Machine learning–based inverse design for electrochemically controlled microscopic gradients of O2 and H2O2

A fundamental understanding of extracellular microenvironments of O₂ and reactive oxygen species (ROS) such as H₂O₂, ubiquitous in microbiology, demands high-throughput methods of mimicking, controlling, and perturbing gradients of O₂ and H₂O₂ at microscopic scale with high spatiotemporal precision. However, there is a paucity of high-throughput strategies of microenvironment design, and it remains challenging to achieve O₂ and H₂O₂ heterogeneities with microbiologically desirable spatiotemporal resolutions. Here, we report the inverse design, based on machine learning (ML), of electrochemically generated microscopic O₂ and H₂O₂ profiles relevant for microbiology. Microwire arrays with suitably designed electrochemical catalysts enable the independent control of O₂ and H₂O₂ profiles with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s. Neural networks aided by data augmentation inversely design the experimental conditions needed for targeted O₂ and H₂O₂ microenvironments while being two orders of magnitude faster than experimental explorations. Interfacing ML-based inverse design with electrochemically controlled concentration heterogeneity creates a viable fast-response platform toward better understanding the extracellular space with desirable spatiotemporal control.

Ubiquitous spatiotemporal heterogeneity of natural environments fosters the diverse and fascinating biology that our world embraces, and motivates researchers to mimic natural environments with high spatiotemporal resolution (1–5). Given their close relevance in biochemical metabolisms, dioxygen (O₂) and hydrogen peroxide (H₂O₂) as a surrogate of reactive oxygen species (ROS) are two ubiquitous biologically relevant species in extracellular medium (1, 6). Their extracellular spatial and temporal distributions, particularly at the microscopic scale ranging from 1 μm to 100 μm (7–11), are critical for signal transduction, protein expression, biochemical redox balance, and regulation for cellular metabolism with extensive ecological, environmental, and biomedical implications (Fig. 1A) (1, 3, 8–13). A programmable creation of the spatiotemporal concentration profiles of O₂ and H₂O₂ offers the freedom to mimic, control, and perturb the microenvironments of O₂ and H₂O₂ and hence advance our understanding in microbiology.Open in a separate windowFig. 1.AI-based inverse design of electrochemically generated O₂ and H₂O₂ heterogeneities. (A) The ubiquitous spatiotemporal heterogeneities of O₂ and H₂O₂ in microbiology and the challenges posed in this research topic. (B) The combination of electrochemistry and ML-based inverse design offers a viable approach to mimicking and controlling the heterogeneities of O₂ and H₂O₂ in microbiology. O, oxidant; R, reductant; E_appl (t), the time-dependent electrochemical voltages applied on electrodes. (C) The design of the electrochemically active microwire array electrodes for the generation of O₂ and H₂O₂ gradients; 4e⁻ ORR & 2e⁻ ORR, four-electron and two-electron oxygen reduction reaction into H₂O and H₂O₂, respectively. (D and E) The 45°-tilting images of SEM for the representative microwire arrays used for the training of the ML model (D) and the ones inversely designed for targeted O₂ and H₂O₂ gradients (E); k = (P, D, L), the morphological vector that includes the P, D, and L of the synthesized wire arrays in units of micrometers. (Scale bars, 20 μm.)Despite recent progress (14–18), there remain major technical challenges, particularly in the achievable spatiotemporal resolution and high-throughput design of concentration profiles to suit a plethora of scenarios in microbiology. Approaches based on microfluidics and hydrogels have been able to achieve concentration gradients of O₂ and H₂O₂ through the provision of either O₂/H₂O₂ source (14, 19–21), O₂/H₂O₂ scavenging agents (15, 22, 23), or a combination of both (24) across liquid-impermeable barriers such as agar layers or polymeric thin films (25, 26). Yet such approaches, dependent on passive mass transport and diffusion across more than 10² μm, are inherently incapable of achieving spatial features of less than 100 μm and temporal resolution smaller than ∼10¹ s, the prerequisites to investigate microbiology at cluster or single-cell levels (10–12). Moreover, the large variations of extracellular O₂ and H₂O₂ gradients in different microbial systems demand an inverse design strategy, which, with minimal expenditure, quickly programs a desired concentration profile catering to a specific biological scenario (2–5). The current lack of inverse design protocol impedes the adoption of controllable extracellular heterogeneity to mimic and investigate microbial systems that are of environmental, biomedical, and sustainability-related significance.We envision that the integration of electrochemically generated concentration gradients with inverse design based on machine learning (ML) will address the aforementioned challenges (Fig. 1B). Electrochemistry offers a venue for transducing electric signals into microscopic concentration profiles within ∼10⁰ μm to ∼10² μm away from electrodes’ surface, following the specific electrode reaction kinetics and the mass transport governing equations in the liquid phase (27). Proper designs of electrodes’ microscopic spatial arrangement and electrochemical kinetics lead to concentration gradients that are spatiotemporally programmable by time-dependent electric signals of varying voltages (28). Such benefits of electrochemically generated concentration gradients lead us to employ electrochemistry as a tool to spatiotemporally control the concentration profiles in the extracellular medium. In one example, we found that wire arrays electrochemically active toward O₂ reduction create anoxic microenvironment about 20 μm away from the aerobic external bulk environments, modulate the size and extent of O₂ depletion in the anoxic microenvironment by the wire array’s morphology and applied electrochemical potential (E_appl), and hence enable O₂-sensitive rhizobial N₂ fixation in ambient air powered by renewable electricity (29). Moreover, while not reported before as far as we know, electrochemically generated concentration heterogeneity is commensurate with ML-based inverse design (30, 31), thanks to the mathematically well-defined electrochemical processes that can be numerically simulated (32, 33). We recently reported neural networks, trained by numerically simulated data, that explore the influence of electrode geometry on electrochemical N₂ fixation and achieve optimized morphologies of wire array electrodes untenable without such an ML-based strategy (34). An inverse design for the electrochemically generated gradients will quickly program desirable microenvironments of O₂ and ROS with high spatiotemporal resolutions, thanks to the well-reported electrochemical transformation related to O₂ and H₂O₂ with high electrochemical selectivity (35, 36).In this work, we report an inverse design based on neural networks for independent electrochemical creation of O₂ and ROS microscopic gradients that are relevant, and mimic their extracellular heterogeneities in microbial systems. We hypothesize that careful design of electrocatalysis of O₂ reduction reaction (ORR) can either facilitate four-electron ORR on Pt electrocatalyst for a controllable O₂ spatiotemporal profile or promote two-electron ORR on Au electrocatalyst for a programmable generation of H₂O₂ gradient without significantly perturbing the O₂ one, thanks to their concentration differences in biological mediums (∼10⁻¹ μM to ∼10¹ μM for H₂O₂ and ∼10¹ μM to ∼10² μM for O₂) (2, 7–11). Electrochemically active microwire array electrodes as exemplary model systems (Fig. 1C) are experimentally shown to achieve tunable heterogeneities of O₂ and H₂O₂ independently, with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s, and are suitable as a platform for independently perturbing biologically relevant O₂ and H₂O₂ profiles in microbial systems. We further established and experimentally validated two neural networks that inversely design the wire array electrodes’ morphologies toward targeted microenvironments of O₂ and H₂O₂, respectively, which is at least one order of magnitude faster than trial-and-error numerical simulation and two orders of magnitude faster than experimental explorations. The demonstrated inverse design of electrochemically generated controlled gradients not only demonstrates a full electrochemical control of concentration profiles in an electrode’s proximity but also establishes an approach of spatiotemporally mimicking and perturbing extracellular space guided by artificial intelligence.     

Extreme ecosystem instability suppressed tropical dinosaur dominance for 30 million years

A major unresolved aspect of the rise of dinosaurs is why early dinosaurs and their relatives were rare and species-poor at low paleolatitudes throughout the Late Triassic Period, a pattern persisting 30 million years after their origin and 10–15 million years after they became abundant and speciose at higher latitudes. New palynological, wildfire, organic carbon isotope, and atmospheric pCO₂ data from early dinosaur-bearing strata of low paleolatitudes in western North America show that large, high-frequency, tightly correlated variations in δ¹³C_org and palynomorph ecotypes occurred within a context of elevated and increasing pCO₂ and pervasive wildfires. Whereas pseudosuchian archosaur-dominated communities were able to persist in these same regions under rapidly fluctuating extreme climatic conditions until the end-Triassic, large-bodied, fast-growing tachymetabolic dinosaurian herbivores requiring greater resources were unable to adapt to unstable high CO₂ environmental conditions of the Late Triassic.One of the major predictions of models of elevated atmospheric CO₂ is the increased frequency and magnitude of events comprising very high temperatures, an enhanced hydrological cycle, and increased precipitation extremes (1, 2). Because such environmental extremes act as limitations on organisms, past time intervals of elevated CO₂ and associated climate extremes might be expected to profoundly influence biogeographic patterns, especially on land, which is relatively unbuffered climatically compared with the oceans. One such time of elevated CO₂ was the Triassic Period, during which both dinosaurs and mammals first appeared. In particular, it has remained an open question why the global ecological dominance of dinosaurs was delayed in the tropics for at least 30 million years after their first appearance and diversification into the three major clades Sauropodomorpha, Theropoda, and Ornithischia (3, 4). Hypotheses proposed to explain this lag have focused largely on competition (or lack thereof) with nondinosaurian archosaurs, principally those on the line to crocodylians (pseudosuchians), but none provide a clear explanation for this unusual and persistent biogeographic pattern.The rise of dinosaurs to ecological dominance was a diachronous evolutionary event (5–8). Small carnivorous early theropod dinosaurs were widespread at low paleolatitudes, whereas evidence for Triassic herbivorous dinosaurs (i.e., sauropodomorphs and ornithischians) in the tropics is completely absent (6, 7, 9, 10) (Fig. 1). In addition, tropical North American theropod dinosaurs were rare and species-poor (5, 7, 10) compared with higher-latitude assemblages. These patterns have been hypothesized to track largely zonal climatic conditions across Pangaea (6, 8, 11, 12) (Fig. 1), but detailed paleoclimatic data and mechanistic explanations have been lacking. Here, we argue these biogeographic patterns are a result of extreme environmental fluctuations in the tropics enhanced by high atmospheric CO₂, which suppressed large-bodied herbivorous dinosaurs until after the end-Triassic mass extinction.Open in a separate windowFig. 1.Late Triassic Pangean map showing latitudinal climate zones (11, 12) and the distribution of major dinosaur clades. See SI Appendix for occurrence data. Question marks indicate geochronologic uncertainty for the Thailand sauropodomorph and Argentine Laguna Colorada heterodontosaurid occurrences (i.e., they may be Early Jurassic in age). Each dinosaur symbol in most cases represents a region with multiple fossiliferous localities containing the illustrated clades.We present, to our knowledge, the first high-resolution paleoenvironmental multiproxy record from the same sedimentary sequences that produce abundant early dinosaur and other vertebrate fossils (6, 8, 10). Specifically, we sampled fluvial and overbank sediments of the Upper Triassic Chinle Formation of the Chama Basin in north central New Mexico (13, 14). This nonmarine succession from low-paleolatitude Pangaea moved from ∼10°N to 14°N during the late Norian and Rhaetian (15), suggesting that this area would have experienced a semiarid climate through the entire sequence (11) (Fig. 1). The formation in this region contains exceptionally diverse and abundant vertebrate assemblages, which allows the early evolution of dinosaurs, their contemporaneous flora, and their paleoenvironment to be examined through time. Furthermore, tight age control is provided by a recent U–Pb radioisotopic age of 211.9 ± 0.7 Ma from the Hayden Quarry (HQ) in the lower portion of the Petrified Forest Member of the Chama Basin (7), and magnetostratigraphic data (16) that are consistent with a late Norian to Rhaetian age for the sequence.     

