Structure, Function, and Thermal Adaptation of the Biotin Carboxylase Domain Dimer from Hydrogenobac
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Buhrman, Greg↗
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Structural Basis for Superoxide Activation of Flavobacterium johnsoniae Class I Ribonucleotide Reductase and for Radical Initiation by Its Dimanganese Cofactor
A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj ) differs fundamentally from known (subclass a–c) class I RNRs, warranting its assignment to a new subclass, Id. Its β subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O 2 – ) for activation, but it does not require the O 2 – -supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj β has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj β directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, we note the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive “met” (III/III) forms seen with I(a–c) βs. This tendency makes Fj β unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O 2 – to the Mn 2 II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj β is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-β.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis
Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate β subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a Mn II 2 cluster by free superoxide to yield a metal-based Mn III Mn IV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia–c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae (Fj) and Actinobacillus ureae (Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive β subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Overall, our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.
59 BASIC BIOLOGICAL SCIENCES↗
Conjugation of Amphiphilic Proteins to Hydrophobic Ligands in Organic Solvent
Protein–ligand conjugations are usually carried out in aqueous media in order to mimic the environment within which the conjugates will be used. Here, we focus on the conjugation of amphiphilic variants of elastin-like polypeptide (ELP), short elastin (sEL), to poorly water-soluble compounds like OPPVs (p-phenylenevinylene oligomers), triarylamines, and polypyridine-metal complexes. These conjugations are problematic when carried out in aqueous phase because hydrophobic ligands tend to avoid exposure to water, which in turn causes the ligand to self-aggregate and/or interact noncovalently with hydrophobic regions of the amphiphile. Ultimately, this behavior leads to low conjugation efficiency and contamination with strong noncovalent “conjugates”. After exploring the solubility of sEL in various organic solvents, we have established an efficient conjugation methodology for obtaining covalent conjugates virtually free of contaminating noncovalent complexes. When conjugating carboxylated ligands to the amphiphile amines, we demonstrate that even when only one amine (the N-terminus) is present, its derivatization is 98% efficient. When conjugating amine moieties to the amphiphile carboxyls (a problematic configuration), protein multimerization is avoided, 98–100% of the protein is conjugated, and the unreacted ligand is recovered in pure form. Our syntheses occur in “one pot”, and our purification procedure is a simple workup utilizing a combination of water and organic solvent extractions. This conjugation methodology might provide a solution to problems arising from solubility mismatch of protein and ligand, and it is likely to be widely applied for modification of recombinant amphiphiles used for drug delivery (PEG-antibodies, polymer-enzymes, food proteins), cell adhesion (collagen, hydrophobins), synthesis of nanostructures (peptides), and engineering of biocompatible optoelectronics (biological polymers), to cite a few.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Mapping Competitive Reduction upon Charging in LiNi 0.8 Co 0.15 Al 0.05 O 2 Primary Particles
Side reactions involving surface reduction play a critical role in the failure of LiNi 0.8 Co 0.15 Al 0.05 O 2 to reach its theoretical capacity as a cathode material for Li-ion batteries. While macroscopic consequences are known, the underlying nanoscopic mechanisms are not fully elucidated. By coupling X-ray spectroscopy with several X-ray microscopy modalities, we have spatially resolved the extent of Ni oxidation at several states of charge and uncovered heterogeneity that is hidden when considering ensemble measurements alone. The use of morphologically controlled particles enabled high-resolution imaging of these materials, uncovering gradients of Ni oxidation states within individual primary particles. At high states of charge, these gradients revealed regions of possible oxygen deficiency extending deeper into the particle than previously observed. Surface-sensitive X-ray coupled scanning tunneling microscopy allows oxidation states to be measured at the material's surface, showing predominantly Ni II in the first atomic layer and mixtures of Ni II with Ni III /Ni IV already appearing 1.5 nm into the particle. These results reveal the subtle interplay between irreversible surface transformations and the bulk reactions that ultimately define function, which will refine strategies of surface passivation that are key to overcoming current performance limitations.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Impact of Transition Metal Doping on the Structural and Optical Properties of Halide Perovskites
