A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H2O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately −240 mV) and low-spin (approximately −110, −190 and −265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (Em=−80 mV) in the presence of NADH (Em=−320 mV) and an NADH–menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.</jats:p

Concentrating Membrane Proteins Using Asymmetric Traps and AC Electric Fields

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a “nested trap” and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins

pH dependent binding in de novo hetero bimetallic coiled coils

Herein the first example of a bimetallic coiled coil featuring a lanthanide binding site is reported, opening opportunities to exploit the attractive NMR and photophysical properties of the lanthanides in multi metallo protein design. In our efforts to fully characterise the system we identified for the first time that lanthanide binding to such sites is pH dependent, with optimal binding at neutral pH, and that the double AsnAsp site is more versatile in this regard than the single Asp site. Our second site featured the structural HgCys3 site, the chemistry of which was essentially unaltered by the presence of the lanthanide site. In fact, both metal binding sites within the hetero bimetallic coiled coil displayed the same properties as their mononuclear single binding site controls, and operated independently of each other. Finally, pH can be used as an external trigger to control the binding of Hg(II) and Tb(III) to the two distinct sites within this coiled coil, and offers the opportunity to “activate” metal binding sites within complex multi metallo and multi-functional designs

One-step fabrication of hollow-channel gold nanoflowers with excellent catalytic performance and large single-particle SERS activity.

Hollow metallic nanostructures have shown potential in various applications including catalysis, drug delivery and phototherapy, owing to their large surface areas, reduced net density, and unique optical properties. In this study, novel hollow gold nanoflowers (HAuNFs) consisting of an open hollow channel in the center and multiple branches/tips on the outer surface are fabricated for the first time, via a facile one-step synthesis using an auto-degradable nanofiber as a bifunctional template. The one-dimensional (1D) nanofiber acts as both a threading template as well as a promoter of the anisotropic growth of the gold crystal, the combination of which leads to the formation of HAuNFs with a hollow channel and nanospikes. The synergy of favorable structural/surface features, including sharp edges, open cavity and high-index facets, provides our HAuNFs with excellent catalytic performance (activity and cycling stability) coupled with large single-particle SERS activity (including ∼30 times of activity in ethanol electro-oxidation and ∼40 times of single-particle SERS intensity, benchmarked against similar-sized solid gold nanospheres with smooth surfaces, as well as retaining 86.7% of the initial catalytic activity after 500 cycles in ethanol electro-oxidation). This innovative synthesis gives a nanostructure of the geometry distinct from the template and is extendable to fabricating other systems for example, hollow-channel silver nanoflowers (HAgNFs). It thus provides an insight into the design of hollow nanostructures via template methods, and offers a versatile synthetic strategy for diverse metal nanomaterials suited for a broad range of applications

Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism

Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5-6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a 'molecular mousetrap', repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.Peer reviewe

Size-dependent activity of carbon dots for photocatalytic H2 generation in combination with a molecular Ni cocatalyst

Carbon dots (CDs) are low-cost light-absorbers in photocatalytic multicomponent systems, but their wide size distribution has hampered rational design and the identification of the factors that lead to their best performance. To address this challenge, we report herein the novel use of gel filtration size exclusion chromatography to separate amorphous, graphitic, and graphitic N-doped CDs depending on their lateral size to study the effect of their size on photocatalytic H2 evolution with a DuBois type Ni cocatalyst. Transmission electron microscopy and dynamic light scattering confirm size-dependent separation, while UV-vis and fluorescence spectroscopy of the more monodisperse fractions show a distinct response which computational modelling attributed to a complex interplay between CD size and optical properties. A size-dependent effect on the photocatalytic H2 evolution performance of the CDs in combination with a molecular Ni cocatalyst is demonstrated with a maximum activity at approximately 2-3 nm CD diameter. Overall, size separation leads to a two-fold increase in the specific photocatalytic activity for H2 evolution using the monodisperse CDs compared to the as synthesized polydisperse samples, highlighting the size-dependent effect on photocatalytic activity towards H2 evolution

Layer-by-Layer Assembly of Supported Lipid Bilayer Poly-l-Lysine Multilayers.

Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play

Supramolecular electrode assemblies for bioelectrochemistry

For more than three decades, the field of bioelectrochemistry has provided novel insights into the catalytic mechanisms of enzymes, the principles that govern biological electron transfer, and has elucidated the basic principles for bioelectrocatalytic systems. Progress in biochemistry, bionanotechnology, and our ever increasing ability to control the chemistry and structure of electrode surfaces has enabled the study of ever more complex systems with bioelectrochemistry. This feature article highlights developments over the last decade, where supramolecular approaches have been employed to develop electrode assemblies that increase enzyme loading on the electrode or create more biocompatible environments for membrane enzymes. Two approaches are particularly highlighted: the use of layer-by-layer assembly, and the modification of electrodes with planar lipid membranes

Enhanced oxygen-tolerance of the full heterotrimeric membrane-bound [NiFe]-hydrogenase of ralstonia eutropha.

Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen-oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane