Abstract One of the unresolved aspects of cellulose-based liquid crystalline phases is their chirality. Although cellulose is intrinsically chiral, both left-handed (LH) and right-handed (RH) chiral nematic phases are reported in cellulose derivatives under different conditions. The origin of these discrepancies?and whether LH and RH twisted structures coexist within a single material?has remained unclear. Here, the first direct evidence of hierarchical LH and RH twisted structures coexisting in a solvent-free, thermotropic cellulose derivative at room temperature is provided. Free-standing cholesteric films exhibit distinct LH and RH twisted domains, whose pitches respond oppositely to uniaxial mechanical strain: the LH pitch increases, while the RH pitch decreases with increasing strain. This contrasting response results from the coexistence of intertwined LH and RH twisted structures, whose optical axes are oriented differently relative to the strain direction. Notably, after stretching beyond their elastic limit, the films spontaneously recover their original shape within minutes. During this recovery, circular dichroism (CD) measurements reveal an increase in RH pitch and a decrease in LH pitch, evidencing reversible, strain-responsive behavior. Multiscale structural characterization confirms the hierarchical chiral organization and its mechanoresponsive nature, providing new insights into the origin of chirality in cellulose-based liquid crystalline materials.

ABSTRACT Mo/W-dependent formate dehydrogenases (Fdhs) catalyze the reversible reduction of CO2 to formate and are key biocatalysts with high potential for CO2 capture/conversion technologies. Although previous studies have suggested the presence of two substrate-access tunnels in Fdhs, experimental evidence for CO2-specific pathways has been lacking. Here, we present an integrated study of Nitratidesulfovibrio vulgaris FdhAB combining crystallography, molecular dynamics simulations, mutagenesis, and kinetic assays. NvFdhAB crystals pressurized with Kr, O2, and CO2 were used to map gas diffusion routes and uncovered a substrate-retention site consistently occupied by small molecules in multiple crystal structures. Our results indicate that both substrates mostly use the main tunnel to reach this retention site, but H2O and CO2 can also enter through a novel side branch before following a shared route to the buried W active site. The retention site, located at the junction of both tunnels, plays a synergistic role in enhancing CO2 reduction by increasing substrate concentration near the catalytic center, thereby improving catalytic efficiency. Notably, variants affecting this site showed a selective effect for CO2 reduction, with no impact on formate oxidation. These findings provide experimental evidence of a CO2-specific pathway and identify structural determinants underpinning efficient CO2 reduction in this enzyme family.

Geobacter bacteria produce multiheme c-type cytochrome nanowires that are involved in long-range extracellular electron transfer. The ability of these protein nanowires to conduct electrical current makes them promising candidates for electronic devices, offering several functional and sustainable advantages over traditional materials. Therefore, this study focused on synthesizing hybrid protein fibers that mimic the natural Geobacter extracellular nanowires. To achieve this, a mutated PpcA triheme protein variant was used as the building block, with thiol-ene coupling employed to bind the protein molecules. This engineered PpcA variant (PpcAK9CK22C) maintained a structure similar to that of the native protein. Thermal denaturation studies revealed a two-state process, with a melting temperature of 62 ± 1 °C and an enthalpy change of 61 ± 2 kcal/mol. The new protein nanowires showed a lower heme group content than the precursor protein and displayed distinct secondary structure features, with a slight reduction in helical content and an increase in β-sheet and unordered structures. Their thermal stability also differed, as it could not be described by the same model applied to the PpcA variant. Despite these differences, the nanowires retained their ability to undergo redox cycling. Morphologically, they consisted of linear single-protein filaments extending over 300 nm in length.

