Purification and Crystallization of Oxygen-Evolving Photosystem II Core Complex from Thermophilic Cyanobacteria

This chapter describes the purification and crystallization of oxygen-evolving photosystem II core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus. Procedures used for purification of photosystem II from the cyanobacterium involves cultivation of cells, isolation of thylakoid membranes, purification of crude and pure photosystem II core complexes by detergent solubilization, followed by differential centrifugation and column chromatography. The purified core dimer particles were successfully used for crystallization, and the methods and conditions used for crystallization are presented. These purification and crystallization procedures can be applied for another thermophilic cyanobacterium T. elongatus

Structure of the catalytic, inorganic core of oxygen-evolving photosystem II at 1.9 Å resolution

The catalytic center for photosynthetic water-splitting consists of 4 Mn atoms and 1 Ca atom and is located near the lumenal surface of photosystem II. So far the structure of the Mn(4)Ca-cluster has been studied by a variety of techniques including X-ray spectroscopy and diffraction, and various structural models have been proposed. However, its exact structure is still unknown due to the limited resolution of crystal structures of PSII achieved so far, as well as possible radiation damages that might have occurred. Very recently, we have succeeded in solving the structure of photosystem II at 1.9 angstrom. which yielded a detailed picture of the Mn(4)CaO(5)-cluster for the first time. In the high resolution structure, the Mn(4)CaO(5)-cluster is arranged in a distorted chair form, with a cubane-like structure formed by 3 Mn and 1 Ca, 4 oxygen atoms as the distorted base of the chair, and 1 Mn and 1 oxygen atom outside of the cubane as the back of the chair. In addition, four water molecules were associated with the cluster, among which, two are associated with the terminal Mn atom and two are associated with the Ca atom. Some of these water molecules may therefore serve as the substrates for water-splitting. The high resolution structure of the catalytic center provided a solid basis for elucidation of the mechanism of photosynthetic water splitting. We review here the structural features of the Mn(4)CaO(5)-cluster analyzed at 1.9 angstrom resolution, and compare them with the structures reported previously

Plasma gelsolin levels and outcomes after aneurysmal subarachnoid hemorrhage

INTRODUCTION: Lower gelsolin levels have been associated with the severity and poor outcome of critical illness. Nevertheless, their link with clinical outcomes of aneurysmal subarachnoid hemorrhage is unknown. Therefore, we aimed to investigate the relationship between plasma gelsolin levels and clinical outcomes in patients with aneurysmal subarachnoid hemorrhage. METHODS: A total of 262 consecutive patients and 150 healthy subjects were included. Plasma gelsolin levels were measured by enzyme-linked immunosorbent assay. Mortality and poor long-term outcome (Glasgow Outcome Scale score of 1-3) at 6 months were recorded. RESULTS: Plasma gelsolin levels on admission were substantially lower in patients than in healthy controls (66.9 (26.4) mg/L vs. 126.4 (35.4) mg/L, P < 0.001), and negatively associated with World Federation of Neurological Surgeons score (r = -0.554, P < 0.001) and Fisher score (r = -0.538, P < 0.001), and identified as an independent predictor of poor functional outcome (odds ratio, 0.957; 95% confidence interval (CI), 0.933-0.983; P = 0.001) and death (odds ratio, 0.953; 95% CI, 0.917-0.990; P = 0.003) after 6 months. The areas under the ROC curve of gelsolin for functional outcome and mortality were similar to those of World Federation of Neurological Surgeons score and Fisher score (all P > 0.05). Gelsolin improved the predictive values of World Federation of Neurological Surgeons score and Fisher score for functional outcome (both P < 0.05), but not for mortality (both P > 0.05). CONCLUSIONS: Gelsolin levels are a useful, complementary tool to predict functional outcome and mortality 6 months after aneurysmal subarachnoid hemorrhage

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

AbstractIn intact PSII, both the secondary electron donor (TyrZ) and side-path electron donors (Car/ChlZ/Cytb559) can be oxidized by P680+ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S1TyrZ EPR signal were independent of the treatment of K3Fe(CN)6, whereas formation and decay of the Car+/ChlZ+ EPR signal correlated with the reduction and recovery of the Fe3+ EPR signal of the non-heme iron in K3Fe(CN)6 pre-treated PSII, respectively. Based on the observed correlation between Car/ChlZ oxidation and Fe3+ reduction, the oxidation of non-heme iron by K3Fe(CN)6 at 0 °C was quantified, which showed that around 50–60% fractions of the reaction centers gave rise to the Fe3+ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of TyrZ oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. TyrZ oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K3Fe(CN)6 takes place only in inactive PSII, which implies that the Fe3+ state is probably not the intermediate species for the turnover of quinone reduction

SHT-based public auditing protocol with error tolerance in FDL-empowered IoVs

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Application of Magnetic Circular Dichroism spectroscopy to the study of the OEC in Photosystem II from cyanobacteria and higher plants.

Absorption from the OEC was expected to be too weak to be measured through basic optical spectroscopy, with a molar extinction coefficient of <100, compared to 106 for chlorophyll. We therefore used circular dichroism (CD) and magnetic circular dichroism (MCD) to search for this absorption

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

Background: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration ( Scope of review: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. Major conclusions: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. General significance: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules