Anders Thapper

Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

BackgroundThe validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.ResultsWith this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution.ConclusionIt was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.

Photochemical water oxidation with visible light using a cobalt containing catalyst

Artificial photosynthesis aims at using solar light energy to oxidatively split water to oxygen, protons and electrons and store the energy in a chemical fuel. Here we present a cobalt phosphonate material that can split water catalytically, driven by visible light in aqueous solutions of pH 7.

Artificial Photosynthesis for Solar Fuels – an Evolving Research Field within AMPEA, a Joint Programme of the European Energy Research Alliance

Abstract On the path to an energy transition away from fossil fuels to sustainable sources, the European Union is for the moment keeping pace with the objectives of the Strategic Energy Technology-Plan. For this trend to continue after 2020, scientific breakthroughs must be achieved. One main objective is to produce solar fuels from solar energy and water in direct processes to accomplish the efficient storage of solar energy in a chemical form. This is a grand scientific challenge. One important approach to achieve this goal is Artificial Photosynthesis. The European Energy Research Alliance has launched the Joint Programme “Advanced Materials & Processes for Energy Applications” (AMPEA) to foster the role of basic science in Future Emerging Technologies. European researchers in artificial photosynthesis recently met at an AMPEA organized workshop to define common research strategies and milestones for the future. Through this work artificial photosynthesis became the first energy research sub-field to be organised into what is designated “an Application” within AMPEA. The ambition is to drive and accelerate solar fuels research into a powerful European field – in a shorter time and with a broader scope than possible for individual or national initiatives. Within AMPEA the Application Artificial Photosynthesis is inclusive and intended to bring together all European scientists in relevant fields. The goal is to set up a thorough and systematic programme of directed research, which by 2020 will have advanced to a point where commercially viable artificial photosynthetic devices will be under development in partnership with industry.

Water Oxidation Catalyzed by a Dinuclear Cobalt–Polypyridine Complex

The dinuclear Co complex [(TPA)Co(μ-OH)(μ-O2 )Co(TPA)](ClO4 )3 (1, TPA=tris(2-pyridylmethyl)amine) catalyzes the oxidation of water. In the presence of [Ru(bpy)3 ](2+) and S2 O8 (2-) , photoinduced oxygen evolution can be observed with a turnover frequency (TOF) of 1.4±0.1 mol(O2 ) mol(1)(-1)  s(-1) and a maximal turnover number (TON) of 58±5 mol(O2 ) mol(1)(-1) . The complex is shown to act as a molecular and homogeneous catalyst and a mechanism is proposed based on the combination of EPR data and light-driven O2 evolution kinetics.

Voltammetric and spectroscopic characterization of early intermediates in the Co(II)–polypyridyl-catalyzed reduction of water

Early intermediates of catalytic water reduction by a Co(II)-polypyridyl species have been characterized. Electrochemical detection of the Co(III)-hydride and time-resolved spectroscopic detection of the Co(I)-ligand intermediates provide an understanding of their reactivity in electrolytic or light-driven reduction of protons to hydrogen.

Two tetranuclear Mn-complexes as biomimetic models of the oxygen evolving complex in Photosystem II. A synthesis, characterisation and reactivity study

In this work we report the preparation of two metallamacrocyclic tetranuclear manganese(II) complexes, [L1(4)Mn4](ClO4)4 and [L2(4)Mn4](ClO4)4 where L1 and L2 are the anions of the heptadentate ligands 2-((2-(bis(pyridin-2-ylmethyl)amino)ethyl)(methyl)amino)acetic acid and 2-(benzyl(2-(bis(pyridin-2-ylmethyl)amino)ethyl)amino)acetic acid), respectively. The complexes have been fully characterized by ESI-MS, elemental analysis, single-crystal X-ray diffraction, magnetic susceptibility, and EPR spectroscopy. Electrochemical reactions as well as reactions with different chemical redox reagents have been performed and a reversible two electron oxidation per manganese ion has been identified. The reaction of [L1(4)Mn4](ClO4)4 with oxone or cerium(IV) results in the evolution of oxygen which makes this system interesting for future studies in the search for a functional mimic of the oxygen evolving complex in Photosystem II.