Biotechnological applications such as bioremediation, bioenergy production, and microbial electrosynthesis are emerging as sustainable alternatives to conventional, environmentally harmful industrial practices. Advancing these technologies requires a deeper understanding of extracellular electron transfer, a process mediated by a network of redox partners bridging the inner membrane and the extracellular environment. The CbcBA complex, a quinol:cytochrome c oxidoreductase from Geobacter sulfurreducens, is essential for the reduction of extracellular metal oxides and electrodes with redox potential values below −210 mV. The complex comprises CbcA, a heptaheme c-type cytochrome anchored to the inner membrane and CbcB, an integral membrane di-heme b-type cytochrome. Additionally, CbcC, a periplasmic dodecaheme cytochrome, and the five periplasmic triheme cytochromes PpcA-E, are proposed to function as redox partners of CbcBA. To investigate their structural and functional properties, the periplasmic domain of CbcA (CbcAsol) and CbcC were heterologously expressed and analyzed using complementary spectroscopic techniques. The crystal structure of CbcAsol was solved at 1.9 Å resolution, revealing a calcium-binding EF-hand motif that may function as a regulatory switch. Circular dichroism and differential scanning calorimetry indicated that CbcAsol and CbcC exhibit high stability, while potentiometric redox titrations demonstrated distinct electrochemical behaviors: CbcAsol has the most negative reduction potential among G. sulfurreducens oxidoreductases, whereas CbcC operates within the redox range of the PpcA-E cytochromes. NMR experiments showed that CbcAsol transfers electrons to CbcC and PpcA-E cytochromes. This result, in agreement with the electrostatic complementarity between the different cytochromes structures, suggests that CbcA may interact with multiple periplasmic cytochromes through distinct surface regions.

Electroactive bacteria mediate electron exchange with external compounds through a process known as extracellular electron transfer (EET). A key step in EET is the transfer of electrons from the menaquinone pool to inner membrane-associated quinol-cytochrome c oxidoreductase complexes, which subsequently relay electrons to periplasmic redox partners. Gene-knockout and proteomic analyses have identified several critical components involved in EET in Geobacter sulfurreducens, including six inner membrane oxidoreductase gene clusters. Of these, three - CbcL, ImcH, and CbcBA - have been linked to specific respiratory pathways depending on the redox potential of the terminal electron acceptor. Cbc4 is one of the other inner membrane oxidoreductase complexes and is composed by three subunits: a membrane-anchored tetraheme c-type cytochrome (CbcS), an iron–sulfur protein containing four [4Fe-4S] clusters (CbcT), and an integral membrane protein (CbcU). In this study, the sequence and AlphaFold model of CbcS were analyzed and its cytochrome domain was produced, and structurally and functionally characterized using Nuclear Magnetic Resonance spectroscopy. CbcS has four bis-histidine low-spin hemes and the structure of its hemecore is homologous to CymA and NrfH from Shewanella and Desulfovibrio species, respectively, despite differences on its axial ligands. Potentiometric titrations showed that the redox active window of CbcS overlaps with those of the triheme periplasmic cytochrome family (PpcA-E), its putative redox partners. Nevertheless, NMR-monitored electron transfer experiments revealed that CbcS transfers electrons to PpcA through the heme group closer to the C-terminal (heme IV). Together, these findings provide insights on a putative new respiratory pathway in G. sulfurreducens.

