,
Bas, 71,7 (1952). 120 (1970).
29. Aronoff, S., and M. Calvin, Plant Physiol., 23, 351 (1948). 71. Zweig, G., and M. Avron, Biochem. Biophys. Res. Commun.,
30. Vishniac, W., and S. Ochoa, Nature, 167, 768 (1951). 19, 397 (1965).
31. Tolmach, L. J., Nature, 167, 946 (1951). 72. Kok, B., H. J. Rurainski, and 0. V. H. Owens, Biochim.
32. Arnon, D. I., Nature, 167, 1008 (1951). Biophys. Acta. 109, 347 (1965).
33. Arnon. D. I., M. B. Allen, and F. R. Whatley, Nature, 174, 73. Black, C. C., Biochim. Biophys. Acta, 120, 332 (1966).
394 (1954). 74. Yocum, C. F., and A. San Pietro, Biochem. Biophys. Re8.
34. Arnon, D. I., Paper presented at the Cell Symposium, Commun., 36, 614 (1969).
Amer. Assoc. Advan. Sci., Berkeley Meeting, 1954; 75. Yocum, C. F., and A. San Pietro, Arch. Biochem. Biophye.,
Science, 122, 9 (1955). 140, 152 (1970).
35. Allen, M. B., D. I. Arnon, J. B. Capindale, F. R. Whatley, 76. Tsujimoto, H. Y., and R. K. Chain, Plant Physiol., 47S,
and L. J. Durham, J. Amer. Chem. Soc., 77, 4149 (1955). 33 (1971).
36. Gibbs, M., and M. A. Cynkin, Nature, 182, 1241 (1958). 77. Shin, M., and D. I. Arnon, J. Biol. Chem., 240, 1405
37. Gibbs, M., and N. Calo, Plant Physiol., 34, 318 (1959). (1965).
38. Tolbert, N. E., in Brookhaven Symp. Biol., No. 11 (1958), 78. Shin, M., K. Tagawa, and D. I. Arnon, Biochem. Z., 338,84
p. 271. (1963).
39. Smillie, R. M., and R. C. Fuller, Plant Physiol., 34, 651 79. Arnon, D. J., H. Y. Tsujimoto, and B. D. McSwain,
(1959). Proc. Nat. Acad. Sci. USA, 51, 1274 (1964).
40. Smillie, R. M., and G. Krotkov, Can. J. Bot., 37, 1217 80. Arnon, D. I., F. R. Whatley, and M. B. Allen, Biochim.
(1959). Biophys. Acta, 16, 607 (1955).
41. Buchner, E., Ber. Deut. Chem. Ges., 30, 117, 1110 (1897). 81. Jagendorf, A. T., and M. Avron, J. Biol. Chem., 231, 277
42. Rubner, M., in Die Ernahrungsphysiologie der Hefezelle bei (1958).
Alkoholischer Gdirung (Veit, Leipzig, 1913), p. 59. 82. Newton, J. W., and M. Kamen, Biochim. Biophys. Acta, 25,
43. Zamecnik, P. C., and E. B. Keller, J. Biol. Chem., 209, 337 462 (1957).
(1954). 83. Anderson, I. C., and R. C. Fuller, Arch. Biochem. Biophy8.,
44. Kornberg, A., Les Prix Nobel en 1959 (Norstedt, Stock- 76, 168 (1958).
holm, 1960). 84. Baltscheffsky, H., Sv. Kem. Tidekr., 72, 310 (1960).
45. Walker, D. A., Plant Physiol., 40, 1157 (1965). 85. Tagawa, K., H. Y. Tsujimoto, and D. I. Arnon, Nature,
46. Walker, D. A., Biochem. J., 92, 22C (1964). 199, 1247 (1963).
47. Jensen, R. G., and J. A. Bassham, Proc. Nat. Acad. Sci. 86. Tagawa, K., H. Y. Tsujimoto, and D. I. Arnon, Proc.
USA, 56, 1096 (1966). Nat. Acad. Sci. USA, 49, 567 (1963).
48. Kalberer, P. P., B. B. Buchanan, and D. I. Arnon, Proc. 87. Anon, D. I., H. Y. Tsujimoto, and B. D. McSwain,
Nat. Acad. Sci. USA, 57, 1542 (1967). Nature, 214, 562 (1967).
49. Arnon, D. I., M. B. Allen, and F. R. Whatley, in Rapports 88. Schurmann, P., B. B. Buchanan, and D. I. Anon, Proc.
et Communications Parvenus Avant le Congres Aux Sections 2nd Intern. Cong. on Photosynthesis Res., Stresa, Italy, 1971,
11 et 12 de 8e Congres International de Botanique, Paris, in press.
July 1964, pp. 1-2. 89. Smith, J. H. C., and C. S. French, Annu. Rev. Plant Physiol.,
50. Arnon, D. I., F. R. Whatley, and M. B. Allen, J. Amer. 14, 181 (1963).
Chem. Soc., 76, 6324 (1954). 90. Boardman, N. K., Annu. Rev. Plant Physiol., 21, 115
51. Vishniac, W., and S. Ochoa, J. Biol. Chem., 198, 501 (1970).
(1952). 91. Vernon, L. P., and W. S. Zaugg, J. Biol. Chem., 235, 2728
52. Frenkel, A., J. Amer. Chem. Soc., 76, 5568 (1954). (1960).
53. Thomas, J. B., and A. J. M. Haans, Biochim. Biophys. 92. Arnon, D. I., H. Y. Tsujimoto, and B. D. McSwain,
Acta, 18, 286 (1955). Nature, 207, 1367 (1965).
