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 photosvnthesis has been ascribed either to a photolysis of carbon
dioxide or to a photolysis of water and a resultant rearrangement of constituent atoms into molecules of oxygen and glucose (or formaldehyde). The discovery of photophosphorylation demonstrated that photosynthesis includes a light-induced phosphorus metabolism that
precedes, and is independent from, a photolysis of water
or CO2. ATP formation could best be accounted for not
by a photolytic disruption of the covalent bonds in C02
or water but by the operation of a light-induced electron
flow that results in a release of free energy which is trapped
in the pyrophosphate bonds of ATP.
Photophosphorylation is now divided into (a) a noncyclic type, in which the formation of ATP is coupled with
a light-induced electron transport from water to ferredoxin
and a concomitant evolution of oxygen and (b) a cyclic
type which yields only ATP and produces no net change in
the oxidation-reduction state of any electron donor or
acceptor. Reduced ferredoxin formed in (a) serves as an
electron donor for the reduction of NADP by an enzymic
reaction that is independent of light. ATP, from both
cyclic and noncyclic photophosphorylation, and reduced
NADP jointly constitute the assimilatory power for the
conversion of C02 to carbohydrates (3 moles of ATP and
2 moles of reduced NADP are required per mole of C02).
Investigations, mainly with whole cells, have shown that
photosynthesis 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 uses
light of "long" wavelength (X > 685 nm). Cyclic photophosphorylation in chloroplasts involves a System I
photoreaction. Noncyclic photophosphorylation is widely
held to involve a collaboration of two photoreactions:
a short-wavelength photoreaction belonging to System II
and a long-wavelength photoreaction belonging to System
I. Recent findings, however, indicate that noncyclic photophosphorylation may include two short-wavelength, System II, photoreactions that operate in series and are joined
by a "dark" electron-transport chain to which is coupled
a phosphorylation site.
Early concepts
The first hypothesis about the role of light in photosynthesis
came very appropriately from Jan Ingenhousz, who some
years earlier had made the epochal discovery that it is "the
influence of the light of the sun upon the plant" (1) that is
responsible for the "restorative" effect of vegetation on
"bad" air-an observation first made in 1771 by Joseph
Priestley without reference to light (2). In 1796, Ingenhousz
wrote that the green plant absorbs from "carbonic acid in
the sunshine, the carbon, throwing out at that time the oxygen
alone, 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 a
century. It seemed to have had a special attraction for some
of the most illustrious chemists in their day, e.g., von Baeyer
2883
(4) and Willsttfiter (5); its last great contemporary protagonist was Otto Warburg (6). After de Saussure (7) showed that
water is a reactant in photosynthesis, the C02 cleavage hypothesis readily accounted for the deceptively simple overall
photosynthesis equation (Eq. i): the C: 2H: 0 proportions
in the carbohydrate product fitted the idea that the carbon
from the photodecomposition of C02 recombines with the
elements of water.
h
0
2
(i)
C02 + H20 (CH20) + 02
A different hypothesis,, one that profoundly influenced research in photosynthesis, was put forward by van Niel (8).
After elucidating the nature of bacterial photosynthesis, he
proposed (8) that bacterial and plant photosynthesis are
special cases of a general process in which light energy is used
to photodecompose a hydrogen donor, H2A, with the released
hydrogen in turn reducing C02 by dark, enzymic reactions:
hp
(ii)
C02 + 2H2A -- (CH20) + H20 + 2A
The hypothesis envisaged that in plant photosynthesis
H2A is water, whereas in green sulfur bacteria (for example)
H2A is H2S, with the results that oxygen becomes the byproduct of plant photosynthesis and elemental sulfur the
by-product of bacterial photosynthesis.
In later formulations (9, 10) van Niel no longer considered
the photodecomposition of water as being unique to plant
photosynthesis but postulated that "the photochemical reaction in the photosynthetic process of green bacteria, purple
bacteria, and green plants represents, in all cases, a photodecomposition of water" (10). According to this concept, the
distinction between plant and bacterial photosynthesis
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
photosynthesis, the acceptor was regenerated when the complex was decomposed by liberating molecular oxygen. In
bacterial photosynthesis, oxygen was not liberated and the
acceptor could be regenerated only when the OH-acceptor
complex was reduced by the special hydrogen donor, H2A,
that is always required in bacterial photosynthesis.