Absence of Jahn?Teller transition in the hexagonal Ba3CuSb2O9 single crystal

With decreasing temperature, liquids generally freeze into a solid state, losing entropy in the process. However, exceptions to this trend exist, such as quantum liquids, which may remain unfrozen down to absolute zero owing to strong quantum entanglement effects that stabilize a disordered state with zero entropy. Examples of such liquids include Bose−Einstein condensation of cold atoms, superconductivity, quantum Hall state of electron systems, and quantum spin liquid state in the frustrated magnets. Moreover, recent studies have clarified the possibility of another exotic quantum liquid state based on the spin–orbital entanglement in FeSc₂S₄. To confirm this exotic ground state, experiments based on single-crystalline samples are essential. However, no such single-crystal study has been reported to date. Here, we report, to our knowledge, the first single-crystal study on the spin–orbital liquid candidate, 6H-Ba₃CuSb₂O₉, and we have confirmed the absence of an orbital frozen state. In strongly correlated electron systems, orbital ordering usually appears at high temperatures in a process accompanied by a lattice deformation, called a static Jahn−Teller distortion. By combining synchrotron X-ray diffraction, electron spin resonance, Raman spectroscopy, and ultrasound measurements, we find that the static Jahn−Teller distortion is absent in the present material, which indicates that orbital ordering is suppressed down to the lowest temperatures measured. We discuss how such an unusual feature is realized with the help of spin degree of freedom, leading to a spin–orbital entangled quantum liquid state.Quantum spin liquids have been widely recognized as a new state of matter, as an increasing number of candidates with quantum spin S = 1/2 have been found recently (1–4), a long time after the first proposal was made for the resonating valence-bond state (5). On the other hand, quantum liquids based on another electronic degree of freedom, orbital, have been theoretically proposed (6). However, this type of liquid state has never been experimentally confirmed because the energy of orbital correlation is normally one order of magnitude stronger than spin exchange coupling, leading to an orbital ordering at a significantly high temperature accompanied by a cooperative Jahn–Teller (JT) distortion. Nevertheless, if we can bring down the orbital energy to the same scale as for the spin coupling, it may lead to a novel spin–orbital entangled state, a “quantum spin–orbital liquid.” A possible spin–orbital entangled liquid state with dimer correlations has been theoretically discussed on a triangular lattice with singly occupied but triply degenerate t_2g orbitals (7). In comparison with the t_2g orbitals’ case, the experimental realization of such a quantum spin–orbital liquid state in the e_g orbital system has been even more challenging (8), because e_g orbitals more strongly couple to the JT modes.Perovskite-type 6H-Ba₃CuSb₂O₉ is a good candidate material for the spin–orbital liquid state that has been theoretically proposed (9–11). Recently, we reported that spin–orbital short-range ordering occurs in the short-range honeycomb lattice of Cu²⁺ with e_g orbital degrees of freedom, as depicted in Fig. 1A (12), sharply contradicting the previously reported crystal structure with a triangular lattice of Cu²⁺ (13). In addition to the confirmation of a dynamic spin state down to 20 mK by muon spin spectroscopy (12, 14, 15), powder X-ray diffraction clearly indicates that even at low temperature, the hexagonal components remain, along with some orthorhombically distorted components. In the hexagonal phase, threefold symmetry exists for the Cu²⁺ sites, which are surrounded by octahedrally coordinated oxygen, indicating the absence of a cooperative JT distortion. To explain this unusual feature, we proposed two possible scenarios. (i) A noncooperative static JT distortion appears. In this scenario, the local symmetry is lowered by a static JT distortion, as schematically shown in Fig.1B, but the overall hexagonal symmetry remains. (ii) The static JT distortion is absent and, instead, a dynamic JT distortion appears, leading to a novel spin–orbital liquid state, as depicted in Fig. 1C. These two possible scenarios cannot be distinguished by experimental results using powder specimens alone, as was reported in the previous paper (12); a thorough structural study is required using a single crystal without orthorhombic components. Here, we report the comprehensive study on a hexagonal single crystal of Ba₃CuSb₂O₉ that exhibits no cooperative JT transition down to low temperatures. This provides, to our knowledge, the first example of a copper 3d⁹ compound with no JT transition. Our results suggest a formation of a spin–orbital liquid state.Open in a separate windowFig. 1.(A) Schematic view of the local structure for hexagonal and orthorhombic samples. (B) Schematic picture of a noncooperative static JT distortion. (C and D) Schematic pictures of spin-singlet formation in short-range honeycomb lattices of Cu²⁺ for (C) hexagonal and (D) orthorhombic samples. For C, a spin–orbital entangled short-range-order state is expected. A pair of up and down arrows indicates a singlet state of the dimer based on the neighboring Cu²⁺ spins. At each site, an unpaired electron of Cu²⁺ occupies the d_x²?y², d_y²?z², or d_z²?x² orbital.     

Nitrogenase-mimic iron-containing chalcogels for photochemical reduction of dinitrogen to ammonia

A nitrogenase-inspired biomimetic chalcogel system comprising double-cubane [Mo₂Fe₆S₈(SPh)₃] and single-cubane (Fe₄S₄) biomimetic clusters demonstrates photocatalytic N₂ fixation and conversion to NH₃ in ambient temperature and pressure conditions. Replacing the Fe₄S₄ clusters in this system with other inert ions such as Sb³⁺, Sn⁴⁺, Zn²⁺ also gave chalcogels that were photocatalytically active. Finally, molybdenum-free chalcogels containing only Fe₄S₄ clusters are also capable of accomplishing the N₂ fixation reaction with even higher efficiency than their Mo₂Fe₆S₈(SPh)₃-containing counterparts. Our results suggest that redox-active iron-sulfide–containing materials can activate the N₂ molecule upon visible light excitation, which can be reduced all of the way to NH₃ using protons and sacrificial electrons in aqueous solution. Evidently, whereas the Mo₂Fe₆S₈(SPh)₃ is capable of N₂ fixation, Mo itself is not necessary to carry out this process. The initial binding of N₂ with chalcogels under illumination was observed with in situ diffuse-reflectance Fourier transform infrared spectroscopy (DRIFTS). ¹⁵N₂ isotope experiments confirm that the generated NH₃ derives from N₂. Density functional theory (DFT) electronic structure calculations suggest that the N₂ binding is thermodynamically favorable only with the highly reduced active clusters. The results reported herein contribute to ongoing efforts of mimicking nitrogenase in fixing nitrogen and point to a promising path in developing catalysts for the reduction of N₂ under ambient conditions.The reduction of atmospheric nitrogen to ammonia is one of the most essential processes for sustaining life. Currently, roughly half of the fixed nitrogen is supplied biologically by nitrogenase, while nearly the other half is from the industrial Haber–Bosch process, which operates under high temperature (400–500 °C) and high pressure (200–250 bar) in the presence of a metallic iron catalyst (1). Nitrogenase, a two-component protein system comprising a MoFe protein and an associated Fe protein, carries out this “fixation” in nature under ambient temperature and pressure (2–4). N₂ substrate binding and activation take place at the iron–molybdenum–sulfur cofactor (FeMoco), and in some cases, Mo-free iron–sulfur cofactor FeFeco and iron–vanadium–sulfur cofactor FeVco cofactors. Electron transfer during this catalytic process is believed to proceed from a [4Fe:4S] cluster located in the Fe protein to another Fe/S cluster (the P cluster) buried in the MoFe protein and finally to the FeMoco (Fig. 1A) (2, 5, 6). Whereas the role of Mo in the reactivity of nitrogenase has been the subject of long debate, iron is now well recognized as the only transition metal essential to all nitrogenases, and recent biochemical and spectroscopic data point to iron as the site of N₂ binding in the FeMoco (7–9). Naturally, understanding and mimicking how the nitrogenase enzyme accomplishes the difficult task of N₂ reduction under ambient conditions is one of the grand challenges in chemistry. To this end, inspired by the molecular structure and function of FeMoco, a number of groups have synthesized transition metal–dinitrogen complexes and examined stoichiometric transformations of their coordinated N₂ into NH₃ and N₂H₄ (9–19). However, the operation of homogeneous transition metal–dinitrogen complexes usually requires organic solvents, strong reducing agents, and often extremely low operation temperatures (5, 10, 12, 14). The prospect of using solar light energy to convert N₂ to ammonia is highly attractive but it represents a great challenge and is a less-investigated line of inquiry. Hamers and co-workers reported that solvated electrons emitted from illuminated diamond can accomplish N₂ reduction (20, 21). Other semiconductor systems such as Fe₂Ti₂O₇ (22), Au/Nb–SrTiO₃/Ru (23), and BiOBr_ov (24) were also reported to perform light-induced N₂ fixation. These systems are not biomimetic and usually exhibit very low conversion efficiency (SI Appendix, Table S1).Open in a separate windowFig. 1.Nitrogenase-inspired biomimetic chalcogels. (A) The two-component proteins of molybdenum nitrogenase: MoFe protein and Fe protein; space filling and stick model structures of the FeMo cofactor and the P cluster. (B) The reaction routes leading to the assembly of FeMoS–SnS, FeMoS–FeS–SnS, and FeMoS–M–SnS (M=Sb³⁺, Sn⁴⁺, Zn²⁺) chalcogel, respectively.Our group has recently developed a new class of porous chalcogenide aerogels by the metathesis reaction, dubbed “chalcogels,” which can be functionalized with biomimetic functionalities (25–27). These materials can easily incorporate Mo₂Fe₆S₈(SPh)₃ or Fe₄S₄ clusters in their structure and have been shown to reduce protons both electrocatalytically and photocatalytically to hydrogen (28, 29). The Mo₂Fe₆S₈(SPh)₃ cluster-based chalcogel was recently demonstrated to be capable of photocatalytically reducing N₂ to NH₃ (30). Inspired by the structure and function of the MoFe protein of nitrogenase which contains both iron–molybdenum–sulfur and iron–sulfur clusters (the P cluster), we prepared a chalcogel that also incorporated two types of clusters: the FeMoco-like Mo₂Fe₆S₈(SPh)₃ and P-cluster–like Fe₄S₄ linked together with units of [Sn₂S₆]^4-, in a 3D superstructure (Fig. 1B) (2, 4, 30). This chalcogel is dubbed “FeMoS–FeS-SnS.” We also prepared two more chalcogels, one with Mo₂Fe₆S₈(SPh)₃ clusters and inert metals such as Sb³⁺, Sn⁴⁺, Zn²⁺ (dubbed “FeMoS–M-SnS”) and a molybdenum-free one, the Fe₄S₄ chalcogel (FeS–SnS) (31, 32). The purpose of using the FeMoS–M–SnS chalcogels was to see if placing the FeMoS clusters farther apart in space would have any effect on the catalytic reaction, whereas that of FeS–SnS was to probe the necessity of Mo. These three chalcogels achieve photocatalytic N₂ reduction but more importantly, and to our surprise, the iron-only FeS–SnS chalcogel is in fact not only capable of N₂ reduction but also with higher rate. Diffuse-reflectance Fourier transform infrared spectroscopy (DRIFTS) experiments performed under light illumination show a clear signature of the N₂ binding process and its subsequent reduction. The results reported here show that the photochemical activation of N₂ using visible light is possible with Mo₂Fe₆S₈(SPh)₃ as well as Fe₄S₄-based materials at room temperature, ambient pressure, and aqueous conditions. Despite the complex progression multielectron/-proton reactions required, we clearly have an unexpectedly viable and robust process that leads to ammonia. Our results also demonstrate that iron rather than molybdenum is the element necessary for photoreduction of N₂ to NH₃.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:1H detection and dynamic nuclear polarization–enhanced NMR of Aβ1-42 fibrils

Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on ¹³C,¹⁵N-enriched, and fully protonated samples of M₀Aβ_1-42 fibrils by high-field ¹H-detected NMR at 23.4 T and 18.8 T, and ¹³C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M₀Aβ_1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than ¹H detection. These results suggest that ¹H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

Amyloid fibrils are highly stable protein deposits found in β-sheet conformations and are notoriously recognized as disruptive agents to cellular function in over 40 human diseases (1, 2). Alzheimer’s disease (AD) is the most pervasive of all known plaque-related diseases and is associated with the presence of amyloid-β (Aβ) peptides in the extracellular space of the brain (3–6). As of 2021, there are ∼6.2 million people in the United States living with Alzheimer’s dementia and ∼50 million worldwide (7), and there is as of yet no cure available for AD. In order to address this epidemic, it is essential that we learn as much as possible about the formation and structure of Aβ plaques, including the detailed features of their catalytic surface, in order to design and develop appropriate treatments to limit the propagation of aggregates and the generation of toxic forms.Aβ is derived from the C-terminal region of the amyloid precursor protein (APP), a membrane protein in neuronal cells, via proteolysis by β- and γ-secretase (8, 9). One of the principal challenges in rationalizing AD etiology is Aβ’s diversity in peptide length, mutations, and posttranslational modifications (10). Their low solubility renders solution NMR ineffective, and high-resolution diffraction analyses have thus far been restricted to shorter peptides with all or most residues being ordered in the fibril core structure (11). Cryogenic electron microscopy (cryo-EM) has made strides in resolution in fibril studies within the past decade (12–18), but faces challenges studying with atomic-level detail due to polymorphism and heterogeneity in the fibril macroassemblies. Studying the individual and collective roles of amyloids at atomic resolution therefore requires alternative, high-resolution, high-throughput techniques for structural analysis. Magic-angle spinning NMR (MAS-NMR) was introduced as a technique with the potential to address these problems (19, 20). Recent technical advances (21, 22) and progress in sample preparation (23) have vastly improved the sensitivity and resolution of the spectra (24). Accordingly, there are now publications describing high-resolution structures of Aβ (25–29) and other amyloid (12, 16, 30–35) fibrils based on distance and torsion angle constraints derived from MAS experiments.To date, all of the known NMR structures of amyloid fibrils were determined using constraints obtained from ¹³C/¹⁵N MAS spectra, which are inhomogeneously broadened and therefore feature well-resolved lines at low spinning frequencies (<25 kHz) (36). However, resolution often remains insufficient for in-depth analysis, and the experiments require relatively large amounts of peptide and extensive signal acquisition periods. Two relatively recent additions to the repertoire of MAS NMR experiments—namely, ¹H detection and dynamic nuclear polarization (DNP)—promise to circumvent these issues by reducing signal acquisition times or, alternatively, the amount of protein required for the experiment (37). ¹H detection offers a factor of (γ_H/γ_S)^3/2 gain in sensitivity, where S is usually a low γ-spin (38–40) such as ¹³C or ¹⁵N. In these two cases it is possible to achieve a factor of ∼8 or ∼32 gain in sensitivity, respectively. Importantly, ¹H detection also introduces an additional spectral dimension and therefore significantly increases the resolution. In parallel, DNP offers a general approach to enhancing sensitivity by factors of ∼100, dramatically reducing signal acquisition times (by ∼10⁴). It does so by exploiting the high spin polarization of unpaired electrons (of gyromagnetic ratio γ_e ∼660 times larger than γ_H) of a paramagnetic polarizing agent to enhance sensitivity by a theoretical factor of γ_e/γ_H. (41–44) Furthermore, DNP experiments are conducted at ∼100 K, thereby increasing the Boltzmann polarization and sensitivity by another factor of ∼3 over experiments conducted at ambient temperature (45).While these arguments are well established for MAS NMR in many systems, it is less obvious that they are applicable to amyloid samples because spectra of amyloids are known to be broad for a variety of reasons, such as sample purity and polymorphism. Furthermore, ¹H-detected NMR at moderate MAS frequencies (∼20 to 60 kHz) needs to be coupled to different levels of deuteration to ensure high sensitivity and narrow linewidths (42). Accordingly, deuteration with partial reprotonation of the amide or Hα sites has been implemented in pioneering studies on Aβ_1-40 at 20 kHz MAS (46), HET-s(218–289) (47), and D76N-β2m at 60 kHz MAS (48). In addition, selective protonation in Aβ_1-40 fibril methyl groups at 18 kHz MAS has led to highly resolved ¹H-detected ¹³C correlations (49). In deuterated samples, however, the amount of potentially available structural information is significantly reduced, which can impair high-resolution structure determinations. The advent of 0.7 mm MAS rotors that achieve ω_r/2π >110 kHz attenuates ¹H-¹H dipole couplings and allows direct acquisition of multidimensional ¹H data without requiring deuteration (50). Furthermore, the spectra provide assignments and structural information. While a proof-of-concept application of this approach was demonstrated on fully protonated highly regular prion fibrils (51, 52), it is not clear whether this methodology is generally applicable and extendable to the detection of resolved inter- and intramolecular contacts in complex amyloid assemblies.In parallel, our MAS DNP studies on M₀Aβ_1-42 (28, 32, 53) report significant broadening of the NMR lines at cryogenic temperatures, which was attributed to distributions of conformations trapped at low temperature and is therefore inhomogeneous in origin. The loss of resolution associated with the MAS DNP methodology is a major obstacle for the detailed structural study of uniformly labeled amyloid samples. Concurrently, reports of well-resolved spectra at high fields and spinning frequencies suggest that the broadening is homogeneous (54–56). The advent of DNP instrumentation operating at high magnetic fields (18.8 T) and faster MAS (ω_r/2π = 40 kHz) provides an approach to alleviate this limitation by attenuating homogeneous couplings (57). However, this comes at the expense of the enhancement factor, potentially compromising the capacity to carry out expeditious multidimensional and multinuclear correlations. Moreover, NMR spectra of amyloid fibrils are known to suffer from additional debilitating broadening associated with their heterogeneous character (sample purity, polymorphism, etc.), which may mitigate the benefits of high magnetic fields.In this work, we show that high resolution and sensitivity are possible for fibrils of M₀-Aβ_1-42. Notably, we demonstrate rapid resonance assignment and site-resolved detection of numerous site-specific internuclear proximities on submilligram sample quantities via ¹H-detected NMR at ω_r/2π ∼110 kHz and high field (23.4 T/1,000 MHz for ¹H) at room temperature and ¹³C-detected DNP MAS NMR at ω_r/2π = 40 kHz and high field (18.8 T/800 MHz for ¹H) at low temperature. While both ¹H detection and DNP afford increased sensitivity, we estimate, using approaches outlined by Ishii and Tycko (40), that DNP, with our current ε = 22, yields a factor of ∼6.5 higher sensitivity. These results therefore illuminate possible paths for the rapid structure elucidation of amyloid fibrils available in limited quantities.     

NhaA antiporter functions using 10 helices,and an additional 2 contribute to assembly/stability

