Rescue of Rare CFTR Trafficking Mutants Highlights a Structural Location-Dependent Pattern for Correction.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Currently, more than 2100 variants have been identified in the gene, with a large number being very rare. The approval of modulators that act on mutant CFTR protein, correcting its molecular defect and thus alleviating the burden of the disease, revolutionized the field of CF. However, these drugs do not apply to all patients with CF, especially those with rare mutations-for which there is a lack of knowledge on the molecular mechanisms of the disease and the response to modulators. In this work, we evaluated the impact of several rare putative class II mutations on the expression, processing, and response of CFTR to modulators. Novel cell models consisting of bronchial epithelial cell lines expressing CFTR with 14 rare variants were created. The variants studied are localized at Transmembrane Domain 1 (TMD1) or very close to the signature motif of Nucleotide Binding Domain 1 (NBD1). Our data show that all mutations analyzed significantly decrease CFTR processing and while TMD1 mutations respond to modulators, those localized in NBD1 do not. Molecular modeling calculations confirm that the mutations in NBD1 induce greater destabilization of CFTR structure than those in TMD1. Furthermore, the structural proximity of TMD1 mutants to the reported binding site of CFTR modulators such as VX-809 and VX-661, make them more efficient in stabilizing the CFTR mutants analyzed. Overall, our data suggest a pattern for mutation location and impact in response to modulators that correlates with the global effect of the mutations on CFTR structure.

Elucidating Film Loss and the Role of Hydrogen Bonding of Adsorbed Redox Enzymes by Electrochemical Quartz Crystal Microbalance Analysis.

The immobilization of redox enzymes on electrodes enables the efficient and selective electrocatalysis of useful reactions such as the reversible interconversion of dihydrogen (H2) to protons (H+) and formate to carbon dioxide (CO2) with hydrogenase (H2ase) and formate dehydrogenase (FDH), respectively. However, their immobilization on electrodes to produce electroactive protein films for direct electron transfer (DET) at the protein-electrode interface is not well understood, and the reasons for their activity loss remain vague, limiting their performance often to hour timescales. Here, we report the immobilization of [NiFeSe]-H2ase and [W]-FDH from Desulfovibrio vulgaris Hildenborough on a range of charged and neutral self-assembled monolayer (SAM)-modified gold electrodes with varying hydrogen bond (H-bond) donor capabilities. The key factors dominating the activity and stability of the immobilized enzymes are determined using protein film voltammetry (PFV), chronoamperometry (CA), and electrochemical quartz crystal microbalance (E-QCM) analysis. Electrostatic and H-bonding interactions are resolved, with electrostatic interactions responsible for enzyme orientation while enzyme desorption is strongly limited when H-bonding is present at the enzyme-electrode interface. Conversely, enzyme stability is drastically reduced in the absence of H-bonding, and desorptive enzyme loss is confirmed as the main reason for activity decay by E-QCM during CA. This study provides insights into the possible reasons for the reduced activity of immobilized redox enzymes and the role of film loss, particularly H-bonding, in stabilizing bioelectrode performance, promoting avenues for future improvements in bioelectrocatalysis.

Fast CO2 hydration kinetics impair heterogeneous but improve enzymatic CO2 reduction catalysis.

The performance of heterogeneous catalysts for electrocatalytic CO2 reduction suffers from unwanted side reactions and kinetic inefficiencies at the required large overpotential. However, immobilized CO2 reduction enzymes-such as formate dehydrogenase-can operate with high turnover and selectivity at a minimal overpotential and are therefore 'ideal' model catalysts. Here, through the co-immobilization of carbonic anhydrase, we study the effect of CO2 hydration on the local environment and performance of a range of disparate CO2 reduction systems from enzymatic (formate dehydrogenase) to heterogeneous systems. We show that the co-immobilization of carbonic anhydrase increases the kinetics of CO2 hydration at the electrode. This benefits enzymatic CO2 reduction-despite the decrease in CO2 concentration-due to a reduction in local pH change, whereas it is detrimental to heterogeneous catalysis (on Au) because the system is unable to suppress the H2 evolution side reaction. Understanding the role of CO2 hydration kinetics within the local environment on the performance of electrocatalyst systems provides important insights for the development of next-generation synthetic CO2 reduction catalysts.

Closing the Gap for Electronic Short-Circuiting: Photosystem†I Mixed Monolayers Enable Improved Anisotropic Electron Flow in Biophotovoltaic Devices.

Well-defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi-artificial energy conversion devices, capable of providing unidirectional electron flow when light-harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short-circuiting processes that typically limit the performance of PSI-based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light-induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]-hydrogenase confirms the possibility to realize light-induced H2 evolution.

Exploring the gas access routes in a [NiFeSe] hydrogenase using crystals pressurized with krypton and oxygen.