The concept of photodecomposition of C02 or photodecomposition of water provided, each in its own historical
period, a broad, general perspective on the role of light in the
overall events of photosynthesis. In the last two decades,
however, the focus in photosynthesis research shifted toward
the isolation, identification, and characterization of the
specific reactions and mechanisms by which light energy
drives the photosynthetic process. This approach has led to
new perspectives on the mechanisms by which light energy is
2884
Amnon
Proc. Nat. Acad. Sci. USA 68 (1971)
EXPERIMENTALLY IDENTIFIED FIRST
PRODUCTS OF PHOTOSYNTHESIS
I1862:
STARCHWHL
11883: SUCROSE, GLUCOSE
1951:
1954:
1957:
1962:
1964:
CELLS
NADPH2
AT P
NADPH2 +ATP CHLOROPLASTS
Fd red
Fd red + AT P
FIG. 1.
used in photosynthesis and to a finding, inadmissible under
either of the two earlier hypotheses, that the photosynthetic
apparatus can convert light energy into a stable form of
chemical energy, independently of the splitting of either water
or C02.
First products of photosynthesis:
experiments with whole cells
In chemical terms, an insight into the role of light in photosynthesis could come from identification of the first chemically
defined products that are formed under the influence of light.
In the 19th century (Fig. 1) this approach established that
starch is the first product of photosynthesis in chloroplasts
(11)-a conclusion that was later revised in favor of soluble
carbohydrates (ref. 12). In the modern period, the powerful
new techniques of 14C (ref. 13), paper chromatography (14),
and radioautography (15) aided in the identification of phosphoglyceric acid (PGA) as the first stable product of photosynthesis, formed only after a few seconds of illumination
(16). Aside from PGA, phosphate esters of two sugars, ribulose
and sedoheptulose, were soon added to the list of early products 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 the
formulation of a photosynthetic carbon cycle (reductive
pentose phosphate cycle), which was convincingly identified
with the dark phase of photosynthesis. The chief importance
of the carbon cycle to the understanding of the role of light in
photosynthesis lay in revealing which of its component enzymic reactions require an input of energy-rich chemical
intermediates that must be formed by the light reactions.
As summarized in Fig. 2, the carbon cycle shows that the conversion of 1 mole of C02 to the level of hexose phosphate requires 3 moles of ATP and 2 moles of reduced NADP. Accordingly, the need for light energy in photosynthesis by green
plants could now be traced to those photochemical reactions
that generate ATP and NADPHE2.
The occurrence of phosphorylated compounds among the
early products of C02 assimilation suggested that lightinduced phosphorus assimilation may, in fact, precede carbon
assimilation but experimental evidence for this conclusion
was lacking in whole cells. When Calvin's group investigated
the photoassimilation of phosphorus by Scenedesmus cells
with the aid of carrier-free KH232PO4, they found that the
shortest exposure to light gave the lowest incorporation of
32p into ATP and, conversely, that the highest incorporation
of 32p into ATP occurred on short, dark exposure (21). The
first compound to be labeled proved to be not the expected
ATP but again PGA (21). Other investigations of direct
photoassimilation of phosphorus by intact cells also gave
results that were at best suggestive [see review (22)1. In
short, experiments with whole cells proved, for reasons discussed below, incapable of yielding evidence for an independent light-induced phosphorus metabolism. Its occurrence in
photosynthesis was discovered not in whole cells but in
isolated chloroplasts.
First products of photosynthesis:
experiments with isolated chloroplasts
Chloroplasts were once widely believed to be the site of complete photosynthesis but this view was not supported by
critical evidence (23, 24) and was largely abandoned after
Hill (25, 26) demonstrated that isolated chloroplasts could
evolve oxygen but could not assimilate C02. [The failure of
isolated chloroplasts to fix C02 was also reported with the
sensitive 14CO2 technique (27).] In the oxygen-producing reaction, which became known as the Hill reaction, isolated
chloroplasts evolved oxygen only in the presence of artificial
oxidants with distinctly positive oxidation-reduction potentials, e.g., ferric oxalate, ferricyanide, benzoquinone.
The Hill reaction established that the photoproduction of
oxygen by chloroplasts is basically independent of C02 assimilation. This provided strong support for the view that the
source of photosynthetic oxygen is water.* Left in doubt was
the role of chloroplasts in the energy-storing reactions needed
for C02 assimilation. The photochemical generation by isolated chloroplasts of a strong reductant capable of reducing
C02 was deemed unlikely on experimental and theoretical
grounds (26, 28). The first experiments with the sensitive 82p
technique to test the ability of isolated chloroplasts to form
ATP, on illumination, also gave negative results (29).
A different perspective on the photosynthetic capacity
of isolated chloroplasts began to emerge in 1951 when three
laboratories (30-32), independently and simultaneously,
found that isolated chloroplasts could photoreduce NADP
despite its strongly electronegative redox potential (Em =
-320 mV, at pH 7). This finding was followed by several
other developments which drastically altered the then prevalent ideas about the photosynthetic capacity of isolated
chloroplasts. These developments (Fig. 1) will now be discussed in chronological order.