Kinetic, structural, and EPR studies reveal that aldehyde oxidoreductase from Desulfovibrio gigas does not need a sulfido ligand for catalysis and give evidence for a direct Mo-C interaction in a biological system.

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH(2) ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

Defining the Far-Red Limit of Photosystem II in Spinach

The far-red limit of photosystem II (PSII) photochemistry was studied in PSII-enriched membranes and PSII core preparations from spinach (Spinacia oleracea) after application of laser flashes between 730 and 820 nm. Light up to 800 nm was found to drive PSII activity in both acceptor side reduction and oxidation of the water-oxidizing CaMn4 cluster. Far-red illumination induced enhancement of, and slowed down decay kinetics of, variable fluorescence. Both effects reflect reduction of the acceptor side of PSII. The effects on the donor side of PSII were monitored using electron paramagnetic resonance spectroscopy. Signals from the S2-, S3-, and S0-states could be detected after one, two, and three far-red flashes, respectively, indicating that PSII underwent conventional S-state transitions. Full PSII turnover was demonstrated by far-red flash-induced oxygen release, with oxygen appearing on the third flash. In addition, both the pheophytin anion and the Tyr Z radical were formed by far-red flashes. The efficiency of this far-red photochemistry in PSII decreases with increasing wavelength. The upper limit for detectable photochemistry in PSII on a single flash was determined to be 780 nm. In photoaccumulation experiments, photochemistry was detectable up to 800 nm. Implications for the energetics and energy levels of the charge separated states in PSII are discussed in light of the presented results.

Water oxidation by manganese oxides formed from tetranuclear precursor complexes: the influence of phosphate on structure and activity

Two types of manganese oxides have been prepared by hydrolysis of tetranuclear Mn(iii) complexes in the presence or absence of phosphate ions. The oxides have been characterized structurally using X-ray absorption spectroscopy and functionally by O2 evolution measurements. The structures of the oxides prepared in the absence of phosphate are dominated by di-μ-oxo bridged manganese ions that form layers with limited long-range order, consisting of edge-sharing MnO6 octahedra. The average manganese oxidation state is +3.5. The structure of these oxides is closely related to other manganese oxides reported as water oxidation catalysts. They show high oxygen evolution activity in a light-driven system containing [Ru(bpy)3](2+) and S2O8(2-) at pH 7. In contrast, the oxides formed by hydrolysis in the presence of phosphate ions contain almost no di-μ-oxo bridged manganese ions. Instead the phosphate groups are acting as bridges between the manganese ions. The average oxidation state of manganese ions is +3. This type of oxide has much lower water oxidation activity in the light-driven system. Correlations between different structural motifs and the function as a water oxidation catalyst are discussed and the lower activity in the phosphate containing oxide is linked to the absence of protonable di-μ-oxo bridges.

Iron Pentapyridyl Complexes as Molecular Water Oxidation Catalysts: Strong Influence of a Chloride Ligand and pH in Altering the Mechanism

The development of molecular water oxidation catalysts based on earth-abundant, non-noble metals is essential for artificial photosynthesis research. Iron, which is the most abundant transition metal in the earths crust, is a prospective candidate for this purpose. Herein, we report two iron complexes based on the polypyridyl ligand Py5OH (Py5OH=pyridine-2,6-diylbis [di(pyridin-2-yl)methanol]) that can catalyse water oxidation to produce O2 in Ru(III) -induced (at pH 8, highest turnover number (TON)=26.5; turnover frequency (TOF)=2.2 s(-1) ), Ce(IV) -induced (at pH≈1.5 highest TON=16; TOF=0.75 s(-1) ) and photo-induced (at pH 8, highest TON=43.5; TOF=0.6 s(-1) ) reactions. A chloride ligand in one of the iron complexes is shown to affect the activity strongly, improve stability and, thereby, the performance at pH 8 but it inhibits oxygen evolution at pH≈1.5. The observations are consistent with a change in mechanism for catalytic water oxidation with the Fe(Py5OH) complexes between acidic (Ce(IV) ) and near-neutral pH (Ru(III) ).