From cytoplasm to lumen-mapping the free pools of protein subunits of three photosynthetic complexes using quantitative mass spectrometry.

The phycobilisome (PBS) captures light energy and transfers it to photosystem I (PSI) and photosystem II (PSII). Which and how many copies of protein subunits in PBSs, PSI, and PSII remain unbound in thylakoids are unknown. Here, quantitative mass spectrometry (QMS) was used to quantify substantial pools of free extrinsic subunits of PSII and PSI.

A real-time analysis of protein transport via the twin arginine translocation pathway in response to different components of the protonmotive force.

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro.

Oxidative modification of LHC II associated with photosystem II and PS I-LHC I-LHC II membranes.

Under aerobic conditions the production of Reactive Oxygen Species (ROS) by electron transport chains is unavoidable, and occurs in both autotrophic and heterotrophic organisms. In photosynthetic organisms both Photosystem II (PS II) and Photosystem I (PS I), in addition to the cytochrome b/f complex, are demonstrated sources of ROS. All of these membrane protein complexes exhibit oxidative damage when isolated from field-grown plant material.

Tocopherol controls D1 amino acid oxidation by oxygen radicals in Photosystem II.

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O) and the hydroxyl (HO) radicals were studied in WT and a tocopherol cyclase () mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol.

Natively oxidized amino acid residues in the spinach PS I-LHC I supercomplex.

Reactive oxygen species (ROS) production is an unavoidable byproduct of electron transport under aerobic conditions. Photosystem II (PS II), the cytochrome  b/f complex and Photosystem I (PS I) are all demonstrated sources of ROS. It has been proposed that PS I produces substantial levels of a variety of ROS including O, O, HO and, possibly, •OH; however, the site(s) of ROS production within PS I has been the subject of significant debate.

Regulation of photosynthetic cyclic electron flow pathways by adenylate status in higher plant chloroplasts.

Cylic electron flow (CEF) around Photosystem I in photosynthetic eukaryotes is likely to be necessary to augment ATP production, rapidly- and precisely balancing the plastid ATP/NADPH energy budget to meet the demands of downstream metabolism. Many regulatory aspects of this process are unclear. Here we demonstrate that the higher plant plastid NADH/Fd:plastoquinone reductase (NDH) and proposed PGR5/PGRL1 ferredoxin:plastoquinone reductase (FQR) pathways of CEF are strongly, rapidly and reversibly inhibited in vitro by ATP with K values of 670 μM and 240 μM respectively, within the range of physiological changes in ATP concentrations.

eIFiso4G Augments the Synthesis of Specific Plant Proteins Involved in Normal Chloroplast Function.

The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis ()-genes encoding the cap binding protein eIFiso4E () and two isoforms of the large subunit scaffolding protein eIFiso4G ( and ). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (, , , , and []) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type.

Natively oxidized amino acid residues in the spinach cytochrome b f complex.

The cytochrome b f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b f is 10-20 fold higher than that observed for the analogous respiratory cytochrome bc complex. The types of ROS produced (OO, and, possibly, HO) and the site(s) of ROS production within the b f complex have been the subject of some debate.

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II.

The Photosystem II reaction center is vulnerable to photoinhibition. The D1 and D2 proteins, lying at the core of the photosystem, are susceptible to oxidative modification by reactive oxygen species that are formed by the photosystem during illumination. Using spin probes and EPR spectroscopy, we have determined that both O and HO are involved in the photoinhibitory process.

Multiple LHCII antennae can transfer energy efficiently to a single Photosystem I.

Photosystems I and II (PSI and PSII) work in series to drive oxygenic photosynthesis. The two photosystems have different absorption spectra, therefore changes in light quality can lead to imbalanced excitation of the photosystems and a loss in photosynthetic efficiency. In a short-term adaptation response termed state transitions, excitation energy is directed to the light-limited photosystem.

N-Terminal Lipid Modification Is Required for the Stable Accumulation of CyanoQ in Synechocystis sp. PCC 6803.

The CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane.

Use of Protein Cross-Linking and Radiolytic Labeling To Elucidate the Structure of PsbO within Higher-Plant Photosystem II.

We have used protein cross-linking with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and radiolytic footprinting coupled with high-resolution tandem mass spectrometry, to examine the structure of higher-plant PsbO when it is bound to Photosystem II. Twenty intramolecular cross-linked residue pairs were identified. On the basis of this cross-linking data, spinach PsbO was modeled using the Thermosynechococcus vulcanus PsbO structure as a template, with the cross-linking distance constraints incorporated using the MODELLER program.

The extrinsic proteins of photosystem II: update.

Recent investigations have provided important new insights into the structures and functions of the extrinsic proteins of Photosystem II. This review is an update of the last major review on the extrinsic proteins of Photosystem II (Bricker et al., Biochemistry 31:4623-4628 2012).

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development.

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation.

Recent advances in the use of mass spectrometry to examine structure/function relationships in photosystem II.

