purification-active-photosystem-i-light-harvesting-complex-i-from

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

Arizona State University

Despite the impressive progress made in recent years in understanding the early steps in charge separation within the photosynthetic reaction centers, our knowledge of how ferredoxin (Fd) interacts with the acceptor side of photosystem I (PSI) is not as well developed. Fd accepts electrons after transiently docking to a binding site on the acceptor side of PSI. However, the exact location, as well as the stoichiometry, of this binding have been a matter of debate for more than two decades. Here, using Isothermal Titration Calorimetry (ITC) and purified components from wild type and mutant strains of the green algae Chlamydomonas reinhardtii we show that PSI has a single binding site for Fd, and that the association consists of two distinct binding events, each with a specific association constant.

Binding of ferredoxin to algal photosystem I involves a single binding site and is composed of two thermodynamically distinct events.

Photochemical conversion in oxygenic photosynthesis takes place in two large protein-pigment complexes named photosystem II and photosystem I (PSII and PSI, respectively). Photosystems associate with antennae in vivo to increase the size of photosynthetic units to hundreds or thousands of pigments. Regulation of the interactions between antennae and photosystems allows photosynthetic organisms to adapt to their environment. In low-iron environments, cyanobacteria express IsiA, a PSI antenna, critical to their survival. Here we describe the structure of the PSI-IsiA complex isolated from the mesophilic cyanobacterium Synechocystis sp. PCC 6803. This 2-MDa photosystem-antenna supercomplex structure reveals more than 700 pigments coordinated by 51 subunits, as well as the mechanisms facilitating the self-assembly and association of IsiA with multiple PSI assemblies.

The structure of the stress-induced photosystem I-IsiA antenna supercomplex.

To identify the molecular composition of the low-energy states in cyanobacterial Photosystem I (PSI) of  PCC6803, we focus on high-resolution (low-temperature) absorption, emission, resonant, and nonresonant hole-burned spectra obtained for wild-type (WT) PSI and three PSI mutants. In the Red_a mutant, the B33 chlorophyll (Chl) is added to the B31-B32 dimer; in Red_b, histidine 95 (His95) on PsaB (which coordinates Mg in the B7 Chl within the His95-B7-A31-A32-cluster) is replaced with glutamine (Gln), while in the Red_ab mutant, both mutations are made. We show that the C706 state (B31-B32) changes to the C710 state (B31-B32-B33) in both Red_a and Red_ab mutants, while the C707 state in WT  (localized on the His95-B7-A31-A32 cluster) is modified to C716 in both Red_b and Red_ab. Excitation energy transfer from C706 to the C714 trap in the WT PSI and Red_b mutant is hampered as reflected by a weak emission at 712 nm. Large electron-phonon coupling strength (exposed via resonant hole-burned spectra) is consistent with a strong mixing of excited states with intermolecular charge transfer states leading to significantly red-shifted emission spectra. We conclude that excitation energy transfer in PSI is controlled by fine-tuning the electronic states of a small number of highly conserved red states. Finally, we show that mutations modify the protein potential energy landscape as revealed by different shapes and shifts of the blue- and red-shifted antiholes.

On the Red Antenna States of Photosystem I Mutants from Cyanobacteria  PCC 6803.

Photosystem I coordinates more than 90 chlorophylls in its core antenna while achieving near perfect quantum efficiency. Low energy chlorophylls (also known as red chlorophylls) residing in the antenna are important for energy transfer dynamics and yield, however, their precise location remained elusive. Here, we construct a chimeric Photosystem I complex in Synechocystis PCC 6803 that shows enhanced absorption in the red spectral region. We combine Cryo-EM and spectroscopy to determine the structurefunction relationship in this red-shifted Photosystem I complex. Determining the structure of this complex reveals the precise architecture of the low energy site as well as large scale structural heterogeneity which is probably universal to all trimeric Photosystem I complexes. Identifying the structural elements that constitute red sites can expand the absorption spectrum of oxygenic photosynthetic and potentially modulate light harvesting efficiency.

The structure of a red-shifted photosystem I reveals a red site in the core antenna.

A Correction to this paper has been published: https://doi-org.remotexs.ntu.edu.sg/10.1038/s41467-020-19953-w.

Author Correction: The structure of a red-shifted photosystem I reveals a red site in the core antenna.

Photosynthetic organisms have adapted to survive a myriad of extreme environments from the earth's deserts to its poles, yet the proteins that carry out the light reactions of photosynthesis are highly conserved from the cyanobacteria to modern day crops. To investigate adaptations of the photosynthetic machinery in cyanobacteria to excessive light stress, we isolated a new strain of cyanobacteria,  0216, from the extreme light environment of the Sonoran Desert. Here we report the biochemical characterization and the 2.7 Å resolution structure of trimeric photosystem I from this high-light-tolerant cyanobacterium. The structure shows a new conformation of the PsaL C-terminus that supports trimer formation of cyanobacterial photosystem I. The spectroscopic analysis of this photosystem I revealed a decrease in far-red absorption, which is attributed to a decrease in the number of long- wavelength chlorophylls. Using these findings, we constructed two chimeric PSIs in  sp. PCC 6803 demonstrating how unique structural features in photosynthetic complexes can change spectroscopic properties, allowing organisms to thrive under different environmental stresses.

The structure of photosystem I from a high-light-tolerant cyanobacteria.

The moss Physcomitrium patens diverged from green algae shortly after the colonization of land by ancient plants. This colonization posed new environmental challenges, which drove evolutionary processes. The photosynthetic machinery of modern flowering plants is adapted to the high light conditions on land. Red-shifted Lhca4 antennae are present in the photosystem I light-harvesting complex of many green-lineage plants but absent in P. patens. The cryo-EM structure of the P. patens photosystem I light-harvesting complex I supercomplex (PSI-LHCI) at 2.8 Å reveals that Lhca4 is replaced by a unique Lhca2 paralogue in moss. This PSI-LHCI supercomplex also retains the PsaM subunit, present in Cyanobacteria and several algal species but lost in vascular plants, and the PsaO subunit responsible for binding light-harvesting complex II. The blue-shifted Lhca2 paralogue and chlorophyll b enrichment relative to flowering plants make the P. patens PSI-LHCI spectroscopically unique among other green-lineage supercomplexes. Overall, the structure represents an evolutionary intermediate PSI with the crescent-shaped LHCI common in vascular plants, and contains a unique Lhca2 paralogue that facilitates the moss's adaptation to low-light niches.

The structure of the Physcomitrium patens photosystem I reveals a unique Lhca2 paralogue replacing Lhca4.

The PSI-IsiA supercomplex is one of the largest and most complicated assemblies in photosynthesis. The IsiA ring, composed of 18 IsiA monomers (IsiA) surrounding the PSI trimer (PSI), forms under iron-deficient conditions in cyanobacteria and acts as a peripheral antenna. Based on the supercomplex structure recently determined via cryo-EM imaging, we model various optical spectra of the IsiA monomers and IsiA ring. Comparison of the absorption and emission spectra of the isolated IsiA monomers and the full ring reveals that about 2.7 chlorophylls (Chls) are lost in the isolated IsiA monomers. The best fits for isolated monomers spectra are obtained assuming the absence of Chl 508 and Chl 517 and 70% loss of Chl 511. The best model describing all three hexamers and the entire ring suggests that the lowest energy pigments are Chls 511, 514, and 517. Based on the modeling results presented in this work, we conclude that there are most likely three entry points for EET from the IsiA hexamer to the PSI core monomer, with two of these entry points likely being located next to each other (i.e., nine entry points from IsiA to the PSI trimer). Finally, we show that excitation energy transfer inside individual monomers is fast (<2 ps at  = 5 K) and at least 20 times faster than intermonomer energy transfer.

Frequency-Domain Spectroscopic Study of the Photosystem I Supercomplexes, Isolated IsiA Monomers, and the Intact IsiA Ring.

The photochemical reaction center (RC) features a dimeric architecture for charge separation across the membrane. In green sulfur bacteria (GSB), the trimeric Fenna-Matthews-Olson (FMO) complex mediates the transfer of light energy from the chlorosome antenna complex to the RC. Here we determine the structure of the photosynthetic supercomplex from the GSB Chlorobaculum tepidum using single-particle cryogenic electron microscopy (cryo-EM) and identify the cytochrome c subunit (PscC), two accessory protein subunits (PscE and PscF), a second FMO trimeric complex, and a linker pigment between FMO and the RC core. The protein subunits that are assembled with the symmetric RC core generate an asymmetric photosynthetic supercomplex. One linker bacteriochlorophyll (BChl) is located in one of the two FMO-PscA interfaces, leading to differential efficiencies of the two energy transfer branches. The two FMO trimeric complexes establish two different binding interfaces with the RC cytoplasmic surface, driven by the associated accessory subunits. This structure of the GSB photosynthetic supercomplex provides mechanistic insight into the light excitation energy transfer routes and a possible evolutionary transition intermediate of the bacterial photosynthetic supercomplex from the primitive homodimeric RC.

Yuval Mazor

School of Molecular Sciences,
Biodesign Center for Applied Structural Discovery

Arizona State University

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

Yuval Mazor

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Molecular Sciences,Biodesign Center for Applied Structural Discovery

Arizona State University

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

School of Molecular Sciences,
Biodesign Center for Applied Structural Discovery