The Escherichia coli Na⁺/H⁺ antiporter (Ec-NhaA) is the best-characterized of all pH-regulated Na⁺/H⁺ exchangers that control cellular Na⁺ and H⁺ homeostasis. Ec-NhaA has 12 helices, 2 of which (VI and VII) are absent from other antiporters that share the Ec-NhaA structural fold. This α-hairpin is located in the dimer interface of the Ec-NhaA homodimer together with a β-sheet. Here we examine computationally and experimentally the role of the α-hairpin in the stability, dimerization, transport, and pH regulation of Ec-NhaA. Evolutionary analysis (ConSurf) indicates that the VI–VII helical hairpin is much less conserved than the remaining transmembrane region. Moreover, normal mode analysis also shows that intact NhaA and a variant, deleted of the α-hairpin, share similar dynamics, suggesting that the structure may be dispensable. Thus, two truncated Ec-NhaA mutants were constructed, one deleted of the α-hairpin and another also lacking the β-sheet. The mutants were studied at physiological pH in the membrane and in detergent micelles. The findings demonstrate that the truncated mutants retain significant activity and regulatory properties but are defective in the assembly/stability of the Ec-NhaA dimer.Living cells are critically dependent on processes that regulate intracellular pH, Na⁺, and volume (1), and Na⁺/H⁺ antiporters play a primary role in these homeostatic mechanisms (reviewed in ref. 2). These antiporters are found in the cytoplasmic and intracellular membranes of most organisms (reviewed in refs. 3–6), and they have long been human drug targets (7).The principal Na⁺/H⁺ antiporter in Escherichia coli, Ec-NhaA, is responsible for intracellular Na⁺ and H⁺ homeostasis (8), and homologs have recently been implicated in the virulence of pathogenic bacteria (9). In humans, orthologs have been suggested to be involved in essential hypertension (10), as well as diabetes (11).Ec-NhaA is characterized by exceptionally high transport activity (12), a stoichiometry of 2H⁺/Na⁺ (13), and strong pH dependence (12), a property shared with other prokaryotic (8) and eukaryotic Na⁺/H⁺ antiporters (reviewed in refs. 3–6). Crystal structures of down-regulated Ec-NhaA at acidic pH (14) (Fig. 1) reveal a unique structural fold shared by a growing number of secondary transporters (15, 16, 17).Open in a separate windowFig. 1.(A) Evolutionary conservation analysis of Ec-NhaA. The crystal structure of Ec-NhaA, in ribbon representation, as seen in the plane of the membrane, represented with gray outlines, with the intracellular side facing up. ConSurf analysis (consurf.tau.ac.il) was carried out using PDB ID code 1ZCD (14). The amino acids are colored by their conservation grades using the color-coding bar, with cyan through maroon indicating variable through conserved. Overall, helices VI and VII, denoted with roman numerals, are of the least conserved TM helices with average conservation scores of 1 and 5, respectively, according to the ConSurf scoring method. The sites of the three single amino acid mutations Q47C, S246C, and V254C and the two sites in which Ec-NhaA was truncated (A182 on and A218 on) are shown as spheres (B) Ec-NhaA dimer. The crystal structure of the Ec-NhaA dimer (27), PDB ID code 4AU5, in ribbon representation as seen from the cytoplasm, with TM helices numbered as in A. The first monomer is colored light blue and the second colored wheat. The α-hairpin (composed of helices VI and VII) and β-sheet of one monomer are highlighted in marine and in the other monomer in orange. The red dashed line marks the boundary between the two functional domains of the transporter, the core domain to the left and the panel/dimer interface to the right. As can be seen the α- and β-hairpins constitute the dimer interface. (C) Close-up view on the interaction between the monomers. The Ec-NhaA dimer interface is shown as ribbon representation, with monomers colored as in B. Although the β-hairpin contributes the majority of interactions between subunits from the extracellular side, there is also a small interface between the cytoplasmic ends of helix VII of one monomer and helix IX of the other. This interaction probably involves hydrogen bond and hydrophobic contacts and forms a zipper-like structure consisting of R204 and L210 of one monomer and V254 and W258 of the other (27). The figures were generated using PyMol (PyMOL Molecular Graphics System, version 1.7.4; Schrödinger, LLC) (47, 48).Omitting transmembrane segments (TMs) VI and VII, the remaining 10 TMs of monomeric Ec-NhaA are organized into a highly conserved, densely packed core domain composed of two structurally related helix bundles (TMs III, IV, and V and TMs X, XI, and XII) that are topologically inverted with respect to each other (Fig. 1B) (14). TMs IV and XI are each interrupted by an unwound chain that crosses the other chain in the middle of the membrane, leaving two short helices oriented toward the cytoplasm (c) or the periplasm (p) (IVc, IVp and XIc, XIp, respectively; Fig. 1) (14). This noncanonical TM assembly—the NhaA fold—creates a delicately balanced electrostatic environment in the middle of the membrane at the ion binding site(s), which likely plays a critical role in cation exchange activity. The other TMs—the dimer interface domain—comprise a bundle along the dimer interface and also contain inverted-topology repeats (TMs I, II and VIII, IX; Fig. 1B) (18).Interestingly, the Na⁺/H⁺ antiporter structures that share the NhaA fold are characterized by different numbers of TMs from 12 to 13 (16, 17, 19) and are dimeric like Ec-NhaA. However, two prokaryotic ASBT (apical sodium-dependent bile acid transporter, also known as SLC10A2) symporters share the NhaA fold but have only 10 TMs; they lack the Ec-NhaA equivalents of helices VI and VII (20, 21). The ASBTs also differ from Ec-NhaA in that they are Na⁺/bile acid symporters. In addition, the ASBTs are monomeric. Taken together, the data raise questions regarding the role of TMs VI and VII in Ec-NhaA.In Ec-NhaA, helices VI and VII, which form an α-hairpin, are located in the dimer interface (Fig. 1 B and C). Therefore, these substructures may contribute to NhaA dimerization and/or stability; however, data in this regard are not available. In the current study, we constructed an NhaA mutant deleted of TMs VI and VII and studied its properties at physiological pH. The mutant devoid of TMs VI and VII is defective with respect to dimerization and/or stability but exhibits decreased but significant transport activity, as well as pH regulation.     

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

Excitation-evoked Ca²⁺ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca²⁺ entry are thought to be functions of voltage-gated calcium (Ca_V) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I–IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of Ca_V1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in Ca_V1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.Voltage-gated Ca²⁺ (Ca_V) channels respond to membrane depolarization by catalyzing Ca²⁺ influx. Ca_V-mediated elevation of intracellular [Ca²⁺] regulates such critical physiological functions as neurotransmitter and hormone release, axonal outgrowth, muscle contraction, and gene expression (1). Their relevance to human physiology is evident from the broad phenotypic consequences of Ca_V channelopathies (2). The voltage dependence of Ca_V-driven Ca²⁺ entry relies on the modular organization of the channel-forming α₁ subunit (Fig. 1), which consists of four repeated motifs (I–IV), each comprising six membrane-spanning helical segments (S1–S6) (Fig. 1A). Segments S1–S4 form a voltage-sensing domain (VSD), whereas segments S5 and S6 contribute to the Ca²⁺-conductive pore (1). The VSDs surround the central pore (Fig. 1B). VSDs are structurally and functionally conserved modules (3–5) capable of transducing a change in the cell membrane electrical potential into a change of ion-specific permeability or enzyme activity. VSDs sense depolarization by virtue of a signature motif of positively charged Arg or Lys at every third position of helix S4 (Fig. 1D), which rearranges in response to depolarization (4, 6–10). In contrast to voltage-gated K⁺ (K_V) channels but similar to pseudotetrameric voltage-gated Na⁺ (Na_V) channels, the amino acid sequences encoding each VSD have evolved independently (Fig. 1D). In addition to their distinct primary structure, the four Ca_V VSDs may also gain distinct functional properties from the asymmetrical association of auxiliary subunits, such as β, α₂δ, and calmodulin (1, 11–16) (Fig. 1C). The structural divergence among VSD-driven channels was foreseen by the classical Hodgkin–Huxley model (17), in which four independent “gating particles” control the opening of homotetrameric K_V channels and only three seem sufficient to open Na_V channels. An early study by Kostyuk et al. (18) suggested that only two gating particles are coupled to Ca_V channel opening. We recognize today that gating particles correspond to VSDs, and in Na_V channels, VSDs I–III control Na⁺ influx, whereas VSD IV is associated with fast inactivation (19–21).Open in a separate windowFig. 1.Ca_V membrane topology, putative structure, and S4 helix homology. (A) Ca_V channel-forming α₁ subunits consist of four concatenated repeats, each encompassing one voltage sensor domain (VSD) and a quarter of the central pore domain (PD) (1). Stars indicate the positions of fluorophore labeling. (B) The atomic structure of an Na_V channel (Protein Data Bank ID code 4EKW; top view) (56) shown as a structural representation for the Ca_V α₁ subunit. (C) The α₁ subunit asymmetrically associates with auxiliary β, α₂δ, and calmodulin (CaM) subunits (11–16). (D) Sequence alignment of VSD helix S4 from each of four Ca_V1.2 repeats and the archetypal homotetrameric Shaker K⁺ channel. Conserved, positively charged Arg or Lys is in blue. Residues substituted by Cys for fluorescent labeling are marked: F231 (VSD I), L614 (VSD II), V994 (VSD II), and S1324 (VSD IV).In this study, we used fluorometry to probe the properties of four individual VSDs in a human L-type calcium channel Ca_V1.2, which is a widely expressed regulator of physiological processes, such as cardiac and smooth muscle contractility (22). Although the collective transition of the Ca_V VSDs and the pore has been investigated in studies measuring total charge displacement (gating currents) (23, 24), the activation properties and functional roles of each VSD are unknown. Evidence for the role of each VSD in L-type Ca_V channel operation has been presented from charge neutralization studies, but a clear picture has yet to emerge. Work on a chimeric L-type channel suggests that VSDs I and III drive channel opening (25), whereas other studies on Ca_V1.2 favored the involvement of VSD II over VSD I, with the roles of VSDs III and IV remaining unclear (26, 27).The individual optical reports of four Ca_V1.2 VSDs revealed that each operates with distinct biophysical parameters. We found that VSDs II and III exhibit voltage- and time-dependent characteristics compatible with channel opening and that they can be considered rate-limiting for activation. We compared the voltage and time dependence of the fluorescent signals and ionic currents with the predictions of thermodynamic models relevant to Ca_V domain organization. We found that Ca_V1.2 activation is compatible with a model of allosteric VSD–pore coupling, where VSDs II and III are the primary drivers of channel opening with a smaller contribution by VSD I. We discuss the mechanism of Ca_V1.2 voltage sensitivity, which exhibits similarities to but also clear differences from the related pseudotetrameric Na_V1.4 channels.     