In 1954, a reinvestigation of photosynthesis in isolated
chloroplasts by different methods yielded evidence for a
light-dependent assimilation of C02 (ref. 33). Chloroplasts
isolated from spinach leaves assimilated 14CO2 to the level
of carbohydrates, including starch, with a simultaneous
evolution of oxygen (34, 35). When the conversion of 14CO2
by isolated chloroplasts to sugars and starch was confirmed
and extended in other laboratories (36-40), the capacity of
chloroplasts to carry on complete extracellular photosynthesis was no longer open to question.
Because of the earlier negative results, special experimental
safeguards were deemed necessary to establish that chloroplasts alone, without other organelles or enzyme systems and
with light as the only energy source, were capable of a total
synthesis of carbohydrates from C02. The chloroplasts were
washed and, to eliminate a possible source of chemical energy
and metabolites, their isolation was performed not as formerly
* Contrary to a widely held belief, this conclusion was not unequivocally documented by experiments with 180 [(see A. H.
Brown and A. W. Frenkel, Annu. Rev. Plant Physiol., 4, 53
(1953)].
Proc. Nat. Acad. Sci. USA 68
(1971)
Light Reactions of Photosynthesis
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 situation similar to the first reconstruction of other cellular processes in vitro, e.g., fermentation (41, 42), protein synthesis
(43), and polymerization of DNA (44). Crucial for the documentation of complete photosynthesis in isolated chloroplasts
were not high rates but the fact that their newly found CO2
assimilation was reproducible and yielded the same intermediate 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
needed, much higher rates of CO2 assimilation by isolated
chloroplasts were obtained with modified procedures (45-48).
Since neither ATP nor reduced NADP was added to isolated chloroplasts that fixed C02, it was clear that these
energy-rich compounds were being photochemically generated
from their respective precursors within the chloroplasts.
Chloroplasts were already known to photoreduce added
NADP but nothing was known about their ability (or that of
any other photosynthetic structures) to form ATP at the
expense of light energy. A renewed attack on this problem in
isolated chloroplasts was therefore undertaken.
Discovery of photosynthetic phosphorylation
The likelihood of detecting a direct role of light in ATP formation was much greater in isolated chloroplasts than in intact
cells. Intact cells contain only catalytic amounts of the precursor adenosine phosphates (AMP, ADP) and these, because
of permeability barriers, could not be increased by external
additions. By contrast, in experiments with isolated chloroplasts, it was possible to supply these normally catalytic
substances in substrate amounts and, with the aid of labeled
inorganic phosphate, determine chemically their light-induced conversion to ATP.
In 1954, work with the same spinach chloroplast preparations that fixed CO2 led to the discovery that they were also
able to convert light energy into chemical energy and trap
it in the pyrophosphate bonds of ATP (49, 33). Several
unique features distinguished this photosynthetic phosphorylation (photophosphorylation), as the process was
named, from substrate-level phosphorylation in fermentation
and oxidative phosphorylation in respiration: (a) ATP formation occurred in the chlorophyll-containing lamellae and
was independent of other enzyme systems or organelles
(including mitochondria, which were previously considered
necessary for photosynthetic ATP formation, ref. 51); (b)
no energy-rich substrate, other than absorbed photons, served
as a source of energy; (c) no oxygen was produced or consumed; (d) ATP formation was not accompanied by a measurable electron transport involving any external electron
donor or acceptor (49, 33, 50). The light-induced ATP formation could be expressed by the equation:
hp
n *ADP + n
-
Pi
n*ATP
(iii)
When photophosphorylation in chloroplasts was followed
by evidence of a similar phenomenon in cell-free preparations
of such diverse types of photosynthetic organisms as photosynthetic bacteria (52) and algae (53, 54), it became evident
that photophosphorylation is not peculiar to plants containing
chloroplasts but is a major ATP-forming process in nature
RNKJILOSF
DIPHOSPHATE
ATP
J
TrI(c
PHOSPHATES
7
PHOSPHATE
7
2885
STARCH
INTERMEDIATES
RIL@UOSE
MONOPHOSPHATE
FIG. 2. Schematic representation of the ATP and reduced
NADP requirements {or CO2 assimilation.
that supplies ATP for the biosynthetic reactions in all types
of photosynthesis.
Soon after the demonstration of photosynthetic phosphorylation in isolated chloroplasts, attempts were made to
evaluate its physiological significance. Since, as with other
cellular processes when first reproduced in vitro, the rates of
photosynthetic phosphorylation were low, there was little
inclination at first to accord this process quantitative importance as a photosynthetic mechanism for converting light
into chemical energy (55). With further improvement in
experimental methods (which included the use of broken
chloroplasts with lowered permeability barriers), rates of photosynthetic 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 of
carbon assimilation in intact leaves. It appeared, therefore,
that isolated chloroplasts retain, without substantial loss,
the enzymic apparatus for photosynthetic phosphorylationa conclusion in harmony with evidence that the phosphorylating system was tightly bound in the water-insoluble lamellar
portion of the chloroplasts.
