Ultrafast X-ray scattering offers a structural view of excited-state charge transfer

Intramolecular charge transfer and the associated changes in molecular structure in N,N′-dimethylpiperazine are tracked using femtosecond gas-phase X-ray scattering. The molecules are optically excited to the 3p state at 200 nm. Following rapid relaxation to the 3s state, distinct charge-localized and charge-delocalized species related by charge transfer are observed. The experiment determines the molecular structure of the two species, with the redistribution of electron density accounted for by a scattering correction factor. The initially dominant charge-localized state has a weakened carbon–carbon bond and reorients one methyl group compared with the ground state. Subsequent charge transfer to the charge-delocalized state elongates the carbon–carbon bond further, creating an extended 1.634 Å bond, and also reorients the second methyl group. At the same time, the bond lengths between the nitrogen and the ring-carbon atoms contract from an average of 1.505 to 1.465 Å. The experiment determines the overall charge transfer time constant for approaching the equilibrium between charge-localized and charge-delocalized species to 3.0 ps.

Understanding the dynamic process of photoinduced charge transfer is expected to lead to many practical applications, including efficient photovoltaic systems, the development of photocatalysts, and better materials for energy storage (1–3). Charge transfer redistributes the electrons in a molecule and is typically associated with changes in the molecular geometry (2). On the fastest time scale, electrons move so rapidly that the nuclei appear frozen, a phenomenon known as charge migration (4–10). When time scales approach the typical vibrational motions of molecules, that is, tens of femtoseconds (10⁻¹⁴ s), the nuclei can adjust their positions, often resulting in localization of electronic charge and permanent changes in molecular geometry (11). While exhibiting a rich phenomenology on different time scales, it is evident that electron charge transfer and nuclear dynamics are intrinsically coupled (12–17). An accurate determination of the changes in molecular structure during charge transfer is therefore of great interest from both applied and fundamental perspectives.New scientific technologies, in particular X-ray free-electron lasers (XFEL) (18, 19) and MeV ultrafast electron diffraction (20), have made it possible to study structural dynamics in the ultrafast regime. Recent femtosecond gas-phase scattering experiments have successfully tracked structural changes during chemical reactions (21–26) and probed specific signatures of excited electronic states (27, 28). Given the emerging ability of ultrafast gas-phase scattering to record both nuclear and electronic structure (25, 27, 28), we use time-resolved gas-phase X-ray scattering to study the photoinduced intramolecular charge transfer in an organic molecule, N,N′-dimethylpiperazine (DMP, C₆H₁₄N₂), shown in Fig. 1. In its ground electronic state, DMP has C_2h symmetry with two equivalent ionization centers, one on each nitrogen atom. Valence ionization, or in the present case excitation to an electronic Rydberg state (29), induces charge transfer between the two nitrogen atoms, making DMP a prototype for exploring electron lone-pair interactions and charge transfer (30–32).Open in a separate windowFig. 1.A schematic illustration of the experimental setup. The ground-state molecules (DMP) were excited by 200 nm ultraviolet pump pulses, and the transient structures were probed by 9.5 keV X-ray pulses at variable time delays. The scattering signals were recorded on a CSPAD detector. The inset shows the calculated spin density, which gives the difference in density of electrons with spin up and spin down, of the charge-localized DMP (3sL) and charge-delocalized DMP (3sD) in the 3s Rydberg states at isovalues of 0.1 electron/ų.Previously, energy relaxation pathways and charge transfer in electronically excited DMP were explored using Rydberg fingerprint spectroscopy (33), a form of photoelectron spectroscopy. As depicted in Scheme 1, the investigations by Deb et al. (33) found that optical excitation at 207 nm prepares the molecule in a 3p Rydberg state, creating a state with a localized charge in the molecular core (3pL). Internal conversion to 3s then leads to charge-localized (3sL) and charge-delocalized (3sD) conformers with 230 and 480 fs time constants, respectively. The charge transfer proceeds as the molecules explore the 3s potential energy surface. An equilibrium between 3sL and 3sD structures is eventually established with an overall time constant of 2.65 ps, with the forward and backward first-order kinetic time constants for the transformation 3.4 and 12.0 ps, respectively (33).Open in a separate windowScheme 1.Reaction pathway for Rydberg-excited DMP as determined previously (33).A limitation in the prior spectroscopic work is that the photoelectron peaks are assigned by comparing measured binding energies with computational results and that the molecular structures that underlie the calculations cannot not be independently determined in the experiments. This is compounded by the fact that theoretical calculation of charge-localized and charge-delocalized excited states is challenging. Widely used computational methods sometimes give unsatisfactory results (34), and even results from high-level computations can be controversial (35–37). This has led to interesting discussions about whether a stable charge-localized structure exists in the DMP cation at all and about the validity of different exchange functionals within density functional theory to study the charge localization in DMP molecules (34). Considering the general experimental and theoretical interest in this system, direct structural measurements would be invaluable. The current study uses ultrafast time-resolved X-ray scattering to observe the structural relaxation dynamics in DMP. This provides a wealth of information, making it possible to test key assumptions of the photoelectron study. Most importantly, we determine the molecular structures of the charge-localized and -delocalized excited states.     

Ultrafast nanometric imaging of energy flow within and between single carbon dots

Time- and space-resolved excited states at the individual nanoparticle level provide fundamental insights into heterogeneous energy, electron, and heat flow dynamics. Here, we optically excite carbon dots to image electron–phonon dynamics within single dots and nanoscale thermal transport between two dots. We use a scanning tunneling microscope tip as a detector of the optically excited state, via optical blocking of electron tunneling, to record movies of carrier dynamics in the 0.1–500-ps time range. The excited-state electron density migrates from the bulk to molecular-scale (∼1 nm²) surface defects, followed by heterogeneous relaxation of individual dots to either long-lived fluorescent states or back to the ground state. We also image the coupling of optical phonons in individual carbon dots with conduction electrons in gold as an ultrafast energy transfer mechanism between two nearby dots. Although individual dots are highly heterogeneous, their averaged dynamics is consistent with previous bulk optical spectroscopy and nanoscale heat transfer studies, revealing the different mechanisms that contribute to the bulk average.

The use of nanomaterials in a wide variety of applications can be hampered or enhanced by the presence of defects (1), which can change the properties of particles from one to the next. For example, catalysis often occurs at defect sites (2, 3), but only a minority of such sites are catalytic (4); likewise, energy transfer from a quantum dot film to another layer can be focused on specific defect sites rather than being uniform throughout the layer (5).Excited-state defects in particular control electron, phonon, and energy flow that is important for chemistry and photophysics at the surface of nanomaterials. For some types of nanoparticles, localized surface excited states, rather than particle size, control multicolor emission dynamics (6, 7). Progress has been made in the imaging and tomography of individual excited-state defects with subnanometer resolution in a range of materials (8–10). These techniques probe spatial resolution in exquisite detail, but they lack the time resolution required for revealing the excited-state dynamics directly.Here we present time-resolved single-molecule absorption scanning tunneling microscopy (trSMA-STM), which uses an STM tip as a photodetector of pump–probe laser excitation, making the spatial resolution independent of wavelength and the time resolution controlled by the laser pulse width. We use trSMA-STM to image the dynamics of single carbon dots and pairs of carbon dots with 100-fs time- and single-nanometer spatial resolution. Carbon dots are a new class of photoluminescent nanomaterials similar in size to quantum dots (<10 nm in diameter) synthesized via hydro-/solvothermal reactions, laser ablation, or microwave treatments. They contain largely sp²-hybridized carbon oxidized by oxygen- and nitrogen-containing defects. Carbon dots have emerged in bioimaging and energy-related applications, owing to their aqueous solubility, low toxicity, and biocompatibility (11–13).We find that excited-state dynamics of carbon dots is highly heterogeneous, with some dots relaxing immediately back to the ground state in a few picoseconds, while in other dots the energy is trapped in highly localized and long-lived surface states. These two classes of dots combine in the bulk to produce the known moderate fluorescence quantum yield of carbon-dot ensembles. We also study colocalized pairs of carbon dots, and for such pairs, we see the transfer of energy in a few square-nanometer regions mediated by plasmonic excitation of the surface, revealing the difference between highly confined and bulk heat conductivity     

Intrinsic electronic conductivity of individual atomically resolved amyloid crystals reveals micrometer-long hole hopping via tyrosines

