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Abstract: Achieving a unified understanding of the mechanism of a multicenter redox enzyme such as [NiFe] hydrogenase is complicated by difficulties in reconciling information obtained by using different techniques and on samples in different physical forms. Measurements of the activity of the enzyme, and of factors which perturb activity, are generally carried out using biochemical assays in solution or with electrode-immobilized enzymes using protein film electrochemistry (PFE). Conversely, spectroscopy aimed at reporting on features of the metalloclusters in the enzyme, such as electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy (XAS), is often conducted on frozen samples and is thus difficult to relate to catalytically relevant states as information about turnover and activity has been lost. To complicate matters further, most of our knowledge of the atomic-level structure of metalloenzymes comes from X-ray diffraction studies in the solid, crystalline state, which are again difficult to link to turnover conditions. Taking [NiFe] hydrogenases as our case study, we show here how it is possible to apply infrared (IR) spectroscopic sampling approaches to unite direct spectroscopic study with catalytic turnover. Using a method we have named protein film IR electrochemistry (PFIRE), we reveal the steady-state distribution of intermediates during catalysis and identify catalytic “bottlenecks” introduced by site-directed mutagenesis. We also show that it is possible to study dynamic transitions between active site states of enzymes in single crystals, uniting solid state and solution spectroscopic information. In all of these cases, the spectroscopic data complement and enhance interpretation of purely activity-based measurements by providing direct chemical insight that is otherwise hidden. The [NiFe] hydrogenases possess a bimetallic [NiFe] active site, coordinated by CO and CN– ligands, linked to the protein via bridging and terminal cysteine sulfur ligands, as well as an electron relay chain of iron sulfur clusters. Infrared spectroscopy is ideal for probing hydrogenases because the CO and CN– ligands are strong IR absorbers, but the suite of IR-based approaches we describe here will be equally valuable in studying substrate- or intermediate-bound states of other metalloenzymes where key mechanistic questions remain open, such as nitrogenase, formate dehydrogenase, or carbon monoxide dehydrogenase. We therefore hope that this Account will encourage future studies which unify information from different techniques across bioinorganic chemistry.

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Abstract: In developing a new compositae air electrode for Solid Oxide Cells (SOCs) it is essential to fully understand the phase chemistry of all components. Ruddlesden-Popper type electrodes such as Pr2NiO4+δ have previously been proposed as attractive alternatives to conventional La0·6Sr0·4Fe0·8Co0·2O3-δ/Ce1-xGdxO2-δ compositae air electrodes for both fuel cell and electrolyser modes of operation. However, Pr2NiO4+δ have been shown to have limited stability, reacting with a Ce1-xGdxO2-δ interlayer to form a Ce1-x-yGdxPryO2-δ (CGPO) phase of unknown stoichiometry. Additionally, Pr2NiO4+δ are known to decompose to Pr4Ni3O10 ± δ under certain conditions. In this work detailed understanding of the chemical reaction between Pr2NiO4+δ and Ce0.9Gd0.1O2-δ (CGO10) under normal solid oxide cell fabrication and operating temperatures was obtained, identifying the composition of the resulting CGPO phase reaction products. It is shown that, in addition to the unreacted CGO10 present after sintering the compositae at 1100 °C for up to 12 h, a series of CGPO chemical compositions were formed with various Ce, Gd and Pr ratios depending on the relative distance of the doped ceria phases from the Pr2NiO4+δ phases. The extent of the chemical reaction was found to depend on the sintering time and the contact area of the two phases. Further thermal treatment of the resulting products under SOC air electrode operating temperature (800 °C) resulted in the initiation of Pr2NiO4+δ decomposition, forming Pr4Ni3O10 ± δ and Pr6O11 with no detectable change in the composition of previously formed Pr-substituted ceria phases. It is apparent that the Pr2NiO4+δ/CGO10 compositae is unsuitable as an air electrode, but there is evidence that the decomposition products, Pr4Ni3O10 ± δ and Ce1-x-yGdxPryO2-δ are stable and suitable candidates for SOC electrodes.

Open Access

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Abstract: The integration of luminescence and chirality in easy-scalable metal-organic frameworks gives rise to the development of advanced luminescent sensors. To date, the synthesis of chiral metal-organic frameworks is poorly predictable and their chirality primarily originates from components that constitute the frameworks. By contrast, the introduction of chirality into the pores of metal-organic frameworks has not been explored to the best of our knowledge. Here, we demonstrate that chirality can be introduced into an anionic Zn-based metal-organic framework via simple cation exchange, yielding dual luminescent centers comprised of the ligand and Tb3+ ions, accompanied by a chiral center in the pores. This bifunctional material shows enantioselectivity luminescent sensing for a mixture of stereoisomers, demonstrated for Cinchonine and Cinchonidine epimers and amino alcohol enantiomers, from which the quantitative determination of the stereoisomeric excess has been obtained. This study paves a pathway for the design of multifunctional metal-organic framework systems as a useful method for rapid sensing of chiral molecules.

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Abstract: Determining the oxidation state of Fe through parameterization of X-ray absorption near-edge structure (XANES) spectral features is highly dependent on accurate and repeatable energy calibration between spectra. Small errors in energy calibration can lead to vastly different interpretations. While simultaneous measurement of a reference foil is often undertaken on X-ray spectroscopy beamlines, other beamlines measure XANES spectra without a reference foil and therefore lack a method for correcting energy drift. Here a method is proposed that combines two measures of Fe oxidation state taken from different parts of the spectrum to iteratively correct for an unknown energy offset between spectra, showing successful iterative self-calibration not only during individual beam time but also across different beamlines.

Open Access

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Abstract: Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.