Role of light in photophosphorylation
Once the main features of photophosphorylation were firmly
established, the next objective was to explain its mechanism,
particularly its absolute dependence on illumination (33, 50).
On the one hand, photophosphorylation was independent of
such classical manifestations of photosynthesis as oxygen
evolution and CO2 assimilation; on the other hand, it seemed
unlikely that light was involved in the formation of ATP itself,
a reaction universally occurring in all cells independently of
photosynthesis. Light energy, therefore, had to be used in
photophosphorylation before ATP synthesis and in a manner
unrelated to CO2 assimilation or oxygen evolution. The most
probable mechanism for such a role seemed to be a lightinduced electron flow (58, 59).
It is often difficult for the student of photosynthesis today
to realize that before the discovery of photophosphorylation
the concept of a light-induced electron transport had no substantial basis in photosynthesis research. The idea that photon
energy is used in photosynthesis to transfer electrons rather
than cumbersome atoms had a few proponents at various
times, for example Katz (60) and Levitt (61) but, as the literature before the late 1950s shows, it did not become a viable
concept in photosynthesis-it was merely one of several specu-
2886
Proc. Nat. Acad. Sci. USA 68 (1971)
Amnon
lative ideas based on model systems. The situation changed
with the discovery of light-induced ATP formation. ATP
is formed in nonphotosynthetic cells at the expense of energy
released by electron transport. The idea that ATP may also
be formed in photosynthesis through a special light-induced
electron flow mechanism in chloroplasts now had a high probability that could be experimentally tested.
The electron flow hypothesis (58, 59) envisaged that a
chlorophyll molecule, on absorbing a quantum of light, becomes excited and promotes an electron to an outer orbital
with a higher energy level. This high-energy electron is then
transferred to an adjacent electron acceptor molecule, a
catalyst (A) with a strongly electronegative oxidation-reduction potential. The transfer of an electron from excited chlorophyll to this first acceptor is the energy conversion step proper
and terminates the photochemical phase of the process. By
transforming a flow of photons into a flow of electrons, it
constitutes a mechanism for generating a strongly electronegative reductant at the expense of the excitation energy of
chlorophyll.
Once the strongly electronegative reductant is formed, no
further input of energy is needed. Subsequent electron transfers within the chloroplast liberate energy, since they constitute
an electron flow from the electronegative reductant to electron acceptors (thought to include chloroplast cytochromes)
with more electropositive redox potentials (58, 59). Several
of the exergonic electron transfer steps, particularly those
involving cytochromes, were thought to be coupled with
phosphorylation. At the end of one cycle, the electron originally emitted by the excited chlorophyll molecule returns
to the electron-deficient chlorophyll molecule and the
quantum absorption process is repeated. A mechanism of
this kind would account for the observed lack of any oxidation-reduction change in any external electron donor or acceptor. Because of the envisaged cyclic pathway traversed by
the emitted electron, the process was named cyclic photo-
phosphorylation (58, 59).
e
chlorophyll
Ihv
A!
cytochrome
I
cytochrome 2
_p
A cyclic electron flow that is driven by light and that
liberates chemical energy, used for the synthesis of the pyrophosphate bonds of ATP, is unique to photosynthetic cells.
The idea has been discussed elsewhere (58, 59) that cyclic
photophosphorylation may be a primitive manifestation of
photosynthetic activity-an activity that is the common
denominator of plant and bacterial photosynthesis.
Noncyclic photophosphorylation
As already alluded to, there was at first no experimental
evidence linking photophosphorylation to the photoreduction
of NADP by chloroplasts. In fact, these two photochemical
activities of chloroplasts appeared to be antagonistic (62).
It was therefore wholly unexpected when a second type of
photophosphorylation was discovered in 1957 (ref. 63) that
provided direct experimental evidence for a coupling between
photoreduction of NADP and the synthesis of ATP. Here, in
contrast to cyclic photophosphorylation, ATP formation was
stoichiometrically coupled with a light-driven transfer of
electrons from water to NADP (or to a nonphysiological
electron acceptor such as ferricyanide) and a concomitant
evolution of oxygen. Moreover, ATP formation in this coupled
system greatly increased the rate of electron transfer from
water to ferricyanide (63-65) or to NADP (66) and the rate
of the concomitant oxygen evolution. It thus became apparent
that the electron transport system of chloroplasts functions
more effectively when it is coupled, as it would be under
physiological conditions, to the synthesis of ATP. The conventional Hill reaction (25, 26) now appeared to measure a
noncoupled electron transport, severed from its normally
coupled phosphorylation (63).
