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Photophosphorylation
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Article Contents
Andrew N Webber, Arizona State University, Tempe, Arizona, USA
. Introduction
Photophosphorylation is the process through which photosynthetic organisms convert
light energy to adenosine triphosphate.
. Physical Organization of the Process in Chloroplasts
. Chlorophylls and Accessory Pigments
. Light Absorption by Antennas
Introduction
. Photosystems I and II: Structures, Organization, Light
Absorption, Mechanisms
Photosynthetic organisms are able to convert light energy
into a chemically useful form (adenosine triphosphate;
ATP) by a process termed photophosphorylation. Photophosphorylation is performed by membrane-associated
protein complexes that serve to capture the light energy
(the reaction centres) and subsequently use the captured
energy to make ATP (the ATP synthase complex). The
unifying principle in all photosynthetic organisms (prokaryotic or eukaryotic) is that they use the captured light
energy to initiate electron transfer, and produce a charge
separation across a membrane. Following the charge
separation process, secondary electron transfer reactions
occur energetically downhill, with some of the energy being
conserved as a proton electrochemical gradient across the
membrane. It is this proton electrochemical gradient that is
the driving force for ATP production.
In oxygenic photosynthetic organisms, two different
reaction centres, termed photosystem II and photosystem
I, act in series to transfer electrons from the ultimate
electron donor, water, to the terminal electron acceptor,
NADP 1 . Photosystem II generates a powerful oxidant
capable of extracting electrons from water, generating
oxygen and protons. Electrons are transferred from
photosystem II to an organic molecule termed plastoquinone to generate the reduced form, plastoquinol. Electrons
from plastoquinol are then transferred to the cytochrome
b6/f complex. Cytochrome f then reduces plastocyanin,
which then transfers its electron to photosystem I. A
second charge separation generates a powerful reductant
that eventually transfers electrons to ferredoxin, and
ultimately to NADP 1 . The proton gradient is generated
by water oxidation and electron transfer between photosystem II and photosystem I.
Physical Organization of the Process in
Chloroplasts
In eukaryotic organisms, photosynthesis occurs inside a
specialized cellular organelle known as the chloroplast
(Figure 1). The chloroplast is delimited by a double
membrane, called the outer and inner envelope membrane.
The outer membrane is porous to most molecules, whereas
the inner membrane serves as a barrier, controlling import
. Establishment of a Proton Electrochemical Potential
and Use for ATP Synthesis
. Production of Oxygen
. Anoxygenic Photosynthetic Organisms
. Reaction Centre of Purple Bacteria
and export of sugars, phosphate and other molecules, using
specialized transport proteins. The inside of the chloroplast is called the stroma. The stroma contains the soluble
enzymes involved in the reduction of carbon dioxide to
sugars. The chloroplast is a semiautonomous organelle
and contains its own DNA. The chloroplast genome is very
small and contains information for the biosynthesis of only
a limited number of chloroplast components. The remainder of the genes required for chloroplast biogenesis are
located in the nucleus (Webber and Baker, 1996).
Inside the chloroplast is an extensive highly structured
membrane, termed the thylakoid, that contains the protein
complexes required for light harvesting, electron transfer,
water oxidation and ATP synthesis. The thylakoid is a
single membrane that forms a large flattened sac enclosing
the lumen. The membrane is highly folded, forming large
stacks (grana) interconnected by regions of unstacked,
nonappressed membrane regions, termed the stromal
lamellae (Figure 1). The physiological reason for the
extensive membrane stacking is still unclear. The highly
structured nature of the thylakoid, however, is thought to
reflect a lateral organization of protein complexes in the
membrane. Photosystem II and associated light-harvesting
complexes are primarily associated with the stacked
regions, whereas photosystem I and the ATP synthase
complex are located in the stromal lamellae. Electron
transfer between photosystem II and photosystem I, via
the cytochrome b6/f complex, is facilitated by lateral
diffusion of plastoquinone and plastocyanin.
Chlorophylls and Accessory Pigments
Light energy is captured by specialized pigments that are
mostly found associated with proteins. In plants and algae,
the major pigments are chlorophyll a, chlorophyll b and
carotenoids. Chlorophylls contain a porphyrin ring
attached to a hydrophobic phytol side-chain. Chlorophylls
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Photophosphorylation
Figure 1 (a) Electron micrograph of an isolated chloroplast. The chloroplast is delimited by a double envelope (CE) membrane. Inside is the stroma (S)
containing the thylakoid membrane. The thylakoid membrane is highly folded, forming granal stack regions (GS) interconnected by stromal exposed
membrane regions.
(b) Schematic of the thylakoid membrane showing the major protein complexes and associated cofactors.
absorb blue and red light, and so appear green to the eye. In
chlorophyll b a formyl group replaces a methyl group on
the porphyrin ring, causing a slight red shift in the
absorption maxima, thus increasing the range of wavelengths of light absorbed. Chlorophylls serve both as lightharvesting antenna pigments and as components of the
electron transfer chain (described below).
