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University of Groningen
Structures of photosynthetic membrane complexes
Semchonok, Dmytro
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Semchonok, D. A. (2016). Structures of photosynthetic membrane complexes [Groningen]: Rijksuniversiteit
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SUMMARY
Some concepts of photosynthesis in a nutshell
Photosynthesis is essential to all life on Earth and all organisms depend on it
in a direct or indirect way. It is the biological process that can capture
energy that originates in outer space (sunlight) and convert it into chemical
compounds (carbohydrates) that every organism uses to power its
metabolism (OpenStax CNX - Biology). This conversion takes many steps,
catalyzed by a series of special proteins. Therefore, photosynthesis is
divided in two stages: the light-dependent reactions and the lightindependent (“dark”) reactions. During the light-dependent reactions,
energy from sunlight is absorbed by pigments and converted into chemical
energy stored in the form of NADPH and ATP. The first steps in this
process are accomplished by protein complexes on which multiple
chlorophyll and carotenoid pigment molecules are bound. The next steps
provide the conversion of the energy into NADPH and ATP. During the
light-independent reactions, the stored energy drives the assembly of sugar
molecules from carbon dioxide. Therefore, although the light-independent
reactions do not use light as a reactant, they require the products of the lightdependent reactions to function. In addition, several enzymes of the lightindependent reactions are activated by light (OpenStax CNX - Biology) .
Light energy is converted into chemical energy in multiprotein complexed
called photosystems. Two types of photosystems, photosystem I (PSI) and
photosystem II (PSII), are found in a special photosynthetic membrane,
called the thylakoid membrane. The heart of the photosystems is the
reaction center. Photosystems function in oxygenic photosynthesis, and
exist in all green plants and some lower organisms such as cyanobacteria.
For some prokaryotes the complex responsible for light conversion is less
complex and is named a reaction center.
Both photosystems and reaction centers need some additional antenna
complexes for light absorption and transfer. These functions are executed by
membrane-bound light harvesting protein complexes and water-soluble
antenna complexes. There is a substantial variation in composition and size
between groups of organisms. Another important point is the association of
photosystems and reaction centers into higher complexes. Unlike the
monomeric reaction centers in green and purple bacteria, PSI forms trimeric
complexes in most cyanobacteria with a 3-fold rotational symmetry. More
recently, in some species a tetrameric PSI complex was observed. However,
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in plants/algae, PSI is monomeric. In contrast, PSII in cyanobacteria and
plants is monomeric or dimeric, but never forms trimers or tetramers.
In chapter 2, we are discussing the attachment of phycobilisomes in an
antenna-photosystem I supercomplex of cyanobacteria. The phycobilisome
(PBS) is an interesting example of a large water-soluble antenna, only found
in algae and cyanobacteria and absent in higher plants. Both photosystem I
and photosystem II in cyanobacteria have a specific internal antenna
complex, but the PBS is the major peripheral antenna protein complex in
cyanobacteria, typically consisting of a core from which several rod-like
subcomplexes protrude. PBSs preferentially transfer light energy to PSII,
whereas a PSI-specific antenna has not been identified. The cores and the
rods of a PBS are hold together by several linker proteins. The
cyanobacterium Anabaena sp. PCC 7120 has rod-core linker genes (cpcG1cpcG2-cpcG3-cpcG4). Their products, except CpcG3, have been detected in
the conventional PBS. Here we report the isolation of a supercomplex that
comprises a PSI tetramer and a second, unique type of a PBS, specific to
PSI. This rod-shaped PBS includes phycocyanin protein copies and CpcG3
(hereafter renamed "CpcL"), but no allophycocyanin copies or CpcGs.
Fluorescence excitation showed efficient energy transfer from PBS to PSI.
The supercomplex was analyzed by electron microscopy and single-particle
averaging. In the supercomplex, one to three rod-shaped CpcL-PBSs
associate to a tetrameric PSI complex. They are mostly composed of two
hexameric plastocyanin units and bind at the periphery of PSI, at the
interfaces of two monomers. Structural modeling indicates, based on twodimensional projection maps, how the PsaI, PsaL, and PsaM subunits link
PSI monomers into dimers and into a rhombically shaped tetramer or
"pseudotetramer." The 3D model further shows where PBSs associate with
the large subunits PsaA and PsaB of PSI. It is proposed that the alternative
form of CpcL-PBS is functional in harvesting energy in a wide number of
cyanobacteria, partially to facilitate the involvement of PSI in nitrogen
fixation.
In chapter 3, we studied another organism, the unicellular cyanobacterium
Chroococcidiopsis sp. TS-821 (TS-821), that also contains a tetrameric PSI
complex. In TS-821, PSI forms tetrameric and dimeric species that were
investigated by Blue Native PAGE, sucrose density gradient centrifugation,
77K fluorescence, circular dichroism, and single-particle analysis.
Transmission electron microscopy analysis of native membranes confirms
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the presence of the tetrameric PSI structure prior to detergent solubilization.