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm

H⁺-transporting F₁F₀ ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F₀ and F₁. H⁺ transport at the subunit a–c interface in transmembranous F₀ drives rotation of a cylindrical c₁₀ oligomer within the membrane, which is coupled to rotation of subunit γ within the α₃β₃ sector of F₁ to mechanically drive ATP synthesis. F₁F₀ functions in a reversible manner, with ATP hydrolysis driving H⁺ transport. ATP-driven H⁺ transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag⁺ and/or Cd⁺² with the substituted sulfhydryl groups. The H⁺ transport pathway mapped via these Ag⁺(Cd⁺²)-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H⁺ release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd⁺² directly inhibits passive H⁺ transport mediated by F₀ reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H⁺ transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H⁺ transport likely interact in a single structural domain, which then functions in gating H⁺ release to the cytoplasm.The F₁F₀-ATP synthase of oxidative phosphorylation uses the energy of a transmembrane electrochemical gradient of H⁺ or Na⁺ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F₁ and F₀ sectors of the enzyme (1). H⁺ transport through the transmembrane F₀ sector is coupled to ATP synthesis or hydrolysis in the F₁ sector at the surface of the membrane. Homologous ATP synthases are found in mitochondria, chloroplasts, and many bacteria. In Escherichia coli and other eubacteria, F₁ consists of five subunits in an α₃β₃γδε stoichiometry. F₀ is composed of three subunits in a likely ratio of a₁b₂c₁₀ in E. coli and Bacillus PS3 (2, 3) or a₁b₂c₁₁ in the Na⁺ translocating Ilyobacter tartaricus ATP synthase (1, 4) and may contain as many as 15 c subunits in other bacterial species (5). Subunit c spans the membrane as a hairpin of two α-helices, with the first transmembrane helix (TMH) on the inside and the second TMH on the outside of the c ring (1, 4). The binding of Na⁺ or H⁺ occurs at an essential, membrane-embedded Glu or Asp on cTMH2. High-resolution X-ray structures of both Na⁺- and H⁺-binding c-rings have revealed the details and variations in the cation binding sites (4–8). In the H⁺-translocating E. coli enzyme, Asp-61 at the center of cTMH2 is thought to undergo protonation and deprotonation, as each subunit of the c ring moves past the stationary subunit a. In the functioning enzyme, the rotation of the c ring is thought to be driven by H⁺ transport at the subunit a/c interface. Subunit γ physically binds to the cytoplasmic surface of the c-ring, which results in the coupling of c-ring rotation with rotation of subunit γ within the α₃β₃ hexamer of F₁ to mechanically drive ATP synthesis (1).E. coli subunit a folds in the membrane with five TMHs and is thought to provide aqueous access channels to the H⁺-binding cAsp-61 residue (9, 10). Interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be critical during the deprotonation–protonation cycle of cAsp-61 (1, 11, 12). At this time, very limited biophysical or crystallographic information is available on the 3D arrangement of the TMHs in subunit a. TMHs 2–5 of subunit a pack in a four-helix bundle, which was initially defined by cross-linking (13), but now, such a bundle, packing at the periphery of the c-ring, has been viewed directly by high-resolution cryoelectron microscopy in the I. tartaricus enzyme (14). Previously published cross-linking experiments support the identification of aTMH4 and aTMH5 packing at the periphery of the c-ring and the identification of aTHM2 and aTMH3 as the other components of the four-helix bundle seen in these images (13, 15, 16). More recently, published cross-linking experiments identify the N-terminal α-helices of two b subunits, one of which packs at one surface of aTMH2 with close enough proximity to the c-ring to permit cross-linking (17). The other subunit b N-terminal helix packs on the opposite peripheral surface of aTMH2 in a position where it can also be cross-linked to aTMH3 (17). The last helix density shown in Hakulinen and colleagues (14) packs at the periphery of the c-ring next to aTMH5 and is very likely to be aTMH1.The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag⁺ and other thiolate-reactive agents (18–20). Two regions of aqueous access were found with distinctly different properties. One region in TMH4, extending from Asn-214 and Arg-210 at the center of the membrane to the cytoplasmic surface, contains Cys substitutions that are sensitive to inhibition by both N-ethylmaleimide (NEM) and Ag⁺ (18–20; Fig. 1). These NEM- and Ag⁺-sensitive residues in TMH4 pack at or near the peripheral face and cytoplasmic side of the modeled four-helix bundle (11, 13). A second set of Ag⁺-sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18, 19), and Ag⁺-sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19, 20). The Ag⁺-sensitive substitutions on the periplasmic side of TMHs 2–5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11, 13, 19, 20). We have proposed that the movement of H⁺ from the periplasmic half-channel and binding to the single ionized Asp61 in the c-ring is mediated by a swiveling of TMHs at the a–c subunit interface (16, 21–24). This gating is thought to be coupled with ionization of a protonated cAsp61 in the adjacent subunit of the c-ring and with release of the H⁺ into the cytoplasmic half-channel at the subunit a–c interface. The route of aqueous access to the cytoplasmic side of the c subunit packing at the a–c interface has also been mapped by the chemical probing of Cys substitutions and, more recently, by molecular dynamics simulations (22, 25, 26).Open in a separate windowFig. 1.The predicted topology of subunit a in the E. coli inner membrane. The location of the most Ag⁺-sensitive Cys substitutions are highlighted in red (>85% inhibition) or orange (66–85% inhibition). The five proposed TMHs are shown in boxes, each with a span of 21 amino acids, which is the minimum length required to span the hydrophobic core of a lipid bilayer. The α-helical segments shown in loops 1–2 and 3–4 are consistent with the predictions of TALOS, based on backbone chemical shifts seen by NMR (29). Others have also predicted extensive α-helical regions in these loops (12, 30), but the possible positions remain largely speculative. aArg210 is highlighted in green. Figure is modified from those shown previously (21, 23, 24, 27).We have also reported Ag⁺-sensitive Cys substitutions in two cytoplasmic loops of subunit a (27) and, more recently, in the cytoplasmic loop of subunit c (28). The mechanism by which Ag⁺ inhibited F₁F₀-mediated H⁺ transport was uncertain. Several of these substitutions were also sensitive to inhibition by Cd⁺², and these substitutions provided a means of testing whether Cd⁺² directly inhibits passive H⁺ transport through F₀ (28). In the case of two subunit c loop substitutions, Cd⁺² was shown to directly inhibit passive F₀-mediated transport activity. In this study, we have extended the survey to Cd⁺²-sensitive Cys substitutions in cytoplasmic loops of subunit a. We report four loop substitutions in which Cd⁺² inhibits passive F₀-mediated H⁺ transport. Further, in two cases, we show cross-linking between pairs of Cys substitutions, which lie in subunits a and c, respectively, and which individually mediate passive H⁺ transport activity. These results suggest that the a and c loops, which gate H⁺ release to the cytoplasm, fold into a single domain at the surface of F₀.     

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H₂O₂ levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H₂O₂ because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H₂O₂ production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H₂O₂ in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H₂O₂ via a previously unidentified mechanism involving H₂O₂-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H₂O₂ detoxification.Cytochrome c peroxidase (Ccp1) is a monomeric nuclear encoded protein with a 68-residue N-terminal mitochondrial targeting sequence (1). This presequence crosses the inner mitochondrial membrane and is cleaved by matrix proteases (2, 3). Mature heme-loaded Ccp1 is found in the mitochondrial intermembrane space (IMS) in exponentially growing yeast (2, 3) but the point of insertion of its single b-type heme is unknown. Under strict anaerobic conditions, Ccp1 is present in mitochondria as the heme-free form or apoform (4). Once cells are exposed to O₂ and heme biosynthesis is turned on, apoCcp1 converts rapidly to the mature holoenzyme by noncovalently binding heme (5).It is well established that mature Ccp1 functions as an efficient H₂O₂ scavenger in vitro (6). Its catalytic cycle involves the reaction of ferric Ccp1 with H₂O₂ (Eq. 1) to form compound I (CpdI) with a ferryl (Fe^IV) heme and a cationic indole radical localized on Trp191 (W191^+•). CpdI is one-electron reduced by the ferrous heme of cytochrome c (Cyc1) to compound II (CpdII) with ferryl heme (Eq. 2), and electron donation by a second ferrous Cyc1 returns CpdII to the resting Ccp1^III form (Eq. 3):Ccp1^III + H₂O₂ → CpdI(Fe^IV, W191^+?) + H₂O[1]CpdI(Fe^IV, W191^+?) + Cyc1^II → CpdII(Fe^IV) + Cyc1^III[2]CpdII(Fe^IV) + Cyc1^II → Ccp1^III + Cyc1^III + H₂O.[3]Because Ccp1 production is not under O₂/heme control (4, 5), CCP activity is assumed to be the frontline defense in the mitochondria, a major source of reactive oxygen species (ROS) in respiring cells (7). Contrary to the time-honored assumption that Ccp1 catalytically consumes the H₂O₂ produced during aerobic respiration (8), recent studies in our group reveal that the peroxidase behaves more like a mitochondrial H₂O₂ sensor than a catalytic H₂O₂ detoxifier (9–11). Notably, Ccp1 competes with complex IV for reducing equivalents from Cyc1, which shuttles electrons from complex III (ubiquinol cytochrome c reductase) to complex IV (cytochrome c oxidase) in the electron transport chain (12).Because CCP activity in the IMS siphons electrons from energy production, an H₂O₂ sensor role for Ccp1 should be energetically more favorable for the cell. Key evidence for a noncatalytic role for Ccp1 in H₂O₂ removal is that the isogenic strain producing the catalytically inactive Ccp1^W191F protein accumulates less H₂O₂ than wild-type cells (10). In fact, this mutant strain exhibits approximately threefold higher catalase A (Cta1) activity than wild-type cells (10) whereas CCP1 deletion results in a strain (ccp1Δ) with negligible Cta1 activity and high H₂O₂ levels (5). Unlike Cta1, which is the peroxisomal and mitochondrial catalase isoform in yeast (13), the cytosolic catalase Ctt1 (14) exhibits comparable activity in the wild-type, Ccp1^W191F, and ccp1Δ strains (10). Given that both Ccp1 and Cta1 are targeted to mitochondria, we hypothesized that Ccp1 may transfer its heme to apoCta1 in respiring cells.Cta1 is nuclear encoded with embedded mitochondrial and peroxisomal targeting sequences (15). Like Ccp1, each monomer noncovalently binds a b-type heme and mature Cta1 is active as a homotetramer. Synthesis of the Cta1 monomer is under O₂/heme control such that the apoenzyme begins to accumulate only during the logarithmic phase of aerobic growth (16). Hence, its O₂/heme independent production (4, 5) allows apoCcp1 to acquire heme while cells are synthesizing apoCta1. This, combined with our observation that Cta1 activity increases in respiring cells producing Ccp1 or Ccp1^W191F but not in ccp1Δ cells (10), led us to speculate that respiration-derived H₂O₂ triggers heme donation from Ccp1 to apoCta1 within mitochondria.What experimental evidence would support heme donation by Ccp1? It has been demonstrated that mutation of the proximal heme Fe ligand, His175, to a residue with weak or no Fe-coordinating ability produces Ccp1 variants (H175P, H175L, H175R, and H175M) that undergo mitochondrial processing but do not accumulate in isolated yeast mitochondria (17). Presumably, reduced heme affinity allows the Ccp1 variants to unfold and cross the outer mitochondrial membrane (17). Hence, we argued that if wild-type Ccp1 donated its heme, the apoprotein would likewise exit mitochondria. Consequently, we examine here age-dependent Ccp1–green fluorescent protein (Ccp1-GFP) localization in live cells chromosomally expressing Ccp1 C-terminally fused to GFP as well as the distribution of wild-type Ccp1 between subcellular fractions. Because weakening or removal of the proximal Fe ligand on His175 mutation reduces heme affinity (17), His175 oxidation in wild-type Ccp1 should have a similar effect, which we investigate here. We further speculated that in the absence of apoCta1 as an acceptor for its heme, more Ccp1 would remain trapped in the IMS so we compare mitochondrial Ccp1 levels in wild-type and cta1∆ cells. Our combined results support triggering of heme donation from Ccp1 to apoCta1 by respiration-derived H₂O₂. Such H₂O₂-activated heme transfer between proteins has not been reported to date and its implications in H₂O₂ signaling are discussed.     