2
Carotenoids absorb blue-green light with absorption
maxima between 420 nm and 480 nm. Carotenoids serve as
accessory light-harvesting pigments, and also fulfil a
photoprotective role in the antenna. Excess light conditions can be potentially damaging to the photosynthetic
apparatus if the energy is not dissipated in a harmless form.
When excited, chlorophyll can undergo a process called
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Photophosphorylation
intersystem conversion, where the excited singlet state
forms a triplet state. The triplet state of chlorophyll is long
lived and may transfer its energy to oxygen. Carotenoids
are able to quench the energy from potentially damaging
excited molecules and dissipate the energy in a harmless
form.
Certain carotenoids, termed xanthophylls, are also able
to quench excitation energy by a phenomena known as
‘nonphotochemical’ quenching (Demmig-Adams, 1990;
Owens, 1996). Under high-light conditions, violozanthin is
rapidly converted to zeazanthin by enzymes associated
with the thylakoid membrane lumen. Zeaxanthin is
thought to have an energy level below chlorophyll, and
so can act as an excited state quencher. Although the
mechanism of quenching is not known in detail, the
xanthophyll cycle, as it is now known, is thought to play a
major role in regulating the dissipation of excitation energy
in antenna complexes, before that energy can be used to
initiate photochemical processes that could lead to
photooxidative damage at high light.
Light Absorption by Antennas
Light energy is captured by pigment molecules in the
antenna complexes and then migrates by excitation energy
transfer to the specialized pigments, termed reaction
centres, where electron transport is initiated. The vast
majority of pigments in photosynthetic organisms function
in light harvesting so that the light energy, which is
relatively dilute, can be efficiently collected and used by the
reaction centre. Photosynthesis is initiated when a photon
of light is absorbed by a pigment molecule of the antenna.
This causes the transition of a delocalized electron from the
ground state to the lowest unoccupied molecular orbital,
producing an excited state molecule. The excited state
energy is then transferred to nearby pigments by a process
termed resonance energy transfer. The rate of transfer is
dependent upon the distance between the donor and
acceptor molecules, the relative orientation of the transition dipoles, and the overlap between the donor emission
spectra and the acceptor absorption spectrum (VanGrondelle and Amesz, 1986). The chlorophyll and other
accessory pigments are thought to be organized by the
protein so as to optimize transfer so that the excitation
energy is rapidly funnelled to the reaction centre. The
three-dimensional structure of several chlorophyll protein
complexes, including the light-harvesting chlorophyll a/b
complex associated with photosystem II (Kuhlbrandt et al.,
1994), are beginning to reveal this precise organization in
the case of chlorophyll a molecules.
Photosystems I and II: Structures,
Organization, Light Absorption,
Mechanisms
Photosystem II uses light energy to oxidize water and
reduce plastoquinone. The photosystem II complex contains several different cofactors that are involved in
mediating the transfer of electrons from water to plastoquinone (Diner and Babcock, 1996; Ruffle and Sayre,
1998). These include manganese (associated with the
water-oxidizing complex described below), a tyrosine, the
reaction centre chlorophyll a (P680), pheophytin a (Pheo;
chlorophyll a lacking the central magnesium) and quinone
(QA and QB). Additional cofactors in photosystem II are
antenna chlorophyll a and cytochrome b559. Electron
transfer is initiated when excitation energy reaches P680.
The excited state reaction centre chlorophyll, P*680,
transfers an electron to pheophytin to form the charge1
Phe 2 . Subsequently, the electron is
separated state P680
rapidly transferred across the photosystem II complex
(and across the thylakoid membrane) to the quinone
acceptor QA. This further separation of the positive and
negative charge across the membrane serves to minimize
the rate of wasteful charge recombination, increasing the
quantum yield of electron transfer to close to unity. The
electron is then transferred from QA to a second quinone,
QB, which serves as a two-electron acceptor, and is only
loosely associated with photosystem II at the QB site. Each
electron transfer to QB is also associated with a protonation of the quinone. The doubly reduced QB molecule
dissociates from photosystem II, and migrates randomly
through the thylakoid membrane until it reaches a
cytochrome b6/f complex. Approximately nine unbound
QB molecules (both reduced and oxidized) per photosystem II complex form the plastoquinone pool.
The photosystem II complex contains numerous ( 4 26)
individual polypeptide components that coordinate cofactors and/or serve a structural role (Ruffle and Sayre, 1998).