In extending the electron flow concept to this new reaction,
it was envisaged (58, 59) that a chlorophyll molecule excited
by a captured photon transfers an electron to NADP (or
to ferricyanide). It was postulated that electrons thus removed from chlorophyll are replaced by electrons from water
(OH-, at pH 7) with a resultant evolution of oxygen. In this
manner, light would induce an electron flow from OH- to
NADP and a coupled phosphorylation. Because of the unidirectional or noncyclic nature of this electron flow, this
process was named noncyclic photophosphorylation (58, 59).
Role of ferredoxin
Further progress in elucidating the role of light in chloroplast
reactions came from investigations of the mechanism of
NADP reduction and of the identity of the catalyst in cyclic
photophosphorylation by chloroplasts.
Investigations of the mechanism of NADP reduction and
cyclic photophosphorylation in chloroplasts led to the recognition of the key role of the iron-sulfur protein, ferredoxin. The
name ferredoxin was introduced in 1962 by Mortenson et al.
(67) and did not, at first, concern photosynthesis. It referred
to a nonheme, iron-containing protein which they isolated
from Clostridium pasteurianum, an anaerobic bacterium
devoid of chlorophyll and normally living in the soil at a
depth to which sunlight does not penetrate. A connection
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
chloroplasts (68). In this reaction, Clostridium ferredoxin
replaced a native chloroplast protein (known by different
names) that up to then was thought to be peculiar to photosynthetic cells. Its replaceability by the bacterial ferredoxin
and other considerations led to renaming the chloroplast protein 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 photosynthesis-a role that includes (but is not limited to) a catalytic
function in both cyclic and noncyclic photophosphorylation.
Ferredoxin is an electron carrier protein whose reversible
oxidation-reduction is accompanied by characteristic changes
in its absorption spectrum (Fig. 3). From the standpoint of
electron transport, the most notable finding (68) was the
strongly electronegative oxidation-reduction potential of
ferredoxin, close to that of hydrogen gas (Em = -420 mV,
at pH 7) and about 100 mV more electronegative than that
of NADP. Thus, ferredoxin (in its reduced state) emerged
as the strongest chemically defined reductant that is photochemically generated by, and is isolable from, chloroplasts.
The existence of stronger reductants in chloroplasts has
been suggested on the basis of observations (71-73) that
Proc. Nat. Acad. Sci. USA 68
(1971)
Light Reactions of Photosynthesis
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. Recent 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
of the reduced ferredoxin-NADP reductase by NADP.
Thus, what was formerly called photoreduction of NADP
turned out to be a photoreduction of ferredoxin, followed by
electron transfer to the flavin component of ferredoxinNADP reductase and thence by hydrogen transfer to NADP
(two reducing equivalents are transferred to NADP+ in the
form of a hydride ion, H-).
Illuminated chloroplasts
ferredoxin
ferredoxin-NADP reductase
H-
NADP
The role assigned to ferredoxin as the terminal electron
acceptor in the photochemical events that lead to NADP
reduction was further documented when the photoreduction
of substrate amounts of ferredoxin was found to be accompanied by stoichiometric oxygen evolution and ATP formation (79). Earlier formulations of noncyclic photophosphorylation (63, 64) were now further refined to show that
the omission of NADP did not affect ATP formation. The
true equation for noncyclic photophosphorylation became:
4 Ferredoxin.. + 2ADP + 2Pj + 2H20
4 Ferredoxinred + 2ATP + 02 + 4H+ (iv)
Turning to cyclic photophosphorylation in chloroplasts,
an unsolved question was its puzzling dependence on an
added catalyst, e.g., menadione (80) or phenazine methosulfate (81), a substance that is foreign to living cells. No
such additions were required for cyclic photophosphorylation
in freshly prepared bacterial chromatophores (82, 83). Moreover, unlike bacterial cyclic photophosphorylation, the process
in chloroplasts was not sensitive to such characteristic inhibitors of phosphorylation as antimycin A, at low concentrations (84).
A possible explanation of this dependence was that chloroplasts lose a soluble constituent like ferredoxin in the process
of chloroplast isolation. Evidence was indeed obtained later
for cyclic photophosphorylation that is dependent only on
catalytic amounts of ferredoxin and proceeds without the
addition of any other catalyst of photophosphorylation (85,
86). Moreover, when catalyzed by ferredoxin, cyclic photophosphorylation in chloroplasts became for the first time sensitive to inhibition by low concentrations of antimycin A and
oligomycin (69), and resembled in this respect cyclic photophosphorylation in bacteria (84).