Single particle analysis shows that the tetramers are similar to those of
Anabaena, studied in chapter 2. They are again pseudo-tetramers, but appear
less elongated than those of Anabaena. It is an open question why tetramers
are formed instead of trimers. Another question is how they are made,
because the trimers and tetramers have the same set of small subunits
involved in association of momomers into multimers. To investigate why
TS-821 forms tetramers instead of trimers, the PsaL gene known to be
responsible for trimer formation was cloned and analyzed. Interestingly, this
gene product contains a short insert between the second and third predicted
transmembrane helices. Phylogenetic analysis based on PsaL protein
sequences shows that TS-821 is closely related to heterocyst-forming
cyanobacteria, some of which also have a tetrameric form of PSI. These
results are discussed in light of chloroplast evolution, and was propose that
PSI evolved stepwise from a trimeric form to tetrameric oligomer en route
to becoming monomeric in plants/algae.
In chapter 4, we are continuing our investigation of the tetrameric form of
PSI from the unicellular cyanobacterium Chroococcidiopsis sp. TS-821
using the cry-electron microscopy (cryo-EM) technique. The aim is to study
the tetramerization ability of TS-821 in more detail. The investigation was
focused on dimer-dimer intermonomer space where some PSI subunits of
each monomer are creating the contact side with the neighboring monomer.
The obtained cryo-EM volume density map at 11.5 Å resolution provides a
general idea about orientation of PSI units inside TS-821. Two different
interfaces created by monomer-monomer interactions were found due to
different contacts of PSI subunits. The crystal structure PSI from
Thermosynechococcus elongatus was fitted in in order to see the contacts
between PSI monomers in the intermonomer space in more detail. The PsaL
subunit of PSI known to participate in the trimerization of PSI in
Thermosynechococcus was modified according to amino acid sequence of
TS-821. The crystal structure of PSI from T. elongatus with modified PsaL
subunit shows better fitting inside obtained 3D volume density map. It is
also concluded that besides PsaL subunit, PsaI, PsaM, PsaA, PsaB subunits
are also involved into the PSI tetramer formation.
In chapter 5, we discuss the organization of a photosynthetic complex in a
bacterium. The principle of a reaction center surrounded by a peripheral
antenna is the same as in green plants and cyanobacteria, but in non194
oxygenic bacteria the reaction center and antenna complexes are
substantially smaller. The reaction center (RC) surrounded by light
harvesting antenna complexes (RC-LH1) of purple bacteria can be either
monomeric or dimeric, depending on the presence of a small protein named
PufX.
Electron microscopy and single-particle averaging were performed on
isolated RC-LH1-PufX complexes of Rhodobaca bogoriensis strain LBB1,
with the aim of establishing the LH1 antenna conformation, and, in
particular, the structural role of the PufX protein in the dimerization of RCLH1 complexes. Projection maps of dimeric complexes were obtained at 13
Å resolution and show the positions of the 2 × 14 LH1 units, each composed
of an α- and β-subunit. This new dimeric complex displays two open, Cshaped LH1 aggregates of 13 αβ polypeptides partially surrounding the RCs
plus two LH1 units forming the dimer interface in the center. Between the
interface and the two half rings are two openings on each side. Next to the
openings, there are four additional densities present per dimer, considered to
be occupied by four copies of PufX. The position of the RC in our model
was verified by comparison with RC-LH1-PufX complexes in membranes.
Our model differs from previously proposed configurations for Rhodobacter
species in which the LH1 ribbon is continuous in the shape of an S, and the
stoichiometry is of one PufX per RC.
One of the important regulatory processes that can occur in plant
photosynthesis is state transition. State transition is a mechanism of
redistribution of the energy between photosystem I and II. In this process
the main players are photosystem I and II and trimeric light-harvesting
complex II (LHCII). LHCII can bind to both PSI and PSII and by
redistribution of LHCII between PSI and PSII the relative excitation of both
photosystems is balanced and optimized. Some aspects of this process are
discussed in chapter 6.
State transitions are driven by reversible LHCII phosphorylation by the
STN7 kinase and PPH1/TAP38 phosphatase. The LHCII trimers are,
however, heterotrimers making the situation more complex. LHCII trimers
are composed of Lhcb1, Lhcb2, and Lhcb3 proteins in various trimeric
configurations. Here, we show that despite their nearly identical amino acid
composition, the functional roles of Lhcb1 and Lhcb2 are different but
complementary. Arabidopsis thaliana plants lacking only Lhcb2 contain
thylakoid protein complexes similar to wild-type plants, where Lhcb2 has
been replaced by Lhcb1. However, these do not perform state transitions, so
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phosphorylation of Lhcb2 seems to be a critical step. In contrast, plants
lacking Lhcb1 had a more profound antenna remodeling due to a decrease in
the amount of LHCII trimers influencing thylakoid membrane structure and,
more indirectly, state transitions. Although state transitions are also found in
green algae, the detailed architecture of the extant seed plant lightharvesting antenna can now be dated back to a time after the divergence of
the bryophyte and spermatophyte lineages, but before the split of the
angiosperm and gymnosperm lineages more than 300 million years ago.
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