Several key proteins in photosystem II make up what is
known as the reaction centre core. In particular, two
related proteins, D1 and D2, coordinate the cofactors
involved in electron transfer, i.e. manganese, P680, pheophytin, QA and QB. The D1 and D2 polypeptides are also
closely associated with the haem-containing cytochrome
b559 complex. While cytochrome b559 is redox active, its
role in photosystem II is unknown, although some
evidence suggests a role in photoprotection of the reaction
centre. Additional chlorophyll a molecules are coordinated
by proteins named CP43 and CP47. CP43 and CP47
function as antenna pigment proteins, funnelling excitation energy to the reaction centre.
The photosystem I complex catalyses the transfer of an
electron from plastocyanin (PC) to ferredoxin (F). Photosystem I consists of approximately 13 individual proteins,
and contains close to 100 chlorophyll a (Webber and
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Bingham, 1998). The structure of the photosystem I
complex has been solved at 0.4 nm resolution, providing
useful information on the organization of the different
cofactors (Krauss et al., 1996). In photosystem I the
antenna chlorophyll a, and chlorophyll a components of
the electron transfer chain (six chlorophyll a), are
associated with two proteins, PsaA and PsaB, that form
the reaction centre heterodimer. Light energy is transferred
from the antenna chlorophyll a to the reaction centre
chlorophyll a dimer (P700). Excitation of P700 results in
electron transfer to the first electron acceptor, a chlorophyll a molecule named A0. An additional chlorophyll a
molecule, termed A, is positioned between P700 and A0, but
has not yet been detected as an electron transfer
intermediate. Electrons are then transferred from A0 to
ferredoxin through several electron carriers, called A1 (a
phylloquinone), FX, FA and FB. FX, FA and FB are iron–
sulfur clusters. FA and FB are 4Fe–4Fe clusters coordinated by a small polypeptide very similar to ferredoxin.
The detailed structure of photosystem I indicates that
there are two potential branches for electron transfer
between P700 and FX. The structure shows that P700 is a
dimer, and that between P700 and FX are four chlorophyll a
molecules. These chlorophylls reside on either side of a
symmetry axis running through P700 and FX. The two
chlorophylls furthest from P700 represent A0. The chlorophyll a molecules between P700 and A0 are named A and
serve an as yet unknown function. Although two quinone
molecules have been identified in the photosystem I crystal
structure, only a single quinone radical (per P700) is
detected spectrophotometrically, raising the intriguing
possibility that only one of the electron transfer branches
is operative. This is similar to the case in reaction centres of
purple sulfur bacteria, described below.
Establishment of a Proton
Electrochemical Potential and Use for
ATP Synthesis
Electron transfer from water to NADP 1 requires the
operation of three complexes – photosystem II, cytochrome b6/f and photosystem I – acting in series. The
doubly reduced QB dissociates from photosystem II and
becomes a part of the plastoquinone pool. The PQH2
diffuses to the cytochrome b6/f complex where it is
oxidized, concomitantly releasing protons that are deposited in the thylakoid lumen. The cytochrome b6/f complex
contains cytochrome b6, cytochrome f, Rieske protein, and
several additional polypeptides. Electrons are next transferred from the cytochrome b6/f complex to photosystem I
by plastocyanin, a small soluble copper-containing protein
located in the thylakoid lumen.
4
Photosynthetic electron transfer and water oxidation
lead to the accumulation of protons on the lumenal side of
the thylakoid membrane. In addition, primary charge
separation generates a charge difference across the
membrane. The combination of a proton concentration
and charge difference across the thylakoid membrane is
termed the electrochemical proton gradient, and drives the
synthesis of ATP from ADP and phosphate.
The proton electrochemical energy is used to make ATP
by a single thylakoid protein complex called the ATP
synthase. The ATP synthase is composed of two major
components termed CF0 and CF1. CF0 is comprised of
several different subunits, and spans the thylakoid
membrane forming a proton channel through the membrane. The CF1 component is made up of five different
proteins termed a, b, d, e and g. Each CF1 contains three ab
dimers, which together form three distinct catalytic sites.
Each interface of an ab dimer can bind ADP and
phosphate, and form ATP. However, each of the three
sites has a different affinity for the nucleotides. Passage of
protons through the CF0 component is thought to change
the conformation of one of the sites, causing ATP to be
released. One model suggests that the passage of protons
causes CF1 to rotate on top of CF0 so that there is rapid
interconversion of the three different nucleotide binding
sites. As the catalytic sites interconvert, ATP is released.
Production of Oxygen
Photosynthesis has produced all the oxygen in the Earth’s
atmosphere. Despite the importance of this reaction, it is
one of the most poorly understood areas of photosynthesis. Water oxidation is mediated by photosystem II, which
is the only complex that can oxidize water. On the lumenal
side of photosystem II are several extrinsic proteins that
form the oxygen-evolving complex, and are required for
water oxidation.
