Martin Winkler

A facility to search for hidden particles at the CERN SPS: the SHiP physics case.

This paper describes the physics case for a new fixed target facility at CERN SPS. The SHiP (search for hidden particles) experiment is intended to hunt for new physics in the largely unexplored domain of very weakly interacting particles with masses below the Fermi scale, inaccessible to the LHC experiments, and to study tau neutrino physics. The same proton beam setup can be used later to look for decays of tau-leptons with lepton flavour number non-conservation, [Formula: see text] and to search for weakly-interacting sub-GeV dark matter candidates. We discuss the evidence for physics beyond the standard model and describe interactions between new particles and four different portals-scalars, vectors, fermions or axion-like particles. We discuss motivations for different models, manifesting themselves via these interactions, and how they can be probed with the SHiP experiment and present several case studies. The prospects to search for relatively light SUSY and composite particles at SHiP are also discussed. We demonstrate that the SHiP experiment has a unique potential to discover new physics and can directly probe a number of solutions of beyond the standard model puzzles, such as neutrino masses, baryon asymmetry of the Universe, dark matter, and inflation.

Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases with High Specific Activities

ABSTRACT Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 μmol H2 min−1 mg−1, while HydA from C. acetobutylicum (HydACa) shows the highest activity (5,522 μmol H2 min−1 mg−1) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydACa protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.

(Fe)-hydrogenases in green algae: photo-fermentation and hydrogen evolution under sulfur deprivation

Abstract Recent studies indicate that [Fe]-hydrogenases and H 2 metabolism are widely distributed among green algae. The enzymes are simple structured and catalyze H 2 evolution with similar rates than the more complex [Fe]-hydrogenases from bacteria. Different green algal species developed diverse strategies to survive under sulfur deprivation. Chlamydomonas reinhardtii evolves large quantities of hydrogen gas in the absence of sulfur. In a sealed culture of C. reinhardtii , the photosynthetic O 2 evolution rate drops below the rate of respiratory O 2 consumption due to a reversible inhibition of photosystem II, thus leading to an intracellular anaerobiosis. The algal cells survive under these anaerobic conditions by switching their metabolism to a kind of photo-fermentation. Although possessing a functional [Fe]-hydrogenase gene, the cells of Scenedesmus obliquus produce no significant amounts of H 2 under S-depleted conditions. Biochemical analyses indicate that S. obliquus decreases almost the complete metabolic activities while maintaining a low level of respiratory activity.

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

Under anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys396 and the C terminus of PetF as well as between the PetF residue Glu122 and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H2 production.

Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae

Hydrogenase expression in Chlamydomonas reinhardtii can be artificially induced by anaerobic adaptation or is naturally established under sulphur deprivation. In comparison to anaerobic adaptation, sulphur-deprived algal cultures show considerably higher expression rates of the [FeFe]-hydrogenase (HydA1) and develop a 25-fold higher in vitro hydrogenase activity. Based on this efficient induction principle we have established a novel purification protocol for the isolation of HydA1 that can also be used for other green algae. From an eight liter C. reinhardtii culture 0.52 mg HydA1 with a specific activity of 741 micromol H2 min(-1) mg(-1) was isolated. Similar amounts were also purified from Chlorococcum submarinum and Chlamydomonas moewusii. The extraordinarily large yields of protein allowed a spectroscopic characterization of the active site of these smallest [FeFe]-hydrogenases for the first time. An initial analysis by EPR spectroscopy shows characteristic axial EPR signals of the CO inhibited forms that are typical for the Hox-CO state of the active site from [FeFe]-hydrogenases. However, deviations in the g-tensor components have been observed that indicate distinct differences in the electronic structure between the various hydrogenases. At cryogenic temperatures, light-induced changes in the EPR spectra were observed and are interpreted as a photodissociation of the inhibiting CO ligand.

Isolation and molecular characterization of the [Fe]-hydrogenase from the unicellular green alga Chlorella fusca☆

[Fe]-hydrogenases are redoxenzymes that catalyze the reversible reduction of protons to hydrogen. Hydrogenase activity was observed in a culture of the unicellular green alga Chlorella fusca after an anaerobic incubation, but not in the related species Chlorella vulgaris. Specific polymerase chain reaction (PCR) techniques lead to the isolation of the cDNA and the genomic DNA of a special type of [Fe]-hydrogenase in C. fusca. The functional [Fe]-hydrogenase was purified to homogeneity and its N-terminus was sequenced. The polypeptide sequence shows a high degree of identity with the amino acid sequence deduced from the respective cDNA region. Structural and biochemical analyses indicate that ferredoxin is the main physiological electron donor.

O2 Reactions at the Six-iron Active Site (H-cluster) in [FeFe]-Hydrogenase

Irreversible inhibition by molecular oxygen (O2) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H2) production. Modification by O2 of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2FeH) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O2 exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O2 reactions. The first phase (τ1 ≤ 4 s) is characterized by the formation of an increased number of Fe–O,C bonds, elongation of the Fe–Fe distance in the binuclear unit (2FeH), and oxidation of one iron ion. The second phase (τ2 ≈ 15 s) causes a ∼50% decrease of the number of ∼2.7-Å Fe–Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ3 ≤ 1000 s) leads to the disappearance of most Fe–Fe and Fe–S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2FeH+ leading to the formation of a reactive oxygen species-like superoxide (O2−), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S]+ unit, leading to 3) complete O2-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O2 tolerance in [FeFe]-hydrogenase.

Molecular basis of [FeFe]-hydrogenase function: an insight into the complex interplay between protein and catalytic cofactor.

The precise electrochemical features of metal cofactors that convey the functions of redox enzymes are essentially determined by the specific interaction pattern between cofactor and enclosing protein environment. However, while biophysical techniques allow a detailed understanding of the features characterizing the cofactor itself, knowledge about the contribution of the protein part is much harder to obtain. [FeFe]-hydrogenases are an interesting class of enzymes that catalyze both, H2 oxidation and the reduction of protons to molecular hydrogen with significant efficiency. The active site of these proteins consists of an unusual prosthetic group (H-cluster) with six iron and six sulfur atoms. While H-cluster architecture and catalytic states during the different steps of H2 turnover have been thoroughly investigated during the last 20 years, possible functional contributions from the polypeptide framework were only assumed according to the level of conservancy and X-ray structure analyses. Due to the recent development of simpler and more efficient expression systems the role of single amino acids can now be experimentally investigated. This article summarizes, compares and categorizes the results of recent investigations based on site directed and random mutagenesis according to their informative value about structure function relationships in [FeFe]-hydrogenases. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

Multiple ferredoxin isoforms in Chlamydomonas reinhardtii – Their role under stress conditions and biotechnological implications

The unicellular green alga Chlamydomonas reinhardtii has at least six plant-type ferredoxins (FDX). Besides the long-known photosynthetic ferredoxin PetF the isoforms Fdx2-Fdx6 have been identified. The FDX genes are differentially expressed under various environmental conditions such as the availability of oxygen, copper, iron and ammonium. Recently, the anaerobically induced Fdx5 as well as Fdx2, which is involved in nitrite reduction were characterized in more detail. Moreover, it was shown that PetF, the central and most abundant FDX of C. reinhardtii, is a suitable partner of the hydrogenase HydA1. Using mutant variants of both PetF and HydA1, amino acid residues essential for the interaction of both proteins could be identified. These findings will help to tailor PetF for achieving an optimized photobiotechnological hydrogen production in C. reinhardtii, which might also benefit from new insights into the mechanism of how oxygen attacks the active site metal cluster of HydA1. This review gives an update on recent advances in understanding the function of ferredoxins and the hydrogenase in C. reinhardtii.

Challenges for large-field inflation and moduli stabilization

A bstractWe analyze the interplay between Kähler moduli stabilization and chaotic inflation in supergravity. While heavy moduli decouple from inflation in the supersymmetric limit, supersymmetry breaking generically introduces non-decoupling effects. These lead to inflation driven by a soft mass term, mφ2 ∼ mm3/2, where m is a supersymmetric mass parameter. This scenario needs no stabilizer field, but the stability of moduli during inflation imposes a large supersymmetry breaking scale, m3/2 ≫ H, and a careful choice of initial conditions. This is illustrated in three prominent examples of moduli stabilization: KKLT stabilization, Kähler Uplifting, and the Large Volume Scenario. Remarkably, all models have a universal effective inflaton potential which is flattened compared to quadratic inflation. Hence, they share universal predictions for the CMB observables, in particular a lower bound on the tensor-to-scalar ratio, r ≳ 0.05.