Fe–N2/CO complexes that model a possible role for the interstitial C atom of FeMo-cofactor (FeMoco)

We report here a series of four- and five-coordinate Fe model complexes that feature an axial tri(silyl)methyl ligand positioned trans to a substrate-binding site. This arrangement is used to crudely model a single-belt Fe site of the FeMo-cofactor that might bind N₂ at a position trans to the interstitial C atom. Reduction of a trigonal pyramidal Fe(I) complex leads to uptake of N₂ and subsequent functionalization furnishes an open-shell Fe–diazenido complex. A related series of five-coordinate Fe–CO complexes stable across three redox states is also described. Spectroscopic, crystallographic, and Density Functional Theory (DFT) studies of these complexes suggest that a decrease in the covalency of the Fe–C_alkyl interaction occurs upon reduction and substrate binding. This leads to unusually long Fe–C_alkyl bond distances that reflect an ionic Fe–C bond. The data presented are contextualized in support of a hypothesis wherein modulation of a belt Fe–C interaction in the FeMo-cofactor facilitates substrate binding and reduction.MoFe-nitrogenase catalyzes the fascinating but poorly understood conversion of nitrogen to ammonia at its iron-molybdenum cofactor (FeMoco) (1, 2). The core of the FeMoco was originally thought to be vacant (3). Later work on Azotobacter vinelandii indicated the presence of a light interstitial atom coordinated to six central, so-called “belt Fe atoms” (4). Crystallographic and spectroscopic studies (5, 6), in addition to studies mapping the biosynthetic pathway of C-atom incorporation (7, 8), establish that carbon is the interstitial atom, as shown in Fig. 1.Open in a separate windowFig. 1.A hypothetical N₂-binding event at a belt iron in FeMoco illustrating a proposed Fe–C elongation. The degree and positions of protonation are unknown under electron loading, but the inorganic sulfides are plausible candidate positions.Although the site(s) of N₂ reduction remain(s) uncertain, a body of evidence that includes biochemical, spectroscopic, and computational studies on FeMoco point to a belt Fe center as a plausible candidate (2, 9–13). In a scenario in which N₂ binds terminally to one of the belt Fe centers, the N₂ ligand would initially be coordinated trans to the interstitial C atom (Fig. 1) (10). This hypothesis calls for model complexes that depict such an arrangement to explore factors that might govern substrate coordination and subsequent reduction. Structurally faithful models of the FeMoco that include an Fe₆C unit stabilized by sulfide or other sulfur-based ligands present a formidable synthetic challenge (14). Moreover, N₂ coordination to synthetic iron–sulfur clusters has yet to be established (15). Model complexes featuring a single Fe site with a C-atom anchor positioned trans to an N₂ binding site are unknown, but would provide a useful tool to evaluate how an Fe–C interaction might respond to N₂ binding and the Fe redox state. Model compounds of this type may facilitate the evaluation of theoretical (10) and spectroscopic studies (16) on FeMoco that suggest a single, flexible Fe–C interaction is observed under turnover conditions.It is in this context that we have pursued mononuclear Fe complexes supported by tripodal, tetradentate ligands featuring three phosphine donor arms tethered to a tertiary alkyl anchor. Although a number of such ligands featuring a central C atom has been described (17–19) we reasoned that an alkyl ligand featuring only electropositive substituents adjacent to the C-atom anchor would provide a crude model of the interstitial carbide of the FeMoco and permit a high degree of ionic bonding to a single Fe–N₂ binding site. To achieve this goal, the C-atom anchor of the auxiliary ligand described is surrounded by three electropositive Si centers, in addition to the Fe site. This model system successfully coordinates N₂ trans to the C-atom anchor and shows how the local Fe geometry and the Fe–C interaction respond as a function of such binding. Using CO instead of N₂, this Fe system also presents an opportunity to study the Fe–C interaction as a function of the Fe redox state. As described below, unusually long Fe–C distances can be accessed that are consistent with a much higher degree of ionic character at the Fe–C interaction than would be anticipated for a prototypical Fe alkyl.     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Evolution of South Atlantic density and chemical stratification across the last deglaciation

Explanations of the glacial–interglacial variations in atmospheric pCO₂ invoke a significant role for the deep ocean in the storage of CO₂. Deep-ocean density stratification has been proposed as a mechanism to promote the storage of CO₂ in the deep ocean during glacial times. A wealth of proxy data supports the presence of a “chemical divide” between intermediate and deep water in the glacial Atlantic Ocean, which indirectly points to an increase in deep-ocean density stratification. However, direct observational evidence of changes in the primary controls of ocean density stratification, i.e., temperature and salinity, remain scarce. Here, we use Mg/Ca-derived seawater temperature and salinity estimates determined from temperature-corrected δ¹⁸O measurements on the benthic foraminifer Uvigerina spp. from deep and intermediate water-depth marine sediment cores to reconstruct the changes in density of sub-Antarctic South Atlantic water masses over the last deglaciation (i.e., 22–2 ka before present). We find that a major breakdown in the physical density stratification significantly lags the breakdown of the deep-intermediate chemical divide, as indicated by the chemical tracers of benthic foraminifer δ¹³C and foraminifer/coral ¹⁴C. Our results indicate that chemical destratification likely resulted in the first rise in atmospheric pCO₂, whereas the density destratification of the deep South Atlantic lags the second rise in atmospheric pCO₂ during the late deglacial period. Our findings emphasize that the physical and chemical destratification of the ocean are not as tightly coupled as generally assumed.The last glacial termination was accompanied by an 80-ppm rise in atmospheric pCO₂ (1, 2), and it is widely believed that this increase in pCO₂ was driven by processes occurring within the Southern Ocean (3–5). These Southern Ocean processes are proposed to have released CO₂ from the deep ocean through a combination of decreased nutrient utilization (6), increased vertical mixing (7), and increased air–sea gas exchange (8). Geochemical records show evidence for an “old” (9) respired dissolved inorganic carbon pool in the glacial Southern Ocean below 2,500 m (10, 11) which became better ventilated over the course of the deglaciation (9, 12), supporting the idea that the deep ocean was isolated from the atmosphere during glacials. Over the deglacial period this chemical stratification between the deep ocean and the overlying intermediate ocean decreased, e.g., ref. 11, implying a change in circulation or ventilation within the Southern Ocean which enabled CO₂ to be upwelled and outgassed to the atmosphere (7). The chemical destratification of the ocean has been attributed either to (i) an increase in air–sea gas exchange, through a decline in the extent of sea ice (8) and/or a decrease in surface ocean stratification (13); or (ii) a breakdown in the density stratification between the poorly ventilated deep ocean and the better-ventilated water masses above (14). Evidence supporting either scenario remains elusive.Pore-water profiles from deep-ocean sediments have provided the first estimates of the density of the deep ocean during the Last Glacial Maximum (LGM) (15). These studies found that the glacial deep ocean was highly saline [∼37 practical salinity units (psu)] and had an in situ density that was 2 kg/m³ denser than modern deep water. These studies lend support to the hypothesis that CO₂ storage within a highly stratified glacial ocean played a significant role in driving lower glacial atmospheric pCO₂. However, pore-water profiles only provide a “snapshot” of the physical properties of the deep ocean at the LGM, and do not provide information about the time-dependent changes in the density of deep water over the deglaciation. Thus, from these studies alone, it is impossible to assess whether the destratification of the deep-ocean density gradients drove the atmospheric pCO₂ increase over the deglacial period.Isotope-enabled intermediate complexity models have been used to suggest a mechanistic link between the physical (density) and chemical (δ¹³C) properties of the ocean over glacial–interglacial timescales (16, 17). These models suggest that deep-ocean stratification, generated by the formation of dense brines during sea ice growth, is required to reconcile the spatial distribution of seawater δ¹³C. This result implies that a decrease in Antarctic sea ice, and therefore reduced brine formation, over the deglacial period will affect both the density of the deep ocean and its chemical properties synchronously. Testing this hypothesis of a mechanistic link between the physical and chemical properties of the ocean requires observational evidence of the density structure evolution of the Southern Ocean over the entire deglacial period.Here, we determine the deglacial evolution of the intermediate-deep density gradient in the high-latitude South Atlantic Ocean by generating temperature and salinity proxy records over the last 20 ka at the intermediate depth site of sediment core GC528 (598 m; 58° 02.43′W, 53° 00.78′S) and the deep site of core MD07-3076Q (3,770 m; 14° 13.7′W, 44° 09.2′S) in the sub-Antarctic Atlantic (Fig. 1). We make the assumption that geochemical changes at a given depth occur synchronously within the South Atlantic (see the Supporting Information). Combined Mg/Ca and δ¹⁸O measurements on the benthic foraminifer Uvigerina spp. are used to estimate benthic seawater temperature (18) and to calculate the δ¹⁸O of deep and intermediate water masses (hereafter referred to as δ_w). Temperature and δ_w (closely related to seawater salinity) are combined to produce a continuous record of the evolution of the density gradient in the South Atlantic over the last deglaciation (Methods). We compare the evolution of the density gradient with benthic δ¹³C and ¹⁴C records from the two sites to assess the hypothesis of a causal link between the physical and chemical properties of the deglacial ocean.Open in a separate windowFig. 1.Location of intermediate (GC528) and deep (MD07-3076Q) sites. Site locations overlain on a schematic map of ocean circulation for (Top) modern ocean and (Bottom) LGM, adapted from Ferrari et al. (41). The grayscale colors indicate the flow path of major water masses. Background colors indicate the relative salinity of water masses (blue, relatively fresh; red, relatively saline).     

Dual role of CO in the stability of subnano Pt clusters at the Fe3O4(001) surface

Interactions between catalytically active metal particles and reactant gases depend strongly on the particle size, particularly in the subnanometer regime where the addition of just one atom can induce substantial changes in stability, morphology, and reactivity. Here, time-lapse scanning tunneling microscopy (STM) and density functional theory (DFT)-based calculations are used to study how CO exposure affects the stability of Pt adatoms and subnano clusters at the Fe₃O₄(001) surface, a model CO oxidation catalyst. The results reveal that CO plays a dual role: first, it induces mobility among otherwise stable Pt adatoms through the formation of Pt carbonyls (Pt₁–CO), leading to agglomeration into subnano clusters. Second, the presence of the CO stabilizes the smallest clusters against decay at room temperature, significantly modifying the growth kinetics. At elevated temperatures, CO desorption results in a partial redispersion and recovery of the Pt adatom phase.Subnanometer metal particles exhibit a range of interesting electronic or catalytic properties that can vary substantially with the removal or addition of a single atom (1–6). Understanding the mechanistic details underlying the rearrangement of the active phase is important because changes in cluster size and shape are known to be commonplace under the conditions used in heterogeneous catalysis (7, 8), and because such processes are associated with deactivation phenomena such as sintering. Although sintering is usually regarded as a thermally activated process, there is increasing evidence that adsorbates influence sintering rates in a reactive environment by formation of mobile metal-molecule intermediates (2, 8–30). Indeed, in a previous study we demonstrated that the formation of highly mobile Pd₁–CO species led to enhanced sintering in the Pd/Fe₃O₄(001) system (31). Here, we turn our attention to Pt. Mobility is induced in the form of Pt₁–CO. In addition, CO stabilizes the smallest clusters. When it desorbs, Pt dimers break up into single atoms; thus, the CO is necessary for preserving nuclei that act as seeds for further growth. Using room-temperature scanning tunneling microscopy (STM), complemented by X-ray photoelectron spectroscopy (XPS) and density functional theory with an on-site Hubbard U (DFT+U), we follow the CO-induced diffusion and coalescence of Pt atom-by-atom, creating catalytically active (32) subnano clusters with a well-defined size distribution. On heating, desorption of CO leads to significant redispersion of Pt into the adatom phase.     