Water oxidation requires the removal of four electrons
from two molecules of water. The oxidation–reduction
potential of water is 1 0.82 mV, which means that it is an
extremely difficult compound to oxidize. In photosystem
1
II, the oxidation of water is driven by P680
, which has a
midpoint potential estimated at 1 1.2 mV, making it a very
1
oxidizes a tyrosine amino acid
strong oxidizing agent. P680
on the D1 protein. The tyrosine radical cation then is able
to oxidize the manganese ions of the oxygen-evolving
complex by an as yet unknown mechanism. There are four
manganese atoms associated with the oxygen-evolving
complex. Four electrons need to be removed from two
molecules of water to produce oxygen. Each electron is
removed sequentially from components of the oxygenevolving complex, with oxygen evolution occurring only
after the fourth electron is removed. This was shown by
subjecting photosystem II to a series of saturating light
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Photophosphorylation
flashes, such that each flash initiates one photochemical
turnover, and following the pattern of oxygen release (Kok
et al., 1970). It was found that oxygen is released every
fourth flash. It is now believed, based on X-ray absorption
spectroscopy, that it is some of the manganese ions that
undergo progressive oxidation following each photosystem II turnover, and oxidize water when sufficient charge
has been accumulated.
Anoxygenic Photosynthetic Organisms
Anoxygenic photosynthetic bacteria are unable to oxidize
water and instead use a range of electron donors with less
positive reduction potentials, such as sulfide, thiosulfate or
succinate. All anoxygenic photosynthetic bacteria contain
only a single type of reaction centre. In the case of the
purple bacteria (either nonsulfur purple bacteria or sulfur
purple bacteria) the reaction centre contains a quinone as
the terminal electron acceptor in the complex. In purple
bacteria electron transfer is cyclic. The reduced quinone
produced by charge separation is reoxidized by the
cytochrome b/c complex. The b/c complex then reduces
cytochrome c2, which then transfers its electron to the
1
. Associated with the reaction
oxidized primary donor, P870
centre are antenna complexes, termed light-harvesting
complexes I and II, that are intrinsic membrane proteins
binding bacteriochlorophyll and carotenoids. The structure of the reaction centre from purple bacteria has been
solved at very high resolution (described below). The
reaction centre of purple bacteria is very similar to
photosystem II, and has served as an excellent model to
Figure 2 Structure of the reaction centre from purple photosynthetic bacteria. The L, M and H subunits making up the reaction centre complex are shown
in yellow, blue and green. Cofactors are shown in red. Reproduced courtesy of Jim Allen.
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Photophosphorylation
guide our understanding of the structure and function of
photosystem II.
Green sulfur bacteria contain reaction centres that have
iron–sulfur centres as the terminal electron acceptor, and
are therefore similar to photosystem I. The iron–sulfur
centre can reduce ferredoxin, which then reduces NAD 1 .
Green sulfur bacteria contain an antenna complex
associated with the surface of the membrane. This complex
contains bacteriochlorophyll and carotenoids and is
termed a chlorosome. Green gliding bacteria also have
chlorosomes, but their reaction centre is similar to that of
purple bacteria. In the 1980s a group of Gram-positive
photosynthetic bacteria, called Heliobacteria, were discovered. These bacteria also have reaction centre complexes similar to photosystem I, but contain
bacteriochlorophyll g as the main photosynthetic pigment.
Reaction Centre of Purple Bacteria
The reaction centre complex from anoxygenic purple
bacteria is the best understood of all reaction centres, and
most of our information on the molecular basis of the early
stages of photosynthesis is based on studies of this system.
In particular, the X-ray structure of this class of reaction
centre has been known for over a decade (Figure 2); this
provides a very good understanding of cofactor organization and protein interaction.
The reaction centre from purple bacteria contains two
related integral membrane proteins called the L and M
subunits. Many also contain an H subunit and a c-type
cytochrome complex. The L and H subunits bind the
cofactors involved in electron transport. These include
four bacteriochlorophyll a, two bacteriopheophytin, two
quinones (either ubiquinone or menaquinone), an iron
atom and, in most cases, a carotenoid.
The reaction centre proteins L and M are very
hydrophobic and each have five membrane-spanning a
helical regions. The H subunit also contains a single
transmembrane a helix. The L and M subunits are
organized such that they show a pseudo-C2 symmetry
around an axis running perpendicular to the membrane
plane. The electron transfer cofactors are similarly
organized about the C2 symmetry axis. The primary
6
electron donor is a dimer of two bacteriochlorophyll a
molecules. The subsequent cofactors, two bacteriochlorophyll a, two bacteriopheophytin and two quinones, are
organized along either side of the symmetry axis running
through the primary donor and the nonhaem iron located
between QA and QB. Thus there are two potential branches
of electron transfer, referred to as the A and B branches.
However, for reasons that remain unclear, electron
transfer occurs predominantly along the A (active) branch
cofactors.
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