Hydrogenases are metalloenzymes that catalyse both H2 evolution and uptake. They are gas-processing enzymes with deeply buried active sites, so the gases diffuse through channels that connect the active site to the protein surface. The [NiFeSe] hydrogenases are a special class of hydrogenases containing a selenocysteine as a nickel ligand; they are more catalytically active and less O2-sensitive than standard [NiFe] hydrogenases. Characterisation of the channel system of hydrogenases is important to understand how the inhibitor oxygen reaches the active site to cause oxidative damage. To this end, crystals of Desulfovibrio vulgaris Hildenborough [NiFeSe] hydrogenase were pressurized with krypton and oxygen, and a method for tracking labile O2 molecules was developed, for mapping a hydrophobic channel system similar to that of the [NiFe] enzymes as the major route for gas diffusion.

Redox-Polymer-Wired [NiFeSe] Hydrogenase Variants with Enhanced O2 Stability for Triple-Protected High-Current-Density H2 -Oxidation Bioanodes.

Variants of the highly active [NiFeSe] hydrogenase from D. vulgaris Hildenborough that exhibit enhanced O2 tolerance were used as H2 -oxidation catalysts in H2 /O2 biofuel cells. Two [NiFeSe] variants were electrically wired by means of low-potential viologen-modified redox polymers and evaluated with respect to H2 -oxidation and stability against O2 in the immobilized state. The two variants showed maximum current densities of (450±84) µA cm-2 for G491A and (476±172) µA cm-2 for variant G941S on glassy carbon electrodes and a higher O2 tolerance than the wild type. In addition, the polymer protected the enzyme from O2 damage and high-potential inactivation, establishing a triple protection for the bioanode. The use of gas-diffusion bioanodes provided current densities for H2 -oxidation of up to 6.3 mA cm-2 . Combination of the gas-diffusion bioanode with a bilirubin oxidase-based gas-diffusion O2 -reducing biocathode in a membrane-free biofuel cell under anode-limiting conditions showed unprecedented benchmark power densities of 4.4 mW cm-2 at 0.7 V and an open-circuit voltage of 1.14 V even at moderate catalyst loadings, outperforming the previously reported system obtained with the [NiFeSe] wild type and the [NiFe] hydrogenase from D. vulgaris Miyazaki F.

Integration of a Hydrogenase in a Lead Halide Perovskite Photoelectrode for Tandem Solar Water Splitting.

Lead halide perovskite solar cells are notoriously moisture-sensitive, but recent encapsulation strategies have demonstrated their potential application as photoelectrodes in aqueous solution. However, perovskite photoelectrodes rely on precious metal co-catalysts, and their combination with biological materials remains elusive in integrated devices. Here, we interface [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough, a highly active enzyme for H2 generation, with a triple cation mixed halide perovskite. The perovskite-hydrogenase photoelectrode produces a photocurrent of -5 mA cm-2 at 0 V vs RHE during AM1.5G irradiation, is stable for 12 h and the hydrogenase exhibits a turnover number of 1.9 × 106. The positive onset potential of +0.8 V vs RHE allows its combination with a BiVO4 water oxidation photoanode to give a self-sustaining, bias-free photoelectrochemical tandem system for overall water splitting (solar-to-hydrogen efficiency of 1.1%). This work demonstrates the compatibility of immersed perovskite elements with biological catalysts to produce hybrid photoelectrodes with benchmark performance, which establishes their utility in semiartificial photosynthesis.

Reversible and Selective Interconversion of Hydrogen and Carbon Dioxide into Formate by a Semiartificial Formate Hydrogenlyase Mimic.

The biological formate hydrogenlyase (FHL) complex links a formate dehydrogenase (FDH) to a hydrogenase (H2ase) and produces H2 and CO2 from formate via mixed-acid fermentation in Escherichia coli. Here, we describe an electrochemical and a colloidal semiartificial FHL system that consists of an FDH and a H2ase immobilized on conductive indium tin oxide (ITO) as an electron relay. These in vitro systems benefit from the efficient wiring of a highly active enzyme pair and allow for the reversible conversion of formate to H2 and CO2 under ambient temperature and pressure. The hybrid systems provide a template for the design of synthetic catalysts and surpass the FHL complex in vivo by storing and releasing H2 on demand by interconverting CO2/H2 and formate with minimal bias in either direction.

Characterization of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough.

The [NiFeSe] hydrogenases are a subgroup of the well-characterized family of [NiFe] hydrogenases, in which a selenocysteine is a ligand to the nickel atom in the binuclear NiFe active site instead of cysteine. These enzymes display very interesting catalytic properties for biological hydrogen production and bioelectrochemical applications: high H2 production activity, bias for H2 evolution, low H2 inhibition, and some degree of O2 tolerance. Here we describe the methodologies employed to study the [NiFeSe] hydrogenase isolated from the sulfate-reducing bacteria D. vulgaris Hildenborough and the creation of a homologous expression system for production of variant forms of the enzyme.

A gas breathing hydrogen/air biofuel cell comprising a redox polymer/hydrogenase-based bioanode.