Downloaded by guest on March 6, 2020
54. Petrack, B., and F. Lipmann, in Light and Life, ed. W. D. 93. Arnon, D. I., Physiol. Rev., 47, 317 (1967).2892 Amono Proc. Nat. Acad. Sci. USA 68 (1971)
94. Levine, R. P., Annu. Rev. Plant Physiol., 20, 523 (1969). ed. J. Peisach, P. Aisen, and W. E. Blumberg (Academic
95. Losada, M., F. R. Whatley, and D. I. Arnon, Nature, 190, Press, New York, 1966), p. 407.
606 (1961). 112. Gorman, D. S., and R. P. Levine, Plant Physiol., 41, 1648
96. Knaff, D. B., and D. I. Arnon, Proc. Nat. Acad. Sci. USA, (1966).
64, 715 (1969). 113. Elstner, E., E. Pistorius, P. Boger, and A. Trebst, Planta,
97. Knaff, D. B., and D. I. Arnon, Biochim. Biophys. Acta, 226, 79, 146 (1968).
400 (1971). 114. Boardman, N. K., and J. Anderson, Biochim. Biophys.
98. Knaff, D. B., and D. I. Arnon, Proc. Nat. Acad. Sci. USA, Acta, 143, 187 (1967).
63,963 (1969). 115. Anderson, J. M., and N. K. Boardman, Biochim. Biophys.
99. Knaff, D. B., and D. I. Arnon, Proc. Nat. Acad. Sci. USA, Acta, 112, 403 (1966).
63, 956 (1969). 116. Arnon, D. I., H. Y. Tsujimoto, B. D. McSwain, and R. K.
100. Knaff, D. B., and D. I. Arnon, Biochim. Biophys. Acta, 223, Chain, in Comparative Biochemistry and Biophysics of
201 (1970). Photosynthesis, ed. K. Shibata, A. Takamiya, A. T.
101. Arnon, D. I., R. K. Chain, B. D. McSwain, H. Y. Tsu- Jagendorf, and R. C. Fuller (University Park Press, State
jimoto, and D. B. Knaff, Proc. Nat. Acad. Sci. USA, 67, College, Pa., 1968), p. 113.
1404 (1970). 117. Cramer, W. A., and W. L. Butler, Biochim. Biophys. Acta,
102. Arnon, D. I., D. B. Knaff, B. D. McSwain, R. K. Chain, 143, 332 (1967).
and H. Y. Tsujimoto, Photochem. Photobiol., 14, 397 118. Fan, H. N., and W. A. Cramer, Biochim. Biophys. Acta,
(1971) 216, 200 (1970).
103. Arnon, D. I., D. B. Knaff, B. D. McSwain, H. Y. Tsuji- 119. Bendall, D. S., and R. Hill, Annu. Rev. Plant Physiol., 19,
moto, R. K. Chain, R. Malkin, and A. J. Bearden, in Energy 167 (1968).
Transduction in Respiration and Photosynthesis, ed. E. 120. Kok, B., Biochim. Biophys. Acta, 48, 527 (1961).
Quagliariello, S. Papa, and E. C. Slater (Adriatica Editrice, 121. Malkin, R., and A. J. Bearden, Proc. Nat. Acad. Sci. USA,
Bari, Italy, in press). 68, 16 (1971).
104. Malkin, R., Biochim. Biophys. Acta, in press. 122. Weaver, E. C., Annu. Rev. Plant Physiol., 10, 283 (1968).
105. Erixon, K., and W. L. Butler, Photochem. Photobiol., 14, 123. Yin, H. C., Y. K. Shen, G. M. Shen, S. Y. Yang, and K. S.
427 (1971). Chiu, Sci. Sinica, 10, 976 (1961).
106. Erixon, K., and W. L. Butler, Biochim. Biophys. Acta, 234, 124. Hoch, G., and I. Martin, Arch. Biochem. Biophys., 102, 430
381 (1971). (1963).
107. Boardman, N. K., J. M. Anderson, and R. G. Hiller, 125. Sauer, K., and R. Park, Biochemistry, 4, 2791 (1965).
Biochim. Biophys. Acta, 234, 126 (1971). 126. Sauer, K., and J. Biggins, Biochim. Biophys. Acta, 102, 55
108. Bendall, D. S., and D. S. Sofrova, Biochim. Biophys. Acta, (1965).
234, 371 (1971). 127. Schwartz, M., Biochim. Biophys. Acta, 131, 559 (1967).
109. Katoh, S., Nature, 186, 533 (1960). 128. Schwartz, M., Biochim. Biophys. Acta, 102, 361 (1965).
110. Katoh, S., and A. Takamiya, Biochim. Biophys. Acta, 99, 129. Avron, M., and G. Ben-Hayyim, in Progress in Photo-
156 (1965). synthesis Research, ed. H. Metzner (Laupp, Tubingen,
111. Katoh, S., and A. San Pietro, in The Biochemistry of Copper, 1969), Vol. 3, p. 1185.
Downloaded by guest on March 6, 2020You can also read
