Reprint of PSII Manganese Cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha

The hard wood-colonizing ascomycete Xylaria polymorpha, that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l⁻¹). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a pI of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 10³ to 7 × 10⁴ M⁻¹ s⁻¹). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic β-O-4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin.     

Electrochemical reduction of ferrous alpha-verdoheme in complex with heme oxygenase-1

The heme oxygenase (HO) reaction consists of three successive oxygenation reactions, i.e. heme to alpha-hydroxyheme, alpha-hydroxyheme to verdoheme, and verdoheme to biliverdin-iron chelate. Of these, the least understood step is the conversion of verdoheme to biliverdin-iron chelate. For the cleavage of the oxaporphyrin ring of ferrous verdoheme, involvement of a verdoheme pi-neutral radical has been proposed. To probe this hypothetical mechanism in the HO reaction, we performed electrochemical reduction of ferrous verdoheme complexed with rat HO-1 under anaerobic conditions. On the basis of the electrochemical spectral changes, the midpoint potential for the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme was found to be -0.47+/-0.01 V vs the normal hydrogen electrode (NHE). Because this potential is far lower than those of both flavins of NADPH-cytochrome P450 reductase, and of NADPH, it is concluded that the one-electron reduction of the oxaporphyrin ring of ferrous verdoheme is unlikely to occur and that the formation of the pi-neutral radical cannot be the initial step in the degradation of verdoheme by HO. Rather, it appears more reasonable to consider an alternative mechanism in which binding of O(2) to the ferrous iron of verdoheme is the first step in the degradation of verdoheme.     

A chromatographic tool for preparing combinatorial quinone-thiol conjugate libraries

Quinones are well established as key players in the production of reactive oxygen species within cellular environments. Many factors govern their cytotoxicity but most studies have been restricted to a few, core, derivatives. A new strategy for the in situ production of quinone derivatives has been developed such that libraries of diverse functionality can be rapidly created without recourse to extensive synthetic procedures. The approach relies upon nucleophilic addition by reduced thiol derivatives to the quinone core within a pre-culture assay mixture and provides a generic strategy that exploits the large reservoir of commercial thiols currently available. A readily accessible chromatographic method has been developed that allows the derivatisation process to be easily monitored and the purity of the resulting one pot preparation to be assessed. The viability of the combinatorial approach has been fully validated through comparison with a range of quinone-S-conjugates prepared using conventional bench synthesis. The latter have been fully characterised.     

Characterization of two new multiforms of Trametes pubescens laccase

Electrochemical properties of two multiforms of laccase from Trametes pubescens basidiomycete (LAC1 and LAC2) have been studied. The standard redox potentials of the T1 sites of the enzymes were found to be 746 and 738 mV vs. NHE for LAC1 and LAC2, respectively. Bioelectroreduction of oxygen based on direct electron transfer between each of the two forms of Trametes pubescens laccase and spectrographic graphite electrodes has been demonstrated and studied. It is concluded that the T1 site of laccase is the first electron acceptor, both in solution (homogeneous case) and when the enzymes are adsorbed on the surface of the graphite electrode (heterogeneous case). Thus, the previously proposed mechanism of oxygen bioelectroreduction by adsorbed fungal laccase was additionally confirmed using two forms of the enzyme. Moreover, the assumed need for extracellular laccase to communicate directly and electronically with a solid matrix (lignin) in the course of lignin degradation is discussed. In summary, the possible roles of multiforms of the enzyme based on their electrochemical, biochemical, spectral, and kinetic properties have been suggested to consist in broadening of the substrate specificity of the enzyme, in turn yielding the possibility to dynamically regulate the process of lignin degradation according to the real-time survival needs of the organism.     