Medial septal GABAergic projection neurons promote object exploration behavior and type 2 theta rhythm

Exploratory drive is one of the most fundamental emotions, of all organisms, that are evoked by novelty stimulation. Exploratory behavior plays a fundamental role in motivation, learning, and well-being of organisms. Diverse exploratory behaviors have been described, although their heterogeneity is not certain because of the lack of solid experimental evidence for their distinction. Here we present results demonstrating that different neural mechanisms underlie different exploratory behaviors. Localized Ca_v3.1 knockdown in the medial septum (MS) selectively enhanced object exploration, whereas the null mutant (KO) mice showed enhanced-object exploration as well as open-field exploration. In MS knockdown mice, only type 2 hippocampal theta rhythm was enhanced, whereas both type 1 and type 2 theta rhythm were enhanced in KO mice. This selective effect was accompanied by markedly increased excitability of septo-hippocampal GABAergic projection neurons in the MS lacking T-type Ca²⁺ channels. Furthermore, optogenetic activation of the septo-hippocampal GABAergic pathway in WT mice also selectively enhanced object exploration behavior and type 2 theta rhythm, whereas inhibition of the same pathway decreased the behavior and the rhythm. These findings define object exploration distinguished from open-field exploration and reveal a critical role of T-type Ca²⁺ channels in the medial septal GABAergic projection neurons in this behavior.When confronted with an unfamiliar environment, or physical or social objects, animals often exhibit behavior patterns that can broadly be termed exploration, such as moving around the environment, touching or sniffing novel objects, and interacting with social stimuli (1). Social exploration involves complex processes that differ from those involved in the nonsocial exploration (2). Several distinctions were proposed to categorize the different forms of nonsocial exploratory behaviors from a motivational perspective (3). Behaviorally, two types of nonsocial exploration are observed in rodents and humans (3–5): object exploration and spatial or environmental exploration in the absence of objects. Object exploration is the behavior to explore discrete novel objects. This activity is elicited and sustained by the physical presence of an object. Several types of preference or “novelty” tests have been developed to investigate object exploration in rodents (3, 5–7). Environmental or spatial exploration in the absence of objects refers to the inquisitive activity of an animal in a new space, where the eliciting and sustaining stimulus is the “place” itself. Various forms of open-field tests have been used to investigate environmental or spatial exploration in rodents (3, 5, 8). Experimentally, however, the distinction can be less obvious because both can occur together (4, 7–9). Spatial exploration is suggested to be hippocampal-dependent (10)—although that is controversial (11)—whereas object exploration is suggested to be hippocampal-independent (12). Thus, it is still a matter of debate whether animal exploration belongs to a unitary category or not (9). To resolve this issue, neural definitions of these two previously proposed exploratory behaviors are needed.Interestingly, the medial septum (MS), where Ca_v3.1 T-type Ca²⁺ channels are highly expressed (13), is suggested to be critical for exploratory behaviors (5, 14–16). Moreover, the MS is also the nodal point for ascending afferent systems involved in the generation of hippocampal theta rhythms, the largest synchronous oscillatory signals in the mammalian brain, which are implicated in diverse brain functions (17, 18). Although the heterogeneity of hippocampal theta rhythms has long been under debate (19), recent studies based on genetic mutations in mice and optogenetics provide strong support for theta rhythm heterogeneity (20–22). However, their exact behavioral correlates are still debated. Ca_v3.1 Ca²⁺ channels play an important role in diverse behaviors, as well as the generation of physiologic and pathophysiologic brain rhythms (23). Notably, T-type, low-threshold Ca²⁺ currents are assumed to be a candidate ionic mechanism of theta rhythm genesis (24), analogous to the role of T-type channels in the generation of oscillations in the reticular nucleus of the thalamus (25). Nevertheless the involvement of T-type Ca²⁺ channels in hippocampal theta rhythms or exploratory behavior has not been examined. Here, we analyzed global KO mice and mice with MS-specific inactivation of the Ca_v3.1 gene encoding T-type Ca²⁺ channels, focusing on finding the neural mechanism that control the exploratory behaviors. Using a combination of tools, we provide evidence that object and open field exploratory behaviors are processed differently in the brain. Furthermore, Ca_v3.1 T-type Ca²⁺ channels in the septo-hippocampal GABAergic projection neurons are critically involved in controlling object exploration through modulating hippocampal type 2 theta rhythm.     

Kinetics of a Criegee intermediate that would survive high humidity and may oxidize atmospheric SO2

Criegee intermediates are thought to play a role in atmospheric chemistry, in particular, the oxidation of SO₂, which produces SO₃ and subsequently H₂SO₄, an important constituent of aerosols and acid rain. However, the impact of such oxidation reactions is affected by the reactions of Criegee intermediates with water vapor, because of high water concentrations in the troposphere. In this work, the kinetics of the reactions of dimethyl substituted Criegee intermediate (CH₃)₂COO with water vapor and with SO₂ were directly measured via UV absorption of (CH₃)₂COO under near-atmospheric conditions. The results indicate that (i) the water reaction with (CH₃)₂COO is not fast enough (k_H2O < 1.5 × 10⁻¹⁶ cm³s⁻¹) to consume atmospheric (CH₃)₂COO significantly and (ii) (CH₃)₂COO reacts with SO₂ at a near–gas-kinetic-limit rate (k_SO2 = 1.3 × 10⁻¹⁰ cm³s⁻¹). These observations imply a significant fraction of atmospheric (CH₃)₂COO may survive under humid conditions and react with SO₂, very different from the case of the simplest Criegee intermediate CH₂OO, in which the reaction with water dimer predominates in the CH₂OO decay under typical tropospheric conditions. In addition, a significant pressure dependence was observed for the reaction of (CH₃)₂COO with SO₂, suggesting the use of low pressure rate may underestimate the impact of this reaction. This work demonstrates that the reactivity of a Criegee intermediate toward water vapor strongly depends on its structure, which will influence the main decay pathways and steady-state concentrations for various Criegee intermediates in the atmosphere.Unsaturated hydrocarbons are emitted into the atmosphere in large quantities from either human or natural sources. Ozonolysis of unsaturated hydrocarbons produces highly reactive Criegee intermediates (CIs) (1), which may (i) decompose to radical species like OH radicals or (ii) react with a number of atmospheric species, for example, with SO₂ to form SO₃ and with NO₂ to form NO₃ (2, 3). The SO₂ oxidation by CIs has gained special attentions because the SO₃ product would be converted into H₂SO₄, an important constituent of aerosols and acid rain (4–8). For example, Mauldin et al. (4) have speculated that Criegee intermediate reactions with SO₂ may account for the discrepancy between the observed and modeled concentrations of H₂SO₄ in a boreal forest region, where various alkenes are emitted by trees.Recently, Welz et al. (2) demonstrated an efficient method to prepare a CI in a laboratory by the reaction of iodoalkyl radical with O₂ (for example, CH₂I + O₂ → CH₂OO + I). This method can produce a CI of high enough concentration that allows direct detection. With photoionization mass spectrometry (PIMS) detection, Welz et al. (2) measured the rate coefficients of the simplest CI (CH₂OO) reactions with SO₂ and NO₂. Notably, these new rate coefficients, confirmed by a few later investigations (9–11), are orders of magnitude larger than those previously used (12, 13) in atmospheric models (e.g., MCM v3.3, available at mcm.leeds.ac.uk/MCM/browse.htt?species=CH2OO), suggesting a greater role of CIs in atmospheric chemistry. This result also indicates previous ozonolysis analyses may be affected by complicated and partly unknown side reactions and may contain errors in some of the reported rate coefficients.Typical water concentration in the troposphere (1.3 × 10¹⁷ to 8.3 × 10¹⁷ cm⁻³ at the dew point of 0–27 °C) is orders of magnitude higher than those of atmospheric trace gases like SO₂, NO₂, and volatile organic compounds (VOC) (on the order of 10¹² cm⁻³ or less). Although it has been shown that CIs may react very fast with SO₂, NO₂, and organic acids (2, 3, 14), the reactions of CIs with atmospheric water vapor would still strongly influence the fates and concentrations of atmospheric CIs (see Fig. 1 for a simplified schematic). As expected, the reactivity of CIs toward water vapor would govern the modeling results of atmospheric H₂SO₄ formation from CIs (8, 15, 16).Open in a separate windowFig. 1.Reaction scheme showing competitions for CIs between reactions with water (monomer and dimer) and with SO₂.However, there had been discrepancies about the reactivity of CIs toward water. Whereas studies (17–20) using C₂H₄ ozonolysis as a CH₂OO source show substantial reactivity of CH₂OO toward water vapor, despite a large scatter (10⁻¹⁷ to 10⁻¹² cm³s⁻¹) in the reported rate coefficient, other studies (2, 10, 21) using the CH₂I+O₂ reaction as a CH₂OO source reported negative observation for the CH₂OO reaction with water vapor.More recently, Chao et al. (22) and Berndt et al. (23) investigated the reaction of CH₂OO with water vapor using the CH₂I+O₂ reaction and the C₂H₄ ozonolysis as their CH₂OO sources, respectively. Both groups observed clear second-order kinetics with respect to the concentration of water and concluded that reaction with water dimer predominates in the decay of CH₂OO under atmospheric conditions and that previous studies may require some reinterpretations. The reported rate coefficient of the CH₂OO reaction with water dimer is large, about 7 × 10⁻¹² cm³s⁻¹ (22), leading to extremely fast decay rate of CH₂OO under typical tropospheric conditions (

Massive and rapid predominantly volcanic CO2 emission during the end-Permian mass extinction

The end-Permian mass extinction event (∼252 Mya) is associated with one of the largest global carbon cycle perturbations in the Phanerozoic and is thought to be triggered by the Siberian Traps volcanism. Sizable carbon isotope excursions (CIEs) have been found at numerous sites around the world, suggesting massive quantities of ¹³C-depleted CO₂ input into the ocean and atmosphere system. The exact magnitude and cause of the CIEs, the pace of CO₂ emission, and the total quantity of CO₂, however, remain poorly known. Here, we quantify the CO₂ emission in an Earth system model based on new compound-specific carbon isotope records from the Finnmark Platform and an astronomically tuned age model. By quantitatively comparing the modeled surface ocean pH and boron isotope pH proxy, a massive (∼36,000 Gt C) and rapid emission (∼5 Gt C yr⁻¹) of largely volcanic CO₂ source (∼−15%) is necessary to drive the observed pattern of CIE, the abrupt decline in surface ocean pH, and the extreme global temperature increase. This suggests that the massive amount of greenhouse gases may have pushed the Earth system toward a critical tipping point, beyond which extreme changes in ocean pH and temperature led to irreversible mass extinction. The comparatively amplified CIE observed in higher plant leaf waxes suggests that the surface waters of the Finnmark Platform were likely out of equilibrium with the initial massive centennial-scale release of carbon from the massive Siberian Traps volcanism, supporting the rapidity of carbon injection. Our modeling work reveals that carbon emission pulses are accompanied by organic carbon burial, facilitated by widespread ocean anoxia.