Proteins are commonly known to transfer electrons over distances limited to a few nanometers. However, many biological processes require electron transport over far longer distances. For example, soil and sediment bacteria transport electrons, over hundreds of micrometers to even centimeters, via putative filamentous proteins rich in aromatic residues. However, measurements of true protein conductivity have been hampered by artifacts due to large contact resistances between proteins and electrodes. Using individual amyloid protein crystals with atomic-resolution structures as a model system, we perform contact-free measurements of intrinsic electronic conductivity using a four-electrode approach. We find hole transport through micrometer-long stacked tyrosines at physiologically relevant potentials. Notably, the transport rate through tyrosines (10⁵ s⁻¹) is comparable to cytochromes. Our studies therefore show that amyloid proteins can efficiently transport charges, under ordinary thermal conditions, without any need for redox-active metal cofactors, large driving force, or photosensitizers to generate a high oxidation state for charge injection. By measuring conductivity as a function of molecular length, voltage, and temperature, while eliminating the dominant contribution of contact resistances, we show that a multistep hopping mechanism (composed of multiple tunneling steps), not single-step tunneling, explains the measured conductivity. Combined experimental and computational studies reveal that proton-coupled electron transfer confers conductivity; both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine influence the hole transport rate through a proton rocking mechanism. Surprisingly, conductivity increases 200-fold upon cooling due to higher availability of the proton acceptor by increased hydrogen bonding.

Many biological processes require electron transport over far longer distances than the 25 Å allowed by single-step tunneling (1). The aromatic amino acids tyrosine and tryptophan, strategically placed between cofactors in proteins, can provide the critical stepping-stones for multistep hopping by acting as intermediaries along the electron transfer route as in Photosystem II and ribonucleotide reductase (1, 2). In addition, chains of aromatic residues have been proposed to move hole carriers from enzyme active sites to the exterior of proteins to avoid oxidative damage caused by redox cofactors, but direct experimental evidence of charge transport beyond the nanometer scale is lacking (3, 4). The ubiquity of such chains observed in proteins, and the established role of aromatic amino acids in the above biological processes, motivates mechanistic studies of how electron transport via aromatic residues can scale over mesoscopic (hundreds of nanometers) to microscopic distances (1, 2).A remarkable example of biological long-distance electron transport is by soil (5, 6) and sediment bacteria (7, 8) that carry electrons to remote acceptors hundreds of micrometers (5, 9) to centimeters away (7–10), 10,000 times the size of the cell. This long-distance transport enables bacteria to get rid of electrons derived from metabolism and survive in harsh environments that lack soluble, membrane-ingestible electron acceptors such as oxygen. Polymerized cytochromes produced by soil bacteria function as “microbial nanowires” by transporting electrons through seamlessly stacked hemes (5, 6). In addition, filamentous proteins produced by both soil (11) and sediment bacteria (8) have been shown to be conductive, even in the absence of cytochromes (8). However, the underlying conductivity mechanism is unclear. Aromatic residues present in these filamentous proteins of soil (11) and sediment bacteria (10) have been proposed to carry electrons over long distances. However, this hypothesis is open to question because previous studies of DNA conductivity have shown that charge transport through aromatic bases is not possible beyond nanometer distances (12, 13), even using a very large driving force (>5 V) and synthetic bases (13).As the structures and composition of the abovementioned filamentous proteins produced by soil and sediment bacteria are unknown, here we use amyloid proteins with stacked tyrosine residues as a model system to evaluate the possibility and mechanism of long-distance electron transport via aromatic residues (Figs. 1 and ​and2A).2A). Many soluble proteins aggregate into an insoluble amyloid state to form elongated, unbranched fibrils that are associated with several fatal diseases, such as Alzheimer’s, type II diabetes, and some types of cancers (14). Amyloids are attractive model systems to evaluate protein conductivity mechanisms due to their unique ability to form several distinct, highly ordered, self-replicating, and stable biomimetic structures (Fig. 1). Short peptide-based amyloids are particularly attractive because of their computationally guided, autonomous, and high-precision synthesis as well as their diverse chemical properties and biocompatibility.Open in a separate windowFig. 