Inhibition of lipoxygenase by soy isoflavones: evidence of isoflavones as redox inhibitors

Hydroperoxides, the products of lipoxygenase mediated pathways, play a major role in the manifestation of chronic inflammatory diseases. Soy isoflavones act as antioxidants due to their ability to scavenge free radicals. Isoflavones inhibit the activity of soy lipoxygenase-1 and 5-lipoxygenase, from human polymorph nuclear lymphocyte in a concentration dependent manner. Spectroscopic and enzyme kinetic measurements have helped to understand the nature and mechanism of inhibition. Genistein is the most effective inhibitor of soy lipoxygenase 1 and 5-lipoxygenase with IC(50) values of 107 and 125 microM, respectively. Genistein and daidzein are noncompetitive inhibitors of soy lipoxygenase 1 with inhibition constants, K(i), of 60 and 80 microM, respectively. Electron paramagnetic resonance and spectroscopic studies confirm that isoflavones reduce active state iron to ferrous state and prevent the activation of the resting enzyme. A model for the inhibition of lipoxygenase by isoflavones is suggested.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

谷氧还蛋白系统及其对细胞氧化还原态势的调控

细胞内氧化还原调控主要是由谷氧还蛋白系统和硫氧还蛋白系统完成。谷氧还蛋白属于硫氧还蛋白超家族,广泛分布在各种生物体内。作为一种巯基转移酶,它能够催化巯基．二硫键交换反应或者还原蛋白质谷胱甘肽二硫化物,以维持胞内的氧化还原态势。谷氧蛋白系统参与氧化胁迫、蛋白修饰、信号转导、细胞调亡和细胞分化等多种生物过程。对其体内作用靶蛋白的研究,有助于阐明谷氧还蛋白在整个细胞氧化还原网络的重要调控作用。     

Oxidation and reduction of actin: Origin,impact in vitro and functional consequences in vivo

Actin is among the most abundant proteins in eukaryotic cells and assembles into dynamic filamentous networks regulated by many actin binding proteins. The actin cytoskeleton must be finely tuned, both in space and time, to fulfill key cellular functions such as cell division, cell shape changes, phagocytosis and cell migration. While actin oxidation by reactive oxygen species (ROS) at non-physiological levels are known for long to impact on actin polymerization and on the cellular actin cytoskeleton, growing evidence shows that direct and reversible oxidation/reduction of specific actin amino acids plays an important and physiological role in regulating the actin cytoskeleton. In this review, we describe which actin amino acid residues can be selectively oxidized and reduced in many different ways (e.g. disulfide bond formation, glutathionylation, carbonylation, nitration, nitrosylation and other oxidations), the cellular enzymes at the origin of these post-translational modifications, and the impact of actin redox modifications both in vitro and in vivo. We show that the regulated balance of oxidation and reduction of key actin amino acid residues contributes to the control of actin filament polymerization and disassembly at the subcellular scale and highlight how improper redox modifications of actin can lead to pathological conditions.     

Adding glutathione to parenteral nutrition prevents alveolar loss in newborn Guinea pig

Bronchopulmonary dysplasia, a main complication of prematurity, is characterized by an alveolar hypoplasia. Oxidative stress is suspected to be a trigger event in this population who has a low level of glutathione, a main endogenous antioxidant, and who receives high oxidative load, particularly ascorbylperoxide from their parenteral nutrition. Hypothesis: the addition of glutathione (GSSG) in parenteral nutrition improves detoxification of ascorbylperoxide by glutathione peroxidase and therefore prevents exaggerated apoptosis and loss of alveoli.Methods: Ascorbylperoxide is assessed as substrate for glutathione peroxidase in Michaelis-Menten kinetics. Three-days old guinea pig pups were divided in 6 groups to receive, through a catheter in jugular vein, the following solutions: 1) Sham (no infusion); 2) PN(-L): parenteral nutrition protected against light (low ascorbylperoxide); 3) PN(+L): PN without photo-protection (high ascorbylperoxide); 4) 180 μM ascorbylperoxide; 5) PN(+L)+10 μM GSSG; 6) ascorbylperoxyde+10 μM GSSG. After 4 days, lungs were sampled and prepared for histology and biochemical determinations. Data were analysed by ANOVA, p<0.05Results: The Km of ascorbylperoxide for glutathione peroxidase was 126±6 μM and Vmax was 38.4±2.5 nmol/min/ U. The presence of GSSG in intravenous solution has prevented the high GSSG, oxidized redox potential of glutathione, activation of caspase-3 (apoptosis marker) and loss of alveoli induced by PN(+L) or ascorbylperoxide.Conclusion: A correction of the low glutathione levels observed in newborn animal on parenteral nutrition, protects lungs from toxic effect of ascorbylperoxide. Premature infants having a low level of glutathione, this finding is of high importance because it provides hope in a possible prevention of bronchopulmonary dysplasia.