The end-Permian mass extinction (EPME) that occurred at 251.941 ± 0.037 Mya is considered the most severe biodiversity loss in Earth history (1, 2). The EPME coincides with the eruption of the Siberian Traps, a voluminous large igneous province (LIP) that occupies 6 million square kilometers (km²) in Siberia, Russia (3–5). The volcanic activity of this LIP is linked to SO₂ and CO₂ degassing generated by sill intrusion (6–10). The large amount of CO₂ injected into the atmosphere is thought to have led to severe global warming (11–14), catastrophic ocean anoxia (15, 16), and extreme ocean and terrestrial acidification (17–21) being lethal for life on land and in the sea (22). To date, no agreement has been reached regarding the source of the ¹³C-depleted carbon that triggered the global carbon cycle perturbation, the decrease in ocean pH, and the global warming across the EPME. Additionally, atmospheric CO₂ levels following the initial pulse of Siberian Traps volcanism and across the EPME remain poorly known (23, 24), limiting our understanding of the climate feedbacks that occur upon greenhouse gas release during this time.To address this critical gap in our knowledge, we constrain the source, pace and total amount of CO₂ emissions using an Earth system model of intermediate complexity (i.e., carbon centric-Grid Enabled Integrated Earth system model [cGENIE]; SI Appendix) forced by new astronomically tuned δ¹³C records from well-preserved lipid biomarkers preserved in sediments from the Finnmark Platform, Norway. The Finnmark Platform is located offshore northern Norway on the Eastern Barents Sea shelf, hosting an expanded shallow marine section (paleo-water depth roughly 50 to 100 m) where two drill cores were collected (7128/12-U-01 and 7129/10-U-01) spanning the Permian–Triassic transition (Fig. 1). A previously generated bulk organic carbon isotope record (δ¹³C_org) from the same core shows a two-step decline with a total carbon isotope excursion (CIE) magnitude of ∼4‰ (25). Although the sedimentary organic carbon was considered primarily of terrestrial origin, small contributions from marine organic carbon production could not be excluded. Here, we use compound-specific carbon isotope analysis of both long-chain and short-chain n-alkanes preserved in marine sediments in the Finnmark Platform to generate separate yet directly comparable records of δ¹³C for the terrestrial and the marine realm, respectively, across the EPME. Long-chain n-alkanes with a strong odd-over-even predominance (n-C₂₇ and n-C₂₉) are produced by higher plant leaf waxes, and their isotopic composition (δ¹³C_wax) relates to their main carbon source (i.e., atmospheric CO₂) (26). On the other hand, short-chain alkanes (n-C₁₇ and n-C₁₉) are derived from marine algae, and their δ¹³C values (δ¹³C_algae) represent carbon in the marine realm (27, 28). To date, only a few EPME compound-specific carbon isotope studies have been reported, all of which are limited by unfavorable sedimentary facies or high thermal maturity of the organic matter (29, 30). In the present study, the exceptionally low thermal maturity of the organic matter is evident from the yellow color of pollen and spores, indicating a color index 2 out of 7 on the thermal alteration scale of Batten (31), which is equivalent to a vitrinite reflectance R₀ of 0.3%. Moreover, the high sedimentation rate (discussed in Carbon Cycle Quantification Using Astrochronology and Earth System Model) of the siliciclastic sediments at the study site allows for studying both marine and terrestrial CIE across the EPME in unprecedented detail. Taken together, the Finnmark sedimentary records enable the reconstruction of individual yet directly comparable carbon isotope records for the terrestrial and the marine realm that can be astronomically tuned and used to quantitatively assess the source, pace, and total amount of ¹³C-depleted carbon released during the Siberian Traps eruption that led to the EPME. Using our new compound-specific carbon isotope records, rather than marine carbonates, has several advantages: 1) new astrochronology enables a 10⁴-year temporal resolution for our paired marine and terrestrial carbon isotope records; 2) we do not need to assume a constant sedimentation rate between tie point or using diachronous biozones to compare age like those used in global compilations (24) (see Fig. 4A); 3) the δ¹³C_algae data are not artificially smoothed as in ref. 32 to avoid underestimation of the CIE magnitude; and 4) our records are not affected by dissolution or truncation, a phenomenon common to shallow marine carbonates due to the presumed ocean acidification occurred during the EPME (18, 33). In addition, the directly comparable records of δ¹³C for the atmosphere and the ocean offer further insights into the size of the true CIE and rate and duration of carbon emissions.Open in a separate windowFig. 1.(A) Paleogeographical map of the Late Permian, with former and current coastlines. Indicated are 1) the location of Finnmark cores 7128/12-U-01 and 7129/10-U-01, 2) the East Greenland site at Kap Stosch discussed in ref. 52, 3) the GSSP site for the base of the Triassic at Meishan, China, and 4) the Kuh-e-Ali Bashi site of Iran (66, 107). The map was modified after ref. 61. (B) Paleogeography and paleobathymetry of the Late Permian used in cGENIE.Open in a separate windowFig. 4.Synthesized proxy records of carbon isotopes from marine carbonates and fossil C₃ land plants remains, sea surface temperature, and pH. (A) Comparison between δ¹³C_algae and global marine carbonate carbon isotopes from sites at Abadeh, Kuh-e-Ali Bashi, Shahreza, and Zal in Iran, Meishan, Wenbudangsang, and Yanggou in South China, at Bálvány North in Hungary, and at Nhi Tao in Vietnam (24). (B) Comparison between δ¹³C_leaf wax and the δ¹³C of sedimentary leaf cuticles and wood of C₃ land plants from South China (24). (C) Reconstructed sea surface temperature data using conodont fossils (circles) (24) and brachiopods (triangles) (14). The conodont-based temperature data are from sites in the Paleo-Tethys, including Chanakhchi, Kuh-e Ali Bashi, Meishan, Shangsi, and Zal. (D) Relative changes in sea surface pH based on boron isotope proxy from ref. 17 and ref. 20. Pink and red circles are data from scenario 1 and scenario 2 in ref. 17, and green and blue diamonds are data from scenario 1 and scenario 2 in ref. 20.     

Erosion during extreme flood events dominates Holocene canyon evolution in northeast Iceland

Extreme flood events have the potential to cause catastrophic landscape change in short periods of time (10⁰ to 10³ h). However, their impacts are rarely considered in studies of long-term landscape evolution (>10³ y), because the mechanisms of erosion during such floods are poorly constrained. Here we use topographic analysis and cosmogenic ³He surface exposure dating of fluvially sculpted surfaces to determine the impact of extreme flood events within the Jökulsárgljúfur canyon (northeast Iceland) and to constrain the mechanisms of bedrock erosion during these events. Surface exposure ages allow identification of three periods of intense canyon cutting about 9 ka ago, 5 ka ago, and 2 ka ago during which multiple large knickpoints retreated large distances (>2 km). During these events, a threshold flow depth was exceeded, leading to the toppling and transportation of basalt lava columns. Despite continuing and comparatively large-scale (500 m³/s) discharge of sediment-rich glacial meltwater, there is no evidence for a transition to an abrasion-dominated erosion regime since the last erosive event because the vertical knickpoints have not diffused over time. We provide a model for the evolution of the Jökulsárgljúfur canyon through the reconstruction of the river profile and canyon morphology at different stages over the last 9 ka and highlight the dominant role played by extreme flood events in the shaping of this landscape during the Holocene.Extreme floods in both terrestrial and extraterrestrial environments can cause abrupt landscape change that can have long-term consequences (1–5), especially when a geomorphic threshold is exceeded (6). The timescale over which this change is visible is controlled by the ability and efficiency of background processes to reshape the landscape. As a result, progress in understanding both short-term and long-term landscape evolution requires better knowledge of bedrock channel erosion processes and thresholds over the different scales at which geomorphological processes operate (7–10).The majority of research into extreme flood events has focused on the interpretation of deposited sediments (e.g., refs. 11 and 12) and the reconstruction of the hydraulic conditions prevailing during such events (e.g., refs. 13–15). Further work has defined the geomorphic impact of extreme flood events in proglacial areas close to the source of the flood water (e.g., refs. 16 and 17). Studies that examine the processes of bedrock erosion, especially large canyon formation, during extreme flood events can help establish a diagnostic link between formation processes and morphology in canyons in both terrestrial and extraterrestrial settings, but they remain scarce (e.g., refs. 18–20). Here, evidence for bedrock landscape change during extreme floods along the course of the Jökulsá á Fjöllum River (northeast Iceland) is used to test whether the contemporary landscape morphology reflects erosion during rare extreme events, or longer-term “background” erosional processes.The Jökulsá á Fjöllum has experienced multiple glacial outburst floods (jökulhlaups) since the Last Glacial Maximum, with peak discharge for the largest flood estimated to be in the order of 0.9 × 10⁶ m³/s (14, 21). The landscape contains many characteristic landforms associated with extreme flood events, including boulder bars and terraces, dry cataracts such as Ásbyrgi, numerous flood overspill channels, and the Jökulsárgljúfur canyon (Fig. 1) (e.g., refs. 16 and 22–25). The canyon has been carved through a volcanic system that was active 8.5 ka B.P. (26) ∼4 km downstream of its head. As the canyon is cut directly through the fissure and associated lava flows and there is no evidence of lava from the fissure flowing into the canyon, the eruption age provides an independent constraint on the maximum age for the formation of the canyon upstream of the fissure (Fig. 1). The impact of the largest flood events has never been tied to the evolution of the bedrock landscape within the Jökulsárgljúfur canyon, as previous studies have focused on sedimentary deposits (e.g., refs. 24 and 25). This study uses topographic analysis and cosmogenic ³He surface exposure dating of fluvial surfaces to determine the erosive impact of extreme flood events and assess the importance, and legacy, of high-magnitude low-frequency events in landscape evolution over multimillennial timescales.Open in a separate windowFig. 1.(A) Location map of Iceland showing the Vatnajökull ice cap, the source of the floodwaters, and the course of the Jökulsá á Fjöllum. The locations of the two study sites—the upper 5 km of Jökulsárgljúfur canyon at Dettifoss and Ásbyrgi, 25 km further downstream—are shown with black stars. The location of the gauging station at Grimsstadir used for hydrological calculations is also shown. (B) Aerial photograph from 1998 of the 5-km study reach at the head of Jökulsárgljúfur canyon. Yellow dashed lines delineate the areas where clear evidence for fluvial erosion is present (landscape outside these areas is shaded to improve clarity). The three large knickpoints are highlighted: Selfoss, Dettifoss, and Hafragilsfoss (height in parentheses), as well as the Sanddalur overspill channel, which contains two cataracts. The volcanic fissure that erupted 8.5 ka ago (black circles show volcanoes) provides an independent constraint on the maximum age of the canyon. Orange stars indicate the locations of the samples collected for surface exposure dating. The upper, middle, and lower terraces are shown in red, green, and yellow, respectively; active fluvial surfaces associated with upper and middle terraces are shown in transparent red and green upstream of Dettifoss. A cross section of the gorge across the line from west to east is inset. (C) A zoomed in image of Dettifoss from 1998, with the yellow line showing the digitized position of the waterfall in 1955. Dettifoss has been mostly stable during the 43-y period between the images, with only a small retreat (maximum 5 m) evident on the western side of the channel. If the Jökulsárgljúfur canyon was formed by the progressive retreat of Dettifoss following the fissure eruption (2,500 m in 8.5 ka, equivalent to a rate of 0.3 m/y), we would expect to see a minimum of 13 m of retreat between 1955 and 1998, shown with the red line. (D) Ásbyrgi canyon and the Klappir scabland area immediately upstream. This landscape exhibits perfectly preserved landforms that were formed during an extreme flood event, with the Jökulsá á Fjöllum now flowing in a deeply incised canyon to the east.