1.Strategy to evaluate protein conductivity mechanism using short peptides as building blocks for model systems. (A) Amino acid sequences of the short peptides used with tyrosine (yellow) and zinc (gray) highlighted. (B) Light microscopy image of microcrystals. (Scale bar, 50 µm.) (C–F) Atomic structures of microcrystals with gray arrows indicating the direction of the fibril axis. (C) X1, Zn²⁺NNQQNY (PDB ID 5K2E); (D) X2, GNNQQNY (PDB ID 5K2G); (E) X3, Zn²⁺GGVLVN (PDB ID 3PPD). (F) X4, KVQIINKKL (PDB ID 6NK4). Tyrosine edge-to-edge distances are 3.5 Å. Chemical elements are shown as follows: green, carbon; blue, nitrogen; red, oxygen; and gray, zinc.Open in a separate windowFig. 2.Intrinsic conductivity measurements of amyloids reveal micrometer-long charge transport through stacked tyrosines. (A) A schematic for measuring the intrinsic conductivity of a protein crystal (black) using four probes. (Inset) Atomic structure of X1 showing stacked tyrosines. (B, Left) AFM image of X1 crystal spanning multiple electrodes and (Right) corresponding height profile at the red dotted line. (Scale bar: B, 1 µm.) (C) Representative current–voltage (I–V) curves taken by two-probe electrical measurements. The dashed lines are linear fits to the data shown. (Inset) Zoomed I–V curve for low-conductive microcrystals. (D) Comparisons of the four-probe (black) and two-probe (gray) conductivity of the microcrystals. (Inset) Zoomed values (below X2 two-probe dashed red line) on a log scale. (E) Average resistance values of microcrystals measured using two-probe, contact, AC impedance spectroscopy (IS), and four-probe methods. Bars represent mean + SEM of multiple replicates [n (two-probe) = 19, 8, 9, 4, and n (four-probe) = 8, 9, 6 for X1, X2, X3, and X4, respectively. n (IS) = 11]. (F) Comparison of current–voltage profile for X1 measured using (Left) four- and (Right) two-probe method.In addition to their biomedical importance, amyloids are attractive biomaterials due to their high stability and capacity to reproduce themselves by seeding, enabling the development of self-repairing and replicating biomimetic materials (14). However, two major bottlenecks remain in the use of amyloids as a model system to evaluate conduction mechanisms and for the development of protein-based multifunctional biomaterials. First, like most proteins, amyloids lack electronic or optical functionality (15, 16), and in prior studies of protein conductivity, high contact resistance—either between proteins or at the protein–electrode interface—has been shown to mask the intrinsic electronic properties (17). Second, in contrast to over 100,000 structures of globular proteins, only a few dozen amyloid fibril structures are available (14), and conformational changes induced by experimental conditions hinder the elucidation of structure–function correlations.To address both these bottlenecks, here we employ a strategy to measure intrinsic conductivity of pristine amyloid microcrystals, with atomic resolution structures (18, 19), aiming to correlate structure with function by avoiding modification or tagged molecules that can induce large conformational changes (20–22). We use individual microcrystals with defined geometry to perform measurements of charge transport through stacked tyrosines, or along the fibril axis (Fig. 1). Notably, we employ a four-electrode system with ionically blocking electrodes and measure steady-state electronic current (9, 17) to avoid artifacts due to contact resistance (23, 24), polarization resistance (25), and ionic currents (23), which are known to mask the intrinsic conductivity of the protein (23, 26). Our studies show that amyloid proteins can efficiently transport charges over micrometer distances under ordinary thermal conditions without any need for redox-active metal cofactors. In contrast to single-step tunneling commonly observed in proteins, we find evidence for a hopping mechanism, previously observed experimentally only in synthetic conjugated molecular wires (27, 28), to account for long-distance conductivity.     

Structure and control of charge density waves in two-dimensional 1T-TaS2

The layered transition metal dichalcogenides host a rich collection of charge density wave phases in which both the conduction electrons and the atomic structure display translational symmetry breaking. Manipulating these complex states by purely electronic methods has been a long-sought scientific and technological goal. Here, we show how this can be achieved in 1T-TaS₂ in the 2D limit. We first demonstrate that the intrinsic properties of atomically thin flakes are preserved by encapsulation with hexagonal boron nitride in inert atmosphere. We use this facile assembly method together with transmission electron microscopy and transport measurements to probe the nature of the 2D state and show that its conductance is dominated by discommensurations. The discommensuration structure can be precisely tuned in few-layer samples by an in-plane electric current, allowing continuous electrical control over the discommensuration-melting transition in 2D.Layered 1T-TaS₂ exhibits a number of unique structural and electronic phases. At low temperature and ambient pressure, the ground state is a commensurate (C) charge density wave (CDW). On heating, it undergoes a sequence of first-order phase transitions to a nearly commensurate (NC) CDW at 225 K, to an incommensurate (IC) CDW at 355 K, and finally to a metallic phase at 545 K. Each transition involves both conduction electron and lattice degrees of freedom—large changes in electronic transport properties occur, concomitant with structural changes to the crystal. By either chemical doping or applying high pressures, it is possible to suppress the CDWs and induce superconductivity (1–3). For device applications, it is desirable to control these phases by electrical means, but this capability is difficult to achieve in bulk crystals due to the high conduction electron density. Recent efforts to produce thin samples by mechanical exfoliation provide a new avenue for manipulating the CDWs in 1T-TaS₂ (4–8). These studies have demonstrated the suppression of CDW phase transitions using polar electrolytes, as well as resistive switching between the different phases. As the material approaches the 2D limit, however, significant changes have been observed in the transport properties (4, 5, 8). However, the microscopic nature of the 2D state remains unclear. In this work, we use transmission electron microscopy (TEM) together with transport measurements to develop a systematic understanding of the CDW phases and phase transitions in ultrathin 1T-TaS₂. We find that charge ordering disappears in flakes with few atomic layers due to surface oxidation. When samples are instead environmentally protected, the CDWs persist and their transitions can be carefully tuned by electric currents.Both the atomic and CDW structure of 1T-TaS₂ can be visualized in reciprocal space by TEM electron diffraction (9, 10). In Fig. 1A, we show diffraction images taken from a bulk-like, 50-nm-thick crystal at low and room temperature (C phase, blue panel; NC phase, red panel). The bright peaks (connected by dashed lines) correspond to Bragg scattering from a triangular lattice of Ta atoms with lattice constant a = 3.36 Å. Additional weaker diffraction peaks appear from the periodic atomic displacements of the CDW. In the low-temperature C phase, Ta atoms displace to make Star-of-David clusters (blue inset, Fig. 1B). The outer 12 atoms within each star displace slightly inward toward the atom at the center, giving rise to a commensurate superstructure with wavelength λ_C = 13a that is rotated ϕ_C = 13.9° with respect to the atomic lattice. The NC phase at room temperature also consists of such 13-atom distortions. Scanning tunneling microscope (STM) measurements have revealed, however, that such ordering is only preserved in quasi-hexagonal domains consisting of tens of stars (11, 12), with domain periodicity 60–90 Å depending on temperature (13, 14). The domains are separated by a discommensuration network forming a Kagome lattice, inside of which the Ta displacements are substantially reduced (15). A schematic of this structure is shown in the red inset of Fig. 1B.Open in a separate windowFig. 