Copper effect on the protein composition of photosystem II

The definitive version is available at: http://www.blackwell-synergy.com/doi/full/10.1111/j.1399-3054.2000.1100419.xWe provide data from in vitro experiments on the polypeptide composition, photosynthetic electron transport and oxygen evolution activity of intact photosystem II (PSII) preparations under Cu(II) toxicity conditions. Low Cu(II) concentrations (Cu(II) per PSII reaction centre unit≤230) that caused around 50% inhibition of variable chlorophyll a fluorescence and oxygen evolution activity did not affect the polypeptide composition of PSII. However, the extrinsic proteins of 33, 24 and 17 kDa of the oxygen-evolving complex of PSII were removed when samples were treated with 300 μM CuCl2 (Cu(II) per PSII reaction centre unit=1 400). The LHCII antenna complex and D1 protein of the reaction centre of PSII were not affected even at these Cu(II) concentrations. The results indicated that the initial inhibition of the PSII electron transport and oxygen-evolving activity induced by the presence of toxic Cu(II) concentrations occurred before the damage of the oxygen-evolving complex. Indeed, more than 50% inhibition could be achieved in conditions where its protein composition and integrity was apparently preserved.This work was supported by the Dirección General de Investigación Científica y Técnica (Grant PB98-1632).Peer reviewe

Non-Photochemical Quenching in Cryptophyte Alga Rhodomonas salina Is Located in Chlorophyll a/c Antennae

Photosynthesis uses light as a source of energy but its excess can result in production of harmful oxygen radicals. To avoid any resulting damage, phototrophic organisms can employ a process known as non-photochemical quenching (NPQ), where excess light energy is safely dissipated as heat. The mechanism(s) of NPQ vary among different phototrophs. Here, we describe a new type of NPQ in the organism Rhodomonas salina, an alga belonging to the cryptophytes, part of the chromalveolate supergroup. Cryptophytes are exceptional among photosynthetic chromalveolates as they use both chlorophyll a/c proteins and phycobiliproteins for light harvesting. All our data demonstrates that NPQ in cryptophytes differs significantly from other chromalveolates – e.g. diatoms and it is also unique in comparison to NPQ in green algae and in higher plants: (1) there is no light induced xanthophyll cycle; (2) NPQ resembles the fast and flexible energetic quenching (qE) of higher plants, including its fast recovery; (3) a direct antennae protonation is involved in NPQ, similar to that found in higher plants. Further, fluorescence spectroscopy and biochemical characterization of isolated photosynthetic complexes suggest that NPQ in R. salina occurs in the chlorophyll a/c antennae but not in phycobiliproteins. All these results demonstrate that NPQ in cryptophytes represents a novel class of effective and flexible non-photochemical quenching

Spectroscopic characterization of intermediate steps involved in donor-side-induced photoinhibition of photosystem II

The reaction center protein D1 in photosystem II shows a high turnover during illumination. The degradation of the DI protein is preceded by photoinhibition of the electron transport in photosystem II. There are two distinct mechanisms for this: acceptor-side- and donor-side-induced photoinhibition. Here, donor-side-induced photoinhibition was studied in photosystem II membranes after Cl- depletion or washing with tris(hydroxymethyl)aminomethane (Tris) which destroys water oxidation, reversibly or irreversibly, respectively. Photoinhibition after these treatments leads to fast degradation of the D1 protein, and the mechanism behind this was investigated, Illumination of Cl- depleted photosystem II membranes resulted in a rapid and simultaneous inhibition of Cl--reconstitutable oxygen evolution, loss of 2 Mn ions per photosystem II center, increase in the electron transfer between the electron donor diphenylcarbazide and electron acceptor 2,6-dichlorophenolindophenol, and an increase in the EPR signal IIfast from tyrosine-Z(ox). The destruction of the Mn cluster leads to the loss of oxygen evolution and to an increased accessibility for diphenylcarbazide to donate electrons to Tyr-Z(ox). The increase in the EPR signal from Tyr-Z(ox) can be explained by slower reduction kinetics of Tyr-Z(ox) due to the Mn release. On a longer photoinhibition time scale, a decrease in the amplitude of Tyr-Z(ox) and inhibition of the electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol occurred simultaneously in both Cl--depleted and Tris-washed photosystem II membranes. These slower photoinhibition reactions were then studied in detail in Tris-washed photosystem II membranes. Compared to photoinhibition of Tyr-Z(ox), the EPR signal from tyrosine-D-ox decreased much slower. Tyr-D-ox was photoinhibited in parallel with the EPRsignals from reduced Q(A), reduced pheophytin, and an oxidized chlorophyll radical (chlorophyll(Z)), This shows that the acceptor side components and the primary charge separation reaction (P680(+) pheophytin(-)) were operational although Tyr-Z was inactivated. The amount of the D1 protein also declined in parallel with Tyr-D-ox, which shows that the D1 protein is not damaged until long after the Mn complex and Tyr-Z have become inactivated. Instead, it is likely that the strongly oxidizing P680(+) is responsible for the damage to the D1 protein

Two-dimensional crystallization and electron crystallography of MAPEG proteins

Members of the membrane-associated proteins in the eicosanoid and glutathione metabolism (MAPEG) superfamily have been subjected to two-dimensional crystallization experiments. A common denominator for successful attempts has been the use of a low lipid/protein ratio in the range of 1-9 (mol/mol). Electron crystallography demonstrated either hexagonal or orthorhombic packing of trimeric protein units. Three-dimensional structure analysis of the MAPEG member microsomal glutathione transferase 1 has shown that the monomer for this protein contains a left-handed bundle of four transmembrane helices. It is likely that this is a common structural motif for MAPEG proteins, because projection maps of all structurally characterized members are very similar

Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.

Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 µmol min–1 mg–1) and high kcat/Km (310 mM–1 s–1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 µmol min–1 mg–1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 µmol min–1 mg–1), 5-hydroperoxyeicosatetraenoic acid (0.043 µmol min–1 mg–1), and 15-hydroperoxy-PGE2 (0.04 µmol min–1 mg–1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 µmol min–1 mg–1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1

Structural basis for induced formation of the inflammatory mediator prostaglandin E2

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH2 to PGE2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided