Discriminating changes in intracellular NADH/NAD+ levels due to anoxicity and H2 supply in R. eutropha cells using the Frex fluorescence sensor

The hydrogen-oxidizing “Knallgas” bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed

Reductive activation and structural rearrangement in superoxide reductase: a combined infrared spectroscopic and computational study

Superoxide reductases (SOR) are a family of non-heme iron enzymes that limit oxidative stress by catalysing the reduction of superoxide to hydrogen peroxide and, thus, represent model systems for the detoxification of reactive oxygen species. In several enzymes of this type, reductive activation of the active site involves the reversible dissociation of a glutamate from the proposed substrate binding site at the iron. In this study we have employed IR spectroscopic and theoretical methods to gain insights into redox-linked structural changes of 1Fe-type superoxide reductases, focusing on the enzyme from the archaeon Ignicoccus hospitalis. Guided by crystal structure data and complemented by spectra calculation for an active site model, the main IR difference signals could be assigned. These signals reflect redox-induced structural changes in the first coordination sphere of the iron centre, adjacent loop and helical regions, and more remote β-sheets. By comparison with the spectra obtained for the E23A mutant of Ignicoccus hospitalis SOR, it is shown that glutamate E23 dissociates reversibly from the ferrous iron during reductive activation of the wild type enzyme. Moreover, this process is found to trigger a global conformational transition of the protein that is strictly dependent on the presence of E23. Similar concerted structural changes can be inferred from the IR spectra of related SORs such as that from Archaeoglobus fulgidus, indicating a widespread mechanism. A possible functional role of this process in terms of synergistic effects during reductive activation of the homotetrameric enzyme is proposed.DFG, EXC 314, Unifying Concepts in Catalysi

Tuning Product Selectivity for Aqueous CO2 Reduction with a Mn(bipyridine)-pyrene Catalyst Immobilized on a Carbon Nanotube Electrode

The development of high-performance electrocatalytic systems for the controlled reduction of CO2 to value-added chemicals is a key goal in emerging renewable energy technologies. The lack of selective and scalable catalysts in aqueous solution currently hampers the implementation of such a process. Here, the assembly of a [MnBr(2,2′-bipyridine)(CO)3] complex anchored to a carbon nanotube electrode via a pyrene unit is reported. Immobilization of the molecular catalyst allows electrocatalytic reduction of CO2 under fully aqueous conditions with a catalytic onset overpotential of η = 360 mV, and controlled potential electrolysis generated more than 1000 turnovers at η = 550 mV. The product selectivity can be tuned by alteration of the catalyst loading on the nanotube surface. CO was observed as the main product at high catalyst loadings, whereas formate was the dominant CO2 reduction product at low catalyst loadings. Using UV–vis and surface-sensitive IR spectroelectrochemical techniques, two different intermediates were identified as responsible for the change in selectivity of the heterogenized Mn catalyst. The formation of a dimeric Mn0 species at higher surface loading was shown to preferentially lead to CO formation, whereas at lower surface loading the electrochemical generation of a monomeric Mn-hydride is suggested to greatly enhance the production of formate. These results emphasize the advantages of integrating molecular catalysts onto electrode surfaces for enhancing catalytic activity while allowing excellent control and a deeper understanding of the catalytic mechanisms.This work was supported by the Christian Doppler Research Association (Austrian Federal Ministry of Science, Research and Economy and the National Foundation for Research, Technology and Development), the OMV Group, the EPSRC (DTA studentship for T.E.R.), the European Union’s Horizon 2020 research and innovation program (Marie SklodowskaCurie IF for K.H.L., GAN 701192), and an ERC Consolidator Grant “MatEnSAP” (GAN 682833). I.Z. is indebted to the German Research Foundation (DFG) for financial support within the cluster of excellence EXC 314: Unifying concepts in catalysis, “UniCat”

An Intermetallic CaFe6Ge6 Approach to Unprecedented Ca Fe O Electrocatalyst for Efficient Alkaline Oxygen Evolution Reaction

Based on the low cost and relatively high catalytic activity, considerable efforts have been devoted towards developing redox active transition metal TM oxygen electrocatalysts for the alkaline oxygen evolution reaction OER while the role of redox inactive alkaline earth metals has often been neglected in OER. Herein, for the first time, we developed a novel ternary intermetallic CaFe6Ge6 precatalyst, whose surface rapidly transforms into a porous ultrathin Ca amp; 8722;Fe amp; 8722;O heteroshell structure during alkaline OER through the oxidative leaching of surficial Ge. Benefiting from synergistic effects, this highly efficient OER active material with distinct Ca amp; 8722;Fe amp; 8722;O layers has a large electrochemical surface area and more exposed active Fe sites than a Ca free FeOx phase. Also, the presence of Ca in Ca amp; 8722;Fe amp; 8722;O is responsible for the enhanced transport and activation of hydroxyls and related OER reaction intermediate as unequivocally illustrated by a combination of quasi in situ Raman spectroscopy and various ex situ method

Host-Guest Chemistry Meets Electrocatalysis: Cucurbit[6]uril on a Au Surface as a Hybrid System in CO2 Reduction.