1.NC-C CDW phase transition in bulk 1T-TaS₂ and CDW suppression by oxidation in thin flakes. (A) TEM diffraction images of 50-nm-thick 1T-TaS₂ at 295 K (red, NC phase) and 100 K (blue, C phase). Weaker peaks are due to CDW distortion. (B) Resistivity vs. temperature of bulk 1T-TaS₂ crystal around the first-order, NC-C transition. (Insets) Real space schematics of CDW structure. (C) (Left) TEM diffraction of few-layer 1T-TaS₂ flake shows absence of CDW order. (Right) High-resolution, cross section electron microscopy image reveals presence of amorphous oxide. (D) Free energy schematic of CDW evolution with temperature. Vertical and horizontal axis represent free energy (E) and reaction coordinate (q), respectively. NC domains grow slowly upon cooling until abrupt transition into the C phase. Energy barrier increases in 2D samples protected from oxidation.When ultrathin 1T-TaS₂ crystals (approximately <5 nm thickness) are exfoliated in an ambient air environment, the CDW structure is not observed by the TEM electron diffraction. In the left panel of Fig. 1C, we show a room temperature electron diffraction pattern taken on a few-layer flake. The presence of Bragg peaks without CDW scattering suggests that the 1T-TaS₂ layers are in a phase that is not observed in bulk crystals at this temperature. High-resolution electron microscopy and energy dispersive spectroscopy on fully suspended samples reveal a strong presence of oxidation as well as an amorphous layer on the surface (Figs. S1 and ​andS2).S2). The amorphous oxide (∼2 nm thickness) can be clearly seen atop both surfaces of the 1T-TaS₂ layers in cross section (Fig. 1C, Right). It is possible that oxidation leads to strong surface pinning, which destroys charge ordering in ultrathin samples. Recent resistivity measurements on exfoliated 1T-TaS₂ crystals have also reported the disappearance of CDWs in sufficiently thin flakes (5). It is not clear, however, whether these are intrinsic effects related to dimensionality or extrinsic consequences of oxidation.Open in a separate windowFig. S1.High-resolution STEM image of ultrathin 1T-TaS₂ prepared in air. (A) Amorphous layers appear on the top and bottom surfaces. (B) Overview of a curled sheet providing in-plane (A) and planar (C) and viewing. (C) High-resolution STEM image of the 1T-TaS₂ sheet shows the high-frequency atomic structure and a lower frequency intensity variation corresponding to the amorphous surface layers. The amorphous surfaces are more clearly visualized in D, which uses Lab Color space to create blue/yellow contrast of the amorphous (low-frequency) intensity variation.Open in a separate windowFig. S2.Chemical analysis with STEM-spectroscopy of ultrathin 1T-TaS₂ exfoliated in air. In addition to the expected presence of Ta and S, oxygen and trace amounts of carbon are present in both the (A) dispersive X-ray and (B) electron energy loss spectroscopy. The sample was suspended such that all detected elements represent chemical species present in the specimen.To prevent surface oxidation, we exfoliated 1T-TaS₂ crystals within a nitrogen-filled glove box with under 2-ppm oxygen concentration. The flakes were protected by a capping layer of thin hexagonal boron nitride (hBN) before transfer out into the ambient environment (Methods). TEM diffraction performed on these protected samples reveals that CDW formation persists down to the lowest thicknesses measured (2 nm), as we discuss in detail in Fig. 4. This finding indicates that the absence of charge order in ultrathin, uncovered flakes is most likely caused by the effects of oxidation. The study and utilization of CDWs in 2D 1T-TaS₂ thus requires careful sample preparation in inert atmosphere.Open in a separate windowFig. 4.Dimensional dependence of phase transition—electron diffraction. (A) Overlaid TEM diffraction images of ultrathin 1T-TaS₂ covered with hBN taken at 295 K (red peaks) and 100 K (blue peaks) for two flake thicknesses. hBN preserves CDW order (circled peaks) but introduces additional diffraction spots. (B) (Upper Right) Zoom-in schematic of CDW diffraction peaks showing temperature evolution. Position of NC spot can be used to estimate commensurate domain periodicity D_NC (Upper Left). (Lower) D_NC vs. temperature with cooling measured for the two covered samples compared with data reproduced from ref. 14. Reduced thickness pushes NC to C phase transition to lower temperature.The different structural phases of 1T-TaS₂ exhibit distinct electronic transport properties that may be exploited for device applications. In the main panel of Fig. 1B, we show temperature-dependent resistivity of a bulk crystal measured across the NC-C phase transition. Resistivity abruptly increases (decreases) by over an order of magnitude on entering the C (NC) phase. The hysteresis loop between cooling and warming defines the temperature region of metastability between the two phases and can be understood by a free energy picture (Fig. 1D). In a first-order transition, an activation barrier separates the stable energy minima corresponding to the NC and C states. With cooling from the NC phase, both the C state energy and the height of the barrier decrease with respect to the NC energy. When the C state has lower energy, the NC phase becomes metastable, but the system only transitions into the C phase when the activation barrier becomes comparable to the thermal energy. The situation is reversed when warming from the C phase. In oxidation-free 2D samples, this electronic transition is qualitatively unchanged.Fig. 2A shows an example of hBN-encapsulated 1T-TaS₂ flakes before (Upper) and after device fabrication (Lower). To make electrical contact to the covered samples, we used a technique of edge metallization developed for graphene/hBN heterostructures (Methods) (16). A side-view device schematic is shown in the Inset of the lower panel. In the main panel of Fig. 2B (I = 0, black curve), we plot resistance as a function of temperature for a 4-nm-thick sample measured across the NC-C phase transition. The behavior is similar to that of the bulk crystal (Fig. 1B); however, the hysteretic region between cooling and warming is substantially widened, indicating that one or both of the CDW phases become more metastable.Open in a separate windowFig. 