The global transition to electric vehicles and renewable energy systems has heightened the demand for lithium-ion batteries (LIBs), creating an urgent need for sustainable battery recycling methods to recover critical raw materials, including lithium. Lithium-selective cation-exchange polymeric membranes are one of the emerging options to achieve such lithium recycling. To make this change even greener, instead of using traditional fossil-origin polymers to produce membranes, this research employed bacterial cellulose acetate (BCA), a bio-derived and eco-friendly polymer. By adding 5 wt% N-methyl-N-propylpiperidinium bis(trifluoromethanesulfonyl)imide (PP13–TFSI), an ionic liquid (IL) which is a plasticizer and lithium-ion conductor, and 20 wt% hydrogen manganese oxide (HMO), which is a lithium-selective inorganic filler, four BCA-based membranes (BCA, BCA-IL, BCA-HMO and BCA-IL-HMO) were prepared. The membranes were extensively characterized for their morphology, thermal stability, chemical, and mechanical properties. Subsequently, they were tested in diffusion cells (without applying any external driving force) for ionic conductivity, lithium selectivity, and lithium flux using binary salt mixtures and synthetic LIB leachate. The BCA-IL membrane outperformed other BCA-based membranes in terms of separation factors, achieving values of 10.50 (Li+/Mn2+), 11.75 (Li+/Ni2+), and 10.95 (Li+/Co2+) with a lithium flux of 0.12 mol m−2 h−1 when processing synthetic LIB leachate. Under the same conditions, the BCA-HMO membranes exhibited a higher lithium flux (0.51 mol m−2 h−1) but with lower separation factor values of 3.39 (Li+/Mn2+), 3.62 (Li+/Ni2+), and 3.36 (Li+/Co2+). The use of plant-derived cellulose acetate (CA) as an alternative to BCA was also assessed; however, despite promising ideal lithium selectivity values (for example, 112 for Li+/Ni2+ in the case of CA-HMO membrane), their conductivity was up to two orders of magnitude lower than that of BCA-based membranes. All these findings highlight the promising potential of BCA-based membranes for lithium recovery from lithium-ion battery leachates.

The rising lithium-ion battery market drives lithium demand and requires efficient and selective lithium recovery methods from aqueous sources. Membrane technologies can address environmental and inherent efficiency issues in conventional lithium extraction methods. This study presents the synthesis of novel lithium-selective mixed matrix membranes (MMMs) by integrating 0–30 wt% of a lithium selective filler named hydrogen manganese oxide (HMO) into a sulfonated polyethersulfone (SPES)-Nafion polymer matrix. The membranes were produced by casting and thoroughly examined to assess their chemical, physical, morphological, thermal, and mechanical characteristics. The transport of lithium across membranes was evaluated in diffusion and electro-diffusion studies. The membrane containing 20 wt% of HMO exhibited the highest ideal selectivity values, which were 1.05 for Li+/K+, 1.20 for Li+/Na+, and 13.36 for Li+/Mg2+; and more than 97% increase in lithium-ion conductivity when compared with the control membrane without HMO. In diffusion experiments, the binary separation factors for Li+/K+, Li+/Na+, and Li+/Mg2+ were 0.71, 1.52, and 11.83, respectively, while under electro-diffusion conditions, the corresponding values were 0.82, 1.55, and 9.88. Above 20 wt% of HMO, membranes lose their separation capacity as HMO aggregates inside the membrane structure. The higher selectivity of membranes towards Li+ in the presence of Mg2+ is due to magnesium's larger hydrated radius and higher hydration energy compared to lithium. Overall, the prepared membranes demonstrated a promising potential for green lithium recovery. This study facilitates the advancement of sustainable lithium-selective MMM synthesis.

The goal of miniaturization in microelectronics catalyzes the evolution of field‐effect transistors (FETs), transitioning from classical scaling approaches to innovative architectures like gate‐all‐around FETs. Among these advancements, nanowire field‐effect transistors (NW‐FETs) emerge as a promising solution to the limitations of traditional FET designs, offering improved electrostatic control, reduction of short‐channel effects, and better overall device performance metrics. Metal oxide nanowires (NWs) provide high mobility, excellent optical transparency, mechanical flexibility, and compatibility with thin‐film technology, making them ideal candidates to be the pillar of a new wave of transparent and flexible electronics with unprecedented integration levels. This review highlights the different configurations of NW‐FETs, exploring their fabrication techniques and different advantages, as well as state‐of‐the‐art progress in metal oxide NW‐FETs, such as zinc oxide (ZnO), indium oxide (In 2 O 3 ), tin oxide (SnO 2 ), and multicomponent materials. To further improve NW‐FET performance, recent developments in doping, surface passivation methods, and post‐fabrication treatments are examined, as well as emerging fabrication methodologies. By addressing material limitations and integrating innovative design strategies, metal oxide NW‐FETs are set to play a pivotal role in sustaining Moore's Law and shaping the future of nanoelectronics.