Sensitivity to antimycin A and to other inhibitors provided
also a sharp distinction between ferredoxin-catalyzed cyclic
2887
wavelength (nm)
FIG. 3. Absorption spectrum of spinach ferredoxin. Insert
shows changes in absorption at 420 and 463 nm upon photoreduction (Buchanan and Arnon, ref. 70).
and noncyclic photophosphorylations. Low concentrations
of antimycin A, oligomycin, and other inhibitors which
sharply inhibited cyclic photophosphorylation had no effect
on noncyclic photophosphorylation (87, 69). By contrast, low
concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea
(DCMU) and o-phenanthroline, which sharply inhibited noncyclic photophosphorylation, actually stimulated cyclic
photophosphorylation (87).
There are now several other lines of evidence that point to
ferredoxin as the endogenous catalyst of cyclic photophosphorylation in chloroplasts: (a) As expected of a true catalyst,
ferredoxin stimulates cyclic photophosphorylation at low
concentrations (1 X 10-4 M), comparable on a molar basis
to those of the other known catalysts of the process; (b) when
light intensity is restricted, ferredoxin catalyzes ATP formation more effectively than any other catalyst; (c) in adequate
light, cyclic photophosphorylation by ferredoxin produces
ATP at a rate comparable with the maximum rates of photosynthesis in vivo (87).
Ferredoxin emerged, therefore, as the physiological catalyst
of cyclic and noncyclic photophosphorylation of chloroplasts.
Other substances that catalyze cyclic or noncyclic photophosphorylation in isolated chloroplasts appear to act as
substitutes for ferredoxins. It is noteworthy that recent evidence (Shanmugam and Arnon, Biochim. Biophys. Acta, in
press) suggests that cyclic photophosphorylation in bacteria]
chromatophores may also be catalyzed by a ferredoxin of a
kind that is membrane-bound.
To recapitulate, cyclic and noncyclic photophosphorylation
account for the basic feature of photosynthesis, i.e., conversion of light energy into chemical energy. Noncyclic photophosphorylation generates part of the needed ATP and all
of the reductant in the form of reduced ferredoxin, which in
turn serves as the electron donor for the reduction of NADP
by an enzymic reaction that is independent of light. Cyclic
photophosphorylation provides the remainder of the required
ATP (literature reviewed in ref. 88). Jointly, cyclic and noncyclic photophosphorylation generate all of the assimilatory
power, made up of ATP and reduced NADP, that is required
for CO2 assimilation.
Two photosystems in plant photosynthesis
Parallel and, in the main, unrelated to investigations of cyclic
and noncyclic photophosphorylation in isolated chloroplasts
were investigations, chiefly at the cellular level, on the effects
2888
Amnon
Proc. Nat. Acad. Sci. USA 68 (1971)
PPS
80"
60
CX 40-
20-I
664nm
714nm
NONCYCLIC
PSP
664 nm
714 nmr
CYCLIC
PSP
664nm
lr4nm
MODIFIED
CYCLIC
1965 (ref. 92)-that the two photosystems must operate in
series and, through such collaboration, bring about the
light-induced reduction of ferredoxin-NADP by water. A
detailed discussion of the relative merits of each concept is
not possible within the space allotted to this survey. The
aim here will be to cover in broad outline some recent findings
pertaining to electron transport in chloroplasts and the interpretation our laboratory placed on them. It may be pertinent to note that different laboratories are already in considerable agreement about some of the new facts even when
they differ about interpretations.
Three light reactions
PSP
(DPIPH7-NADP)
FIG. 4. Photophosphorylation (PSP) relative to equal light
absorption at 664 and 714 nm. Effectiveness of red (664 nm)
and far-red (714 nm) monochromatic light for cyclic and noncyclic photophosphorylation, expressed on the basis of equal
light absorption at each wavelength (Arnon et al., ref. 102).
DPIPH2, reduced 2,6-dichlorophenol indophenol.
of monochromatic light on plant photosynthesis. This work
has led to wide agreement (see reviews, 89, 90) that photosynthesis in green plants includes two photosystems, one involving light reactions that proceed best in "short" wavelength (X < 700 nm) light (System II) and another-known
as System I-that proceeds best in "long" wavelength (X >
700 nm) light. It became important, therefore, to determine
the relation of cyclic and noncyclic photophosphorylation to
these two photosystems.
Soon after ferredoxin-catalyzed cyclic photophosphorylation was discovered, it was found to proceed most effectively
in long-wavelength light associated with System I. By contrast, noncyclic photophosphorylation (and oxygen evolution) was found to proceed best in short-wavelength light and
to come to an almost complete halt at wavelengths above 700
nm (87). Expressed on the basis of equal absorption by chloroplasts of light at each wavelength, the sharp decline or "red
drop" 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 wavelengths (Fig. 4). Thus, on the basis of their response to
monochromatic light, noncyclic photophosphorylation was
identified with System II and cyclic photophosphorylation
with System I.