Tandem mass spectrometry often coupled with chemical modification techniques, is developing into increasingly important tool in structural biology. These methods can provide important supplementary information concerning the structural organization and subunit make-up of membrane protein complexes, identification of conformational changes occurring during enzymatic reactions, identification of the location of posttranslational modifications, and elucidation of the structure of assembly and repair complexes. In this review, we will present a brief introduction to Photosystem II, tandem mass spectrometry and protein modification techniques that have been used to examine the photosystem.

High Yield Non-detergent Isolation of Photosystem I-Light-harvesting Chlorophyll II Membranes from Spinach Thylakoids: IMPLICATIONS FOR THE ORGANIZATION OF THE PS I ANTENNAE IN HIGHER PLANTS.

Styrene-maleic acid copolymer was used to effect a non-detergent partial solubilization of thylakoids from spinach. A high density membrane fraction, which was not solubilized by the copolymer, was isolated and was highly enriched in the Photosystem (PS) I-light-harvesting chlorophyll (LHC) II supercomplex and depleted of PS II, the cytochrome b6/f complex, and ATP synthase. The LHC II associated with the supercomplex appeared to be energetically coupled to PS I based on 77 K fluorescence, P700 photooxidation, and PS I electron transport light saturation experiments.

Use of protein cross-linking and radiolytic footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II.

Protein cross-linking and radiolytic footprinting coupled with high-resolution mass spectrometry were used to examine the structure of PsbP and PsbQ when they are bound to Photosystem II. In its bound state, the N-terminal 15-amino-acid residue domain of PsbP, which is unresolved in current crystal structures, interacts with domains in the C terminus of the protein. These interactions may serve to stabilize the structure of the N terminus and may facilitate PsbP binding and function.

Integration of apo-α-phycocyanin into phycobilisomes and its association with FNRL in the absence of the phycocyanin α-subunit lyase (CpcF) in Synechocystis sp. PCC 6803.

Phycocyanin is an important component of the phycobilisome, which is the principal light-harvesting complex in cyanobacteria. The covalent attachment of the phycocyanobilin chromophore to phycocyanin is catalyzed by the enzyme phycocyanin lyase. The photosynthetic properties and phycobilisome assembly state were characterized in wild type and two mutants which lack holo-α-phycocyanin.

The PsbP domain protein 1 functions in the assembly of lumenal domains in photosystem I.

Photosystem I (PS I) is a multisubunit membrane protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase. The PsbP domain protein 1 (PPD1; At4g15510) is located in the thylakoid lumen of plant chloroplasts and is essential for photoautotrophy, functioning as a PS I assembly factor. In this work, RNAi was used to suppress PPD1 expression, yielding mutants displaying a range of phenotypes with respect to PS I accumulation and function.

Photoheterotrophic growth of Physcomitrella patens.

Physcomitrella patens is a model bryophyte representing an early land plant in the green plant lineage. This organism possesses many advantages as a model organism. Its genome has been sequenced, its predominant life cycle stage is the haploid gametophyte, it is readily transformable and it can integrate transformed DNA into its genome by homologous recombination.

Radiolytic mapping of solvent-contact surfaces in Photosystem II of higher plants: experimental identification of putative water channels within the photosystem.

Photosystem II uses water as an enzymatic substrate. It has been hypothesized that this water is vectored to the active site for water oxidation via water channels that lead from the surface of the protein complex to the Mn4O5Ca metal cluster. The radiolysis of water by synchrotron radiation produces amino acid residue-modifying OH(•) and is a powerful technique to identify regions of proteins that are in contact with water.

The PsbP family of proteins.

The PsbP family of proteins consists of 11 evolutionarily related thylakoid lumenal components. These include the archetypal PsbP protein, which is an extrinsic subunit of eukaryotic photosystem II, three PsbP-like proteins (CyanoP of the prokaryotic cyanobacteria and green oxyphotobacteria, and the PPL1 and PPL2 proteins found in many eukaryotes), and seven PsbP-domain (PPD) proteins (PPD1-PPD7, most of which are found in the green plant lineage). All of these possess significant sequence and structural homologies while having very diverse functions.

Oxidized amino acid residues in the vicinity of Q(A) and Pheo(D1) of the photosystem II reaction center: putative generation sites of reducing-side reactive oxygen species.

Under a variety of stress conditions, Photosystem II produces reactive oxygen species on both the reducing and oxidizing sides of the photosystem. A number of different sites including the Mn4O5Ca cluster, P680, PheoD1, QA, QB and cytochrome b559 have been hypothesized to produce reactive oxygen species in the photosystem. In this communication using Fourier-transform ion cyclotron resonance mass spectrometry we have identified several residues on the D1 and D2 proteins from spinach which are oxidatively modified and in close proximity to QA (D1 residues (239)F, (241)Q, (242)E and the D2 residues (238)P, (239)T, (242)E and (247)M) and PheoD1 (D1 residues (130)E, (133)L and (135)F).