Metal oxide nanostructures have recently gained high attention due to advances in their synthesis, particularly hydrothermal techniques, which allow precise control over their morphology, composition, and crystallinity, as well as integration into devices. Zinc-tin oxide (ZTO) nanostructures, in particular, are notable for their sustainability and multifunctional applications, including catalysis, electronics, sensors, and energy harvesting. Their ternary oxide nature supports a broad range of functionalities. The use of seed layers during synthesis has proven to be beneficial, particularly for binary systems such as ZnO, as it not only impacts the growth of nanostructures but is also advantageous for applications requiring nanostructures supported on substrates, such as in photocatalysis and sensor technologies. This work investigates the effect of various seed layers (e.g., Cu, stainless steel, Cr, Ni) on the hydrothermal synthesis of ZTO nanostructures. Compared to seed layer free methods under similar conditions, the presence of seed layers significantly influenced the resulting structures. The study produced diverse morphologies, including ZnSnO₃ nanowires and Zn₂SnO₄ nanoparticles, octahedrons, and nanowires. Findings suggest a relationship between the seed layer's phase and the resulting nanostructure phase. Furthermore, shorter synthesis durations favored discrete nanostructures, while longer durations facilitated the formation of thin films with nanostructured surfaces. These observations underscore the dual role of seed layers in influencing both the structural phase and growth kinetics of ZTO nanostructures.

Summary Mammalian aldehyde oxidases (AOXs) are complex enzymes dependent on a molybdopterin-active site (Moco), two [2Fe?2S] spectroscopically distinct centers, and one flavin adenine dinucleotide (FAD) cofactor. AOXs belong to the xanthine oxidase (XO) family of mononuclear molybdenum enzymes. AOXs and XO share a high degree of structural similarity forming homodimers that encompass in each subunit a chain of redox centers involved in the transfer of reducing equivalents from substrate oxidation (Moco ? [2Fe?2S] I ? [2Fe?2S] II? FAD) to molecular oxygen. However, AOXs and XO differ in substrate specificity and, while XO has a clear role in the last steps of purine catabolism, AOXs are very promiscuous enzymes. They catalyze a wide diversity of reactions, accept diverse substrates, and play an important role in drug and xenobiotic metabolism. Despite numerous studies, the physiological substrates of AOX and its physiological relevance are still unclear.

Metal-dependent formate dehydrogenases (FDHs) catalyze, under mild conditions, the reversible reduction of CO2 to formate, a versatile C1 feedstock that can contribute to a carbon-neutral economy. Metal-dependent FDHs are the most widespread selenoproteins found in bacteria, and around 44% of them include selenocysteine (Sec) as a ligand to the Mo/W active site. In the sulfate-reducer Nitratidesulfovibrio vulgaris Hildenborough, the main FDH responsible for CO2 reduction is the W/Sec-dependent FdhAB, which is among the most active CO2 reductases reported so far. In contrast to most metal-dependent FDHs, this enzyme is relatively O2-tolerant and can be purified aerobically. In this work, we evaluated the role of Sec in the catalytic and stability properties of the W/Sec-FdhAB. For that, a Sec-to-Cys variant (U192C) was created, its catalytic and spectroscopic properties were characterized, and its crystal structure was determined. Sec substitution by Cys strongly affects activity, decreases the KM for formate, and increases susceptibility to O2. While Sec-to-Cys replacement induces only weak changes of the WV EPR signals, using 77Se-labeled enzyme, we could show that Sec undoubtedly coordinates the W metal in the WV redox state. The crystal structure of U192C confirmed previous findings on the redox switch mechanism of activation and protection of FdhAB, while revealing a putative catalytic intermediate of FdhAB with Arg441 orienting a CO2 substrate analog (probably SO2) in the active site. Overall, the results indicate that Sec plays a critical role in the high activity displayed by W/Sec-FdhAB, and that it may also be involved in or modulate the proton transfer to and from the active site.Metal-dependent formate dehydrogenases (FDHs) catalyze, under mild conditions, the reversible reduction of CO2 to formate, a versatile C1 feedstock that can contribute to a carbon-neutral economy. Metal-dependent FDHs are the most widespread selenoproteins found in bacteria, and around 44% of them include selenocysteine (Sec) as a ligand to the Mo/W active site. In the sulfate-reducer Nitratidesulfovibrio vulgaris Hildenborough, the main FDH responsible for CO2 reduction is the W/Sec-dependent FdhAB, which is among the most active CO2 reductases reported so far. In contrast to most metal-dependent FDHs, this enzyme is relatively O2-tolerant and can be purified aerobically. In this work, we evaluated the role of Sec in the catalytic and stability properties of the W/Sec-FdhAB. For that, a Sec-to-Cys variant (U192C) was created, its catalytic and spectroscopic properties were characterized, and its crystal structure was determined. Sec substitution by Cys strongly affects activity, decreases the KM for formate, and increases susceptibility to O2. While Sec-to-Cys replacement induces only weak changes of the WV EPR signals, using 77Se-labeled enzyme, we could show that Sec undoubtedly coordinates the W metal in the WV redox state. The crystal structure of U192C confirmed previous findings on the redox switch mechanism of activation and protection of FdhAB, while revealing a putative catalytic intermediate of FdhAB with Arg441 orienting a CO2 substrate analog (probably SO2) in the active site. Overall, the results indicate that Sec plays a critical role in the high activity displayed by W/Sec-FdhAB, and that it may also be involved in or modulate the proton transfer to and from the active site.