Fig. 4 also shows that the wavelength dependence of the
phosphorylation associated with electron flow from an artificial electron donor (reduced 2,6-dichlorophenol indophenol) to NADP (91) resembles the cyclic system. This
similarity suggests that electron transport involved in the
photoreduction of NADP by DPIPH2 proceeds via (a portion
of) System I and is not involved in the photoreduction of
NADP by water via System II (92, 93). In this view (further
elaborated below), ferredoxin-NADP could be reduced either
by water via System II or by an artificial electron donor via
System I. System I identified with cyclic, and System II
identified with noncyclic, electron transport could thus be
regarded as parallel processes in chloroplasts, each basically
capable 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
Until recently there was general agreement that System II
included only one short-wavelength light reaction. But recent
experiments in our laboratory on electron transport in isolated
chloroplasts have yielded evidence (96-104) for two shortwavelength photoreactions (Ila and Ilb) in the noncyclic
electron transport from water to ferredoxin-NADP. These
two photoreactions of System II are linked in series; together
with 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 Component
550 (C550), while photoreaction Ila oxidizes plastocyanin
(PC) and reduces ferredoxin. These two light reactions are
joined by a "dark" electron transfer chain which includes
(but is not limited to) cytochrome b559 and is coupled to the
noncyclic phosphorylation site.
Component 550 is a newly discovered photoreactive chloroplast component, distinct from cytochromes, which undergoes
photoreduction by electrons from water (or a substitute
electron donor) only when chloroplasts are illuminated by
System II (short-wavelength) light (96-98). The photoreduction of Component 550 is measured by a decrease in absorbance at 550 nm (546 nm at 770K) but its chemical nature is
otherwise still unknown. The existence of Component 550
has now been confirmed by Erixon and Butler (105, 106),
Boardman et al. (107), and Bendall and Sofrova (108). Erixon
and Butler (106) have also added the significant observation
that Component 550 acts as the previously postulated
quencher (Q) of fluorescence of System II.
Plastocyanin is a copper-containing protein in chloroplasts
discovered by Katoh (109) and implicated in noncyclic
electron transport from water to NADP (110-113). Cytochrome b559 is one of the three cytochromes native to chloroplasts, the other two being cytochromes f and bN. Cytochrome
b559 has been associated with chloroplast fragments enriched
in System II (refs. 114-116). Some investigators (117-119)
have proposed that it serves as an electron donor to cytochrome f in an electron transport chain that joins Systems
II and I. In this formulation, plastocyanin is a requlirement
for the photooxidation by System I of both cytochromes
b559 andf:
H20-*- hvip,, Q cyt. b559 - cyt. f->PC
hvi Fd -NADP
Our recent experiments show that removal of plastocyanin
from chloroplasts abolished their capacity to photooxidize
cytochrome b599 but not that of cytochrome f. The addition
of plastocyanin restored the photooxidation of cytochrome
Proc. Nat. Acad. Sci. USA 68
-0.4
(1971)
Light Reactions of Photosynthesis
-
2889
-0.4
NADP '
-0.2
-0.2
0
0
en
0
0
-0
+0.2
+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
effect on the photoreduction of cytochrome b559 by Component 550, nor on the photoreduction of Component 550
itself by photoreaction IIb (97). These findings, buttressed
by the observation (99, 96, 100) that cytochrome b5i9, unlike
cytochrome f, is photooxidized effectively only by System II
(short-wavelength) light, account for the proposed sequence
of electron carriers in System II (Fig. 5).
Cytochromes b6 and f have previously been assigned to
cyclic photophosphorylation (92, 93) and are now also considered components of System I. The present concept places
cytochrome f and P700 in System I and, in contrast to the
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
energy absorbed by the "bulk" chlorophyll of System I and
to participate directly in photochemical reactions (120). ]
Experimental verification of this prediction was sought
from experiments with chloroplast fragments (101, 104). The
concept of three photoreactions arranged in two parallel
photosystems was greatly strengthened when chloroplasts
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 pertinent demonstration was the photoreduction of ferredoxinNADP 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
impossible according to the old hypothesis.
no
of System I. A primary electron acceptor would be expected to
undergo reduction by an electron transferred solely as a result
of photon capture at temperatures low enough to inhibit
chemical reactions. This expectation was fulfilled for photoreaction 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-a
technique that, unlike absorption spectrophotometry, would
permit detection of ferredoxin reduction without the interference of chlorophyll or chloroplast cytochromes. When whole
spinach chloroplasts were illuminated at 770K from 10 to 50
Ul
H
~~548-
540
55056
0
\
K
546
540
Photoreduction of primary electron acceptors at 770K
The new scheme for electron transport envisages two primary
electron acceptors: Component 550 in photoreaction lIb and
ferredoxin in photoreaction Ila and in the single photoreaction
ASPINA
s
-2-
/
M
LETTUCE
546
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; reference
wavelength, 538 nm, Knaff and Arnon, ref. 98).