Hydrogen is one of the most promising alternatives for fossil fuels. However, the power output of hydrogen/oxygen fuel cells is often restricted by mass transport limitations of the substrate. Here, we present a dual-gas breathing H2/air biofuel cell that overcomes these limitations. The cell is equipped with a hydrogen-oxidizing redox polymer/hydrogenase gas-breathing bioanode and an oxygen-reducing bilirubin oxidase gas-breathing biocathode (operated in a direct electron transfer regime). The bioanode consists of a two layer system with a redox polymer-based adhesion layer and an active, redox polymer/hydrogenase top layer. The redox polymers protect the biocatalyst from high potentials and oxygen damage. The bioanodes show remarkable current densities of up to 8 mA cm-2. A maximum power density of 3.6 mW cm-2 at 0.7 V and an open circuit voltage of up to 1.13 V were achieved in biofuel cell tests, representing outstanding values for a device that is based on a redox polymer-based hydrogenase bioanode.

A fully protected hydrogenase/polymer-based bioanode for high-performance hydrogen/glucose biofuel cells.

Hydrogenases with Ni- and/or Fe-based active sites are highly active hydrogen oxidation catalysts with activities similar to those of noble metal catalysts. However, the activity is connected to a sensitivity towards high-potential deactivation and oxygen damage. Here we report a fully protected polymer multilayer/hydrogenase-based bioanode in which the sensitive hydrogen oxidation catalyst is protected from high-potential deactivation and from oxygen damage by using a polymer multilayer architecture. The active catalyst is embedded in a low-potential polymer (protection from high-potential deactivation) and covered with a polymer-supported bienzymatic oxygen removal system. In contrast to previously reported polymer-based protection systems, the proposed strategy fully decouples the hydrogenase reaction form the protection process. Incorporation of the bioanode into a hydrogen/glucose biofuel cell provides a benchmark open circuit voltage of 1.15 V and power densities of up to 530 µW cm-2 at 0.85 V.

Protection and Reactivation of the [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough under Oxidative Conditions.

We report on the fabrication of bioanodes for H2 oxidation based on [NiFeSe] hydrogenase. The enzyme was electrically wired by means of a specifically designed low-potential viologen-modified polymer, which delivers benchmark H2 oxidizing currents even under deactivating conditions owing to efficient protection against O2 combined with a viologen-induced reactivation of the O2 inhibited enzyme. Moreover, the viologen-modified polymer allows for electrochemical co-deposition of polymer and biocatalyst and, by this, for control of the film thickness. Protection and reactivation of the enzyme was demonstrated in thick and thin reaction layers.

H2 -Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration.

ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1 F0 -ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.

Induction of a proton gradient across a gold-supported biomimetic membrane by electroenzymatic H2 oxidation.

Energy-transduction mechanisms in living organisms, such as photosynthesis and respiration, store light and chemical energy in the form of an electrochemical gradient created across a lipid bilayer. Herein we show that the proton concentration at an electrode/phospholipid-bilayer interface can be controlled and monitored electrochemically by immobilizing a membrane-bound hydrogenase. Thus, the energy derived from the electroenzymatic oxidation of H2 can be used to generate a proton gradient across the supported biomimetic membrane.

Structural basis for Z-DNA binding and stabilization by the zebrafish Z-DNA dependent protein kinase PKZ.

The RNA-dependent protein kinase PKR plays a central role in the antiviral defense of vertebrates by shutting down protein translation upon detection of viral dsRNA in the cytoplasm. In some teleost fish, PKZ, a homolog of PKR, performs the same function, but surprisingly, instead of dsRNA binding domains, it harbors two Z-DNA/Z-RNA-binding domains belonging to the Zalpha domain family. Zalpha domains have also been found in other proteins, which have key roles in the regulation of interferon responses such as ADAR1 and DNA-dependent activator of IFN-regulatory factors (DAI) and in viral proteins involved in immune response evasion such as the poxviral E3L and the Cyprinid Herpesvirus 3 ORF112. The underlying mechanism of nucleic acids binding and stabilization by Zalpha domains is still unclear. Here, we present two crystal structures of the zebrafish PKZ Zalpha domain (DrZalpha(PKZ)) in alternatively organized complexes with a (CG)6 DNA oligonucleotide at 2 and 1.8 Å resolution. These structures reveal novel aspects of the Zalpha interaction with DNA, and they give insights on the arrangement of multiple Zalpha domains on DNA helices longer than the minimal binding site.

Crystal structure of a poxvirus-like zalpha domain from cyprinid herpesvirus 3.

Zalpha domains are a subfamily of the winged helix-turn-helix domains sharing the unique ability to recognize CpG repeats in the left-handed Z-DNA conformation. In vertebrates, domains of this family are found exclusively in proteins that detect foreign nucleic acids and activate components of the antiviral interferon response. Moreover, poxviruses encode the Zalpha domain-containing protein E3L, a well-studied and potent inhibitor of interferon response. Here we describe a herpesvirus Zalpha-domain-containing protein (ORF112) from cyprinid herpesvirus 3. We demonstrate that ORF112 also binds CpG repeats in the left-handed conformation, and moreover, its structure at 1.75 Å reveals the Zalpha fold found in ADAR1, DAI, PKZ, and E3L. Unlike other Zalpha domains, however, ORF112 forms a dimer through a unique domain-swapping mechanism. Thus, ORF112 may be considered a new member of the Z-domain family having DNA binding properties similar to those of the poxvirus E3L inhibitor of interferon response.