Polysaccharides in plant cell walls serve as a rich carbon and energy source, yet their structural complexity presents a barrier to efficient degradation. To address this, anaerobic microorganisms like R. flavefaciens have developed sophisticated multi-enzyme complexes known as cellulosomes, which enable the efficient breakdown of these recalcitrant polysaccharides. These complexes are assembled through high-affinity interactions between cohesin (Coh) modules in scaffoldin proteins and dockerin (Doc) modules in cellulosomal enzymes. R. flavefaciens FD-1 harbors one of the most intricate cellulosomes described to date, comprising over 200 Doc-containing proteins encoded in its genome. Despite substantial research on this cellulosome, the role of a group of truncated but functional dockerins, known as group-2 Docs, remains unclear. In this study, we present a detailed structural and binding analysis of a Coh-Doc complex involving the cohesin from the cell-anchoring scaffoldin ScaE and a group-2 Doc that bears only one of the two Ca+2-coordinating loops that characterise the canonical Docs. Our findings reveal a novel tripartite binding mechanism, in which the cohesin can simultaneously bind two distinct dockerin units in three alternative conformations. This discovery provides new insights into the modular versatility of the R. flavefaciens cellulosome and sheds light on the mechanisms that enhance its efficiency in polysaccharide degradation.

The bacterium Geotalea uraniireducens, commonly found in uranium-contaminated environments, plays a key role in bioremediation strategies by converting the soluble hexavalent form of uranium (UVI) into less soluble forms (e.g. UIV.). While most of the reduction and concomitant precipitation of uranium occur outside the cells, there have been reports of important reduction processes taking place in the periplasm. In any case, the triheme periplasmic cytochromes are crucial players, either by ensuring an effective interface between the cell´s interior and exterior or by directly participating in the reduction of the metal. Therefore, understanding the functional mechanism of the highly abundant G. uraniireducens’ triheme cytochromes is crucial to assist the elucidation on the respiratory pathways in this bacterium. In this work, a detailed functional characterization of the triheme cytochromes PpcA and PpcB from G. uraniireducens was conducted using NMR and visible spectroscopy techniques. Despite sharing high amino acid sequence and structural homology with their counterparts from G. sulfurreducens, the results obtained showed that the heme reduction potential values are less negative, the order of oxidation of the hemes is distinct, and the redox and redox-Bohr network of interactions revealed unprecedented functional mechanisms of the G. uraniireducens cytochromes. In these cytochromes, the reduction potential values of the three heme groups are much more similar, hence covering a narrow range of values, features that facilitate the directional electron flow from the inner membrane, thereby favouring the optimal reduction of uranium.