2.Electrical control of NC-C transition in oxidation-free, 2D devices. (A) Optical images of 1T-TaS₂ flakes on a SiO₂/Si wafer covered by hBN in inert atmosphere before (Upper) and after (Lower) side electrical contact. (Inset) Side-view device schematic. (B) ac resistance vs. temperature for 4-nm-thick device as a function of dc current. Continuous current flow stabilizes NC phase at low temperature. Normalized resistance difference between cooling and warming is plotted as a function of dc current in Inset. (C) (Upper) Current vs. voltage sweep at 150 K starting in NC phase shows abrupt decreases in current and transition to the C phase. (Lower) Same at 200 K starting in C phase shows abrupt increase in current and transition to NC phase. Sweep rate is 3–6 V/min. Free energy schematics of electrically induced transitions are plotted in Insets.Metastable phases of a CDW system are generally more susceptible to electronic perturbations, because CDWs directly couple to electric field (6–8, 17). In our device, we observe that continuous current flow stabilizes the NC phase at low temperatures. In Fig. 2B (main panel), we show ac resistance with temperature while also applying a continuous, in-plane dc current, starting at room temperature (300 K). As the dc current I is increased, the final resistance at low temperature is monotonically lowered. Concomitant with this trend, the resistance jump resulting from the NC-C phase transition also decreases with increasing I. In the Inset, we plotted the ratio of the resistance difference between cooling and warming, ∆R, to resistance R in the more conducting state at T = 180 K, the temperature in the middle of the hysteresis region, as a function of the dc current level. For I = 35 μA (blue curve in main panel), the NC-C phase transition is completely absent. This measurement indicates that C phase formation in the current driven sample is very different compared with the zero-current, equilibrium condition. Current flow hinders the formation of the C phase and maintains the sample in the more conductive NC state at low temperature. We exclude Joule heating of the sample as a possible explanation by slowly turning off the current at low temperature and verifying that the resistance does not change. We also note that cooling and warming the sample again without dc current flow reproduces the original phase transitions (Fig. S3), indicating that the currents have not damaged the flake irreversibly.Open in a separate windowFig. S3.Resistance vs. temperature before and after dc current measurements. Trace for I = 0 (after) reproduces original phase transitions suppressed by large dc current.Our observation suggests that it is possible to maintain the NC phase in a temperature region where it is not thermodynamically stable. We now show that the opposite phenomenon is also possible, i.e., we can drive a transition toward the thermodynamically stable state, if we apply an in-plane current after cooling or warming the sample in equilibrium. Fig. 2C shows the current induced phase transitions in the same device (4 nm thickness). Here, we start in the NC phase at room temperature and cool the sample down to 150 K without current flow. At this temperature, although the sample remains in the NC state, the NC phase is now metastable, and the C phase is the thermodynamically stable state. As we increase the voltage across the device (upper panel, dark green curve), the measured current through the device decreases in abrupt steps (marked by red arrows) when it reaches a critical current I_c ∼ 30 μA (marked by red dashed line). On sweeping the bias current back to zero (light green curve), the device remains in a more insulating state. Warming up the sample after this point produces a temperature curve similar to the C phase, and a transition to the NC phase is observed. We have demonstrated that a bias current applied to the sample can be used to drive the metastable NC phase toward the thermodynamically preferred C state. The dashed green arrow in Fig. 2B marks the direction of this current-induced NC to C phase transition and a free energy schematic of this process is shown in the