Metal ion doping into halide perovskite materials is a promising approach to enhance long-term film stability and improve solar cell performance. Moreover, the idea of doping metals into halide perovskites is also driven by applications in heterogeneous catalysis. Here, we investigate the incorporation of the transition metals Pt and Pd into mixed-halide perovskite materials. We find that Pt aggregates in large clusters in iodine- and bromine-rich perovskite film compositions, whereas Pd incorporation depends on the underlying bromine to iodine ratio. Furthermore, by using optical and structural characterization methods, we observe that phase segregation in the perovskite layer is suppressed upon doping of either of these metals into the material. This approach further shows the potential to obtain catalytically active halide perovskite films in which the perovskite can serve as a host matrix.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Physics-Informed Machine-Learning Prediction of Curie Temperatures and Its Promise for Guiding the Discovery of Functional Magnetic Materials
High-performance permanent magnets with a high Curie temperature, containing less critical materials, are integral to zero-carbon energy solutions. We built a machine-learning model trained over available experimentally measured Curie temperature values to predict the T C of multicomponent magnetic materials. We chose two compositions from a pseudo-binary (Zr 1–x Ce x )Fe 2 system, namely, (Zr 0.16 Ce 0.84 )Fe 2 and (Zr 0.94 Ce 0.06 )Fe 2 , to experimentally validate the ability of our model to predict the Curie temperature of novel compounds. We also provided a detailed discussion on the correlation of the Curie temperature with the de Gennes scaling factor in rare-earth intermetallic compounds and its breakdown below a certain rare-earth content. The electronic structure calculations (density of states and Fermi surface) were performed using the density functional theory on selected compounds (Zr 0.16 Ce 0.84 )Fe 2 and (Zr 0.94 Ce 0.06 )Fe 2 to understand the electronic origin of a strong magnetic exchange. We found that the change in the electronic density of states and electron/hole fillings at the Fermi level directly correlate with the Curie temperature. Notably, our model was able to capture these key electronic structure trends, which show that physics-informed machine learning can play a crucial role in designing new high-performance magnets with improved properties for environmentally sustainable applications.
36 MATERIALS SCIENCE↗
Cooling Mixed A-Site Halide Perovskites: Impact of Temperature on Optical and Structural Properties
The widespread utilization of perovskite-based photovoltaics requires probing both the structural and optical properties under extreme operating conditions to gain a holistic understanding of the material behavior under stressors. Here, in this study, we investigate the temperature-dependent behavior of mixed A-site cation lead triiodide perovskite thin films (85% methylammonium and 15% formamidinium) in the range from 300 to 20 K. Through a combination of optical and structural techniques, we find that the tetragonal-to-orthorhombic phase transition occurs at similar to 110 K for this perovskite composition, as indicated by the change in the diffraction pattern. With decreasing temperature, the quantum yield increases with a concurrent elongation of the carrier lifetimes, indicating suppression of nonradiative recombination pathways. Interestingly, in contrast to single A-site cation perovskites, an additional optical transition appears in the absorption spectrum when the phase transition is approached, which is also reflected in the emission spectrum. We propose that the splitting of the optical absorption and emission is due to local segregation of the mixed cation perovskite during the phase transition.
36 MATERIALS SCIENCE↗
Role of Ammonium Hydroxide on Glucose Oxidase Immobilized in Metal–Azolate Framework-7 Enzyme Activity
Enzymes are nature’s catalysts, but their instability outside native environments limits practical applications. Metal–organic frameworks (MOFs) offer a promising platform for enzyme immobilization, enhancing stability, and reusability through their tunable porosity and crystallinity. While ZIF-based MOFs have been extensively studied, metal azolate framework-7 (MAF-7) remains largely unexplored for biomimetic mineralization. Its hydrophilic character makes it a promising alternative for enzyme encapsulation; however, the role of essential basic modulators such as ammonium hydroxide (NH 4 OH) on enzyme structure and function has not been investigated. Here, in this study, we examine the NH 4 OH mediated synthesis of glucose oxidase within MAF-7 using powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), cryogenic transmission electron microscopy (cryo-TEM), and enzyme assays. Our results show that NH 4 OH modulates crystal morphology, enzyme activity, and nucleation behavior, with higher concentrations leading to partial enzyme denaturation and reduced catalytic performance. By integrating time-resolved cryo-TEM with activity assays, we uncover how modulator concentration impacts early stage crystallization and enzyme folding, a mechanistic insight not previously demonstrated in MOF systems. These findings highlight modulator chemistry as a critical, underexplored variable in MOF synthesis, advancing rational design strategies for enzyme@MOFs (E@MOF) beyond ZIFs and toward more tunable, biocompatible frameworks like MAF-7.
36 MATERIALS SCIENCE↗
Synthesis and Electrochemical and Structural Investigations of Oxidatively Stable Li 2 MoO 3 and x Li 2 MoO 3 ·(1 – x )LiMO 2 Composite Cathodes
We report the structural evolution of Li 2 MoO 3 during electrochemical delithiation/lithiation is reported. Li 2 MoO 3 undergoes an irreversible crystalline to amorphous transformation which starts during the first delithiation step and gradually proceeds throughout subsequent cycles. This observation is supported by complementary data obtained from X-ray diffraction (XRD), Raman spectroscopy, and transmission electron microscopy (TEM). The amorphization does not prevent reversible Li storage, and the Li 2 MoO 3 cathodes exhibit initial capacities of 147 mAh/g (corresponding to 0.87 Li cycled per formula unit) at an average operating potential ~2.5 V vs Li/Li + with reasonably good cycling stability (81% capacity retention after 50 cycles). In-situ mass spectrometry studies reveal the amorphization of Li 2 MoO 3 does not involve the release of oxygen gas even when charged to very positive potentials (i.e., 4.8 V vs Li/Li + ). The Li storage properties and excellent oxidative stability of Li 2 MoO 3 make it a promising candidate to improve the performance of traditional LiMO 2 compounds in layered–layered composite cathodes with the general formula x Li 2 MoO 3 ·(1 – x)LiMO 2 . Multiple synthesis routes were explored to prepare composites with x = 0.10–0.15, and their structure was characterized using XRD and time-of-flight neutron diffraction. Lastly, preliminary characterization of these materials shows that Li 2 MoO 3 improves the cycling stability of an NMC cathode, presumably by mitigating detrimental structural rearrangement at high states of charge.
36 MATERIALS SCIENCE↗
Atomically Layered and Ordered Rare-Earth i -MAX Phases: A New Class of Magnetic Quaternary Compounds
In 2017, we discovered quaternary i -MAX phases—atomically layered solids, where M is an early transition metal, A is an A group element, and X is C—with a (M 1 2/3 M 2 1/3 ) 2 AC chemistry, where the M 1 and M 2 atoms are in-plane ordered. Herein, we report the discovery of a class of magnetic i -MAX phases in which bilayers of a quasi-2D magnetic frustrated triangular lattice overlay a Mo honeycomb arrangement and an Al Kagomé lattice. The chemistry of this family is (Mo 2/3 RE 1/3 ) 2 AlC, and the rare-earth, RE, elements are Ce, Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, and Lu. Here, the magnetic properties were characterized and found to display a plethora of ground states, resulting from an interplay of competing magnetic interactions in the presence of magnetocrystalline anisotropy.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater
Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.
59 BASIC BIOLOGICAL SCIENCES↗
Benchmarking Concentration and Extraction Methods for Wastewater-Based Surveillance of Eight Human Respiratory Viruses: Implications for Rapid Application to Novel Pathogens
To provide early warning and support a rapid response to a novel virus through wastewater surveillance, it would be ideal to understand in advance which concentration and extraction methods are likely to be effective for dPCR-based methods, depending on virus characteristics. In this study, we spiked raw wastewater samples with eight human respiratory viruses and processed them with four methods that concentrate and/or extract nucleic acids from both liquid and solid fractions (Promega, Nanotrap, and InnovaPrep) or only the solid fraction of wastewater (Solids). Our findings provide encouraging evidence that all four methods combined with dPCR could detect an emerging virus in wastewater, although they differed in sensitivity. The pattern of recovery efficiency for adenoviruses, coronaviruses, and influenza A viruses was consistent across methods, with Promega producing higher median recovery efficiencies, while distinct patterns were observed for coxsackieviruses. We also normalized the concentration data with two endogenous fecal indicators, PMMoV and Carjivirus (formerly crAssphage). We found that normalization could reduce method-associated differences if the indicator exhibited a recovery pattern similar to that of the target virus. These findings can guide the selection of concentration and extraction methods for wastewater monitoring based on the properties of target viruses, thus enhancing pandemic preparedness.