The rational control of forming and stabilizing reaction intermediates to guide specific reaction pathways remains to be a major challenge in electrocatalysis. In this work, we report a surface active-site engineering approach for modulating electrocatalytic CO2 reduction using the macrocycle cucurbit[6]uril (CB[6]). A pristine gold surface functionalized with CB[6] nanocavities was studied as a hybrid organic-inorganic model system that utilizes host-guest chemistry to influence the heterogeneous electrocatalytic reaction. The combination of surface-enhanced infrared absorption (SEIRA) spectroscopy and electrocatalytic experiments in conjunction with theoretical calculations supports capture and reduction of CO2 inside the hydrophobic cavity of CB[6] on the gold surface in aqueous KHCO3 at negative potentials. SEIRA spectroscopic experiments show that the decoration of gold with the supramolecular host CB[6] leads to an increased local CO2 concentration close to the metal interface. Electrocatalytic CO2 reduction on a CB[6]-coated gold electrode indicates differences in the specific interactions between CO2 reduction intermediates within and outside the CB[6] molecular cavity, illustrated by a decrease in current density from CO generation, but almost invariant H2 production compared to unfunctionalized gold. The presented methodology and mechanistic insight can guide future design of molecularly engineered catalytic environments through interfacial host-guest chemistry

Analyzing the catalytic processes of immobilized redox enzymes by vibrational spectroscopies

Analyzing the structure and function of redox enzymes attached to electrodes is a central challenge in many fields of fundamental and applied life science. Electrochemical techniques such as cyclic voltammetry which are routinely used do not provide insight into the molecular structure and reaction mechanisms of the immobilized proteins. Surface-enhanced infrared absorption (SEIRA) and surface-enhanced resonance Raman (SERR) spectroscopy may fill this gap, if nanostructured Au or Ag are used as conductive support materials. In this account, we will first outline the principles of the methodology including a description of the most important strategies for biocompatible protein immobilization. Subsequently, we will critically review SERR and SEIRA spectroscopic approaches to characterize the protein and active site structure of the immobilized enzymes. Special emphasis is laid on the combination of surface-enhanced vibrational spectroscopies with electrochemical methods to analyze equilibria and dynamics of the interfacial redox processes. Finally, we will assess the potential of SERR and SEIRA spectroscopy for in situ investigations on the basis of the first promising studies on human sulfite oxidase and hydrogenases under turnover conditions. Copyright © 2012 Wiley Periodicals, Inc

Photoorientation of a liquid crystalline polyester with azobenzene side groups:1. Effects of irradiation with linearly polarized blue light

The photoorientation process in a polyester with 4-cyano-4'-alkoxyazobenzene side group and long methylene spacers in the side and the main-chain was studied as a function of irradiation with linearly polarized light of 488 nm under systematic variation of the power density and temperature. This model polymer is characterized by liquid crystallinity (g 24 S-X 26 S-A 34 n 47 i) and a strong aggregation tendency. The photoorientation is cooperative, i.e., the orientation of the photochromic side group induces the alignment of the ester unit (which is a part of the main-chain) and both methylene segments in the side- and main-chain. The very high values of the normalized linear dichroism up to 0.8 and the birefringence (above 0.3) are due to the interaction of photoorientation and thermotropic self-organization. The induction of anisotropy shows a pronounced dependence on the power density and the working temperature. This is related to the thermal properties of the LC polymer and the resulting ordering behavior. Surprisingly, the photoorientation process, is not restricted by J-aggregation of the azobenzene groups even at low power densities. The highest anisotropy at 27 T (slightly above T-g) is achieved by a low power density (1 mW/cm(2) and dose of 0.5 mW/cm(2)s). Higher power densities result in a decrease of the maximum dichroism and, finally, the initially induced small anisotropy is erased during continuing light exposure. At a constant high power density of about 700 mW/cm(2), the saturation value of the dichroism first increases with the irradiation temperature, then passes through a maximum of about 0.8 at 18 degreesC and finally the film is transferred to the isotropic state at 27 T. Both effects indicate a significant thermal effect of the laser beam on the polymer film. Taking into account the thermal properties of the investigated polymer it is demonstrated that the photoinduced anisotropy depends,,strongly on the irradiation conditions

Raw data supporting publication: Tuning Product Selectivity for Aqueous CO2 Reduction with a Mn(bipyridine)-pyrene Catalyst Immobilized on a Carbon Nanotube Electrode

raw data supporting article: uning Product Selectivity for Aqueous CO2 Reduction with a Mn(bipyridine)-pyrene Catalyst Immobilized on a Carbon Nanotube Electrod