2890
Amnon
Proc. Nat. Acad. Sci. USA 68 (1971)
241
System II
System I
(H20-NADP)
(DPIPH2-NADP)
I24
8
,6
k.)
S0
0)
k
4
w
1
550
600
650
700 730
550
600
v
--""
19~~~
650
700 730
3
2
wavelength (nm)
FIG. 8. Quantum requirements of light-induced electron
NADP) and System I (DPIPH2
- NADP) in monochromatic light of different wavelengths.
(Data of B. D. McSwain.)
transport by System II (H20
3200
3300
34
Magnetic field (gauss)
FIG. 7. Low-temperature light-induced EPR signals in spinach
chloroplasts. Illumination: 770K; EPR: 250K (Malkin and
Bearden, ref. 121).
min and examined by EPR spectroscopy at 250K, light-induced changes in the EPR spectrum were observed (121).
Fig. 7 shows the EPR spectrum of chloroplasts in the dark
and when illuminated for 20 min at 770K. Illumination induced increased EPR absorption at g-values of 1.86, 1.94, and
2.05. No other light-induced absorptions were detected when
the scanning range was widened to include g-values from 1.5
to 6. The large (off-scale) signal- in the g = 2.00 region, which
occurred in both the light and dark samples, was due to free
radicals (not associated with ferredoxin) previously observed
in photosynthetic systems by the EPR technique (122).
It appears that the light-induced EPR spectrum of chloroplasts was produced by a bound type of plant ferredoxin different from the soluble ferredoxin contained in whole chloroplasts.
Illumination at 770K of broken chloroplasts, prepared by
osmotically disrupting whole chloroplasts and washing to remove any remaining soluble ferredoxin, gave an EPR spectrum
similar to that observed with whole chloroplasts (121). The
absence of soluble ferredoxin in this chloroplast preparation
was evidenced by its inability (without added ferredoxin) to
reduce NADP photochemically with either water or reduced
dye as the hydrogen donor.
Quantum efficiency of photosynthesis
Few questions in photosynthesis have received more intensive
theoretical and experimental study and led to more controversy than the efficiency of the quantum conversion process.
The extensive literature on this subject, which is beyond the
scope of this article, concerns quantum efficiency of complete
photosynthesis in whole cells, as measured by oxygen evolution or CO2 fixation. A different approach to the bioenergetics
of photosynthesis emerged from the electron flow theory that
was invoked to explain cyclic and noncyclic photophosphorylation. Transfer of one electron from excited chlorophyll to a
primary electron acceptor, Component 550 or ferredoxin, is
depicted as a one-quantum photochemical act. Since two
photoacts are involved in transfer of an electron from water
to ferredoxin-NADP (Fig. 5), the theoretical quantum requirement for System II becomes two quanta per electron or
eight quanta per molecule of 02 (Eq. iv). Likewise, with only
one photoact in System I, its light-induced electron transport
would have a theoretical quantum requirement of one quantum per electron.
Fig. 8 shows experimental measurements close to two quanta
per electron for System II and one quantum per electron for
System I. The quantum requirement measurements in Fig. 8
demonstrate again the contrasting responses of Systems II and
I to monochromatic light: increasing quantum efficiency for
System II and decreasing quantum efficiency for System I at
the shorter wavelengths and a reverse situation at the longer
wavelengths. Similar results, despite many variations in conditions and experimental design, have been obtained by other
investigators (123-129).
Since the assimilation of one molecule of CO2 to the level of
hexose via the reductive pentose phosphate cycle requires two
molecules of NADPH2 and three molecules of ATP (Fig. 2), it
becomes possible to arrive at the maximum theoretical quantum efficiency of photosynthesis from measurements of the
respective quantum efficiencies for cyclic and noncyclic electron transport. A requirement of two quanta per electron for
System II means that eight quanta would be required to
produce two NADPH2 and two ATP (accompanied by evolution of one 02) via noncyclic photophosphorylation. Two additional quanta assigned to generate one ATP via cyclic photophosphorylation would give a total requirement of about ten
quanta per molecule of CO2 assimilated or 02 liberated-a
value in good agreement with measurements of the overall
quantum efficiency of photosynthesis in intact cells.
The experimental work from the author's laboratory cited
herein was supported, in part, by National Science Foundation
Grant GB-23796.
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