Biological and medical sciences↗
Seeding Advanced Treated Wastewater for Purposes of Direct Potable Reuse
Direct potable reuse (DPR) is a promising solution to address water scarcity. However, a better understanding of how introducing advanced treated water (ATW) affects microbial communities present in distribution systems is needed. Here, in this study, we measured changes to the microbial water quality in simulated distribution systems that were conditioned using treated, unimpaired surface water (SW) and then transitioned to ATW. In addition, we investigated whether adding a biological filtration step would seed the microbial community of the ATW and whether the influence would persist in the simulated distribution systems. We found that the bulk water in the ATW-fed distribution systems had lower cell counts and ATP concentrations and a distinct microbial community (based on 16S amplicon sequencing) compared to the SW-fed or the seeded ATW-fed systems. However, biofilm community composition and biomass remained consistent regardless of the feedwater. Increased microbial biomass and diversity were present in the seeded ATW, with several amplicon sequence variants identified as being introduced by the biological filter. Our results suggest that directly introducing ATW to distribution systems could disturb the existing microbial community. Preparing ATW for distribution via biological filtration may deliver more predictable and stable microbial water quality than introducing unseeded ATW.
16S↗
Genome-Resolved Meta-Omics Ties Microbial Dynamics to Process Performance in Biotechnology for Thiocyanate Degradation
Remediation of industrial wastewater is important for preventing environmental contamination and allowing water reuse. Biological treatment for one industrial contaminant, thiocyanate (SCN - ), relies upon microbial hydrolysis, but this process is sensitive to high loadings. To examine the activity and stability of a microbial community over increasing SCN - loadings, we established and operated a continuous-flow bioreactor fed increasing loadings of SCN - . A second reactor was fed ammonium sulfate to mimic breakdown products of SCN - . Biomass was sampled from both reactors for metagenomics and metaproteomics, yielding a set of genomes for 144 bacteria and one rotifer that constituted the abundant community in both reactors. We analyzed the metabolic potential and temporal dynamics of these organisms across the increasing loadings. In the SCN - reactor, Thiobacillus strains capable of SCN - degradation were highly abundant, whereas the ammonium sulfate reactor contained nitrifiers and heterotrophs capable of nitrate reduction. Key organisms in the SCN - reactor expressed proteins involved in SCN - degradation, sulfur oxidation, carbon fixation, and nitrogen removal. Lower performance at higher loadings was linked to changes in microbial community composition. This work provides an example of how meta-omics can increase our understanding of industrial wastewater treatment and inform iterative process design and development.
60 APPLIED LIFE SCIENCES↗
Accelerating Innovation that Enhances Resource Recovery in the Wastewater Sector: Advancing a National Testbed Network
This article examines significant challenges and opportunities to spur innovation and accelerate adoption of reliable technologies that enhance integrated resource recovery in the wastewater sector through the creation of a national testbed network. The network is a virtual entity that connects appropriate physical testing facilities, and other components needed for a testbed network, with researchers, investors, technology providers, utilities, regulators, and other stakeholders to accelerate the adoption of innovative technologies and processes that are needed for the water resource recovery facility of the future. Here we summarize and extract key issues and developments, to provide a strategy for the wastewater sector to accelerate a path forward that leads to new sustainable water infrastructures.
54 ENVIRONMENTAL SCIENCES↗
Second-Sphere Effects in Dinuclear Fe III Zn II Hydrolase Biomimetics: Tuning Binding and Reactivity Properties
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36 MATERIALS SCIENCE↗