Inset of the upper panel of Fig. 2C.Similarly, the metastable C state can also be driven toward the NC phase with current. Here, we start in the C phase at 50 K and warm up to 200 K. The sample remains in the C phase, but now the NC phase is the thermodynamic ground state. As shown in the lower panel of Fig. 2C, sweeping the voltage in this case results in a sharp increase in current and drives the sample toward the more conducting NC state. We have used the dashed orange arrow in Fig. 2B and the free energy picture in the inset of the lower panel of Fig. 2C to represent this opposite C to NC transition. Interestingly, both induced transitions occur when the current reaches about I_c ∼ 30 μA, indicating that indeed current flow rather than electric field is the underlying mechanism that drives the transition. We repeated this measurement at various temperatures and initial conditions. In all cases, whenever the initial system is metastable, reaching a current threshold of 30 to 40 μA drives the system toward the thermodynamically stable state, regardless of device resistance. In contrast, we observe no induced transition up to 45 μA at 260 K, where a metastable phase ceases to exist.Taken together, the results of Fig. 2 demonstrate that it is possible to electrically control the NC-C transition in 2D 1T-TaS₂, where the temperature region of metastability is significantly enhanced. A more detailed study of this phase transition in 2D samples, however, can provide a better understanding of our experimental observations. The key structural difference between the two CDW phases is the presence of the discommensuration network in the NC phase (Fig. 1B, red inset). The NC-C transition can then be interpreted as a discommensuration-melting transition, which can be significantly affected by dimensionality (18, 19). The discommensurations have a striking effect on the electronic transport properties in 1T-TaS₂. The NC phase is an order of magnitude more conductive than the C phase. If we assume that the interior of each commensurate domain has similar transport properties as the C phase, this then implies the discommensuration regions in the NC phase are at least 10 times more conductive than the domain interior (3). Such a view is supported by the fact that the atomic structure within the discommensurations is close to the high-temperature metallic phase (15). With this interpretation, we can use transport measurements to better understand the role of dimensionality on the discommensuration-melting transition.As the number of 1T-TaS₂ layers decreases, the resistivity change corresponding to the NC-C phase transition evolves in a continuous manner down to 2 nm thickness in environmentally protected samples. Fig. 3A shows resistivity as a function of temperature for four hBN-covered 1T-TaS₂ flakes, all measured using a 1 K/min sweep rate. Their thicknesses are 2, 4, 6, and 8 nm as determined using an atomic force microscope. For comparison, we show data from an unprotected, 20-nm-thick flake, which exhibits characteristics similar to the bulk crystal, indicating that the effects of oxidation are less pronounced in thicker samples. The temperature hysteresis associated with the phase transition between cooling and warming is substantially increased in thinner samples, consistent with our earlier observations of the device in Fig. 2A. The progressive widening of the hysteresis loop continues down to the 4-nm-thick device, below which there is no longer a detectable transition. A guide to the eye for the expansion of this metastable region is shown by the colors in Fig. 3A. In the upper panel of Fig. 3B, we plot ∆T = T_c,warm – T_c,cool as a function of flake thickness, where T_c,warm and T_c,cool are the experimentally observed NC to C or C to NC transition temperature during the warming or cooling process, respectively. Here, T_c is determined by the temperature at which the first derivative peaks in the temperature sweep. ∆T is 60 K for the 20-nm flake, slightly larger than that for the bulk crystal (40 K), and grows to 120 K for the 4-nm device. In the same panel, we also plot the average temperature T_c,avg = (T_c,warm + T_c,cool)/2 of the transition. T_c,avg does not change substantially with thickness and remains between 180 and 190 K, which then implies that lower dimensionality does not stabilize either the NC or C phase. Instead, the NC (C) phase becomes increasingly metastable during cooling (warming) for thinner samples, indicating that the size of the energy barrier separating the NC and C phases increases (Fig. 1D).Open in a separate windowFig. 3.Dimensional dependence of phase transition—electron transport. (A) Thickness evolution of temperature-dependent resistivity around NC-C phase transition measured on hBN-covered ultrathin samples and 20-nm-thick flake. (B) Average transition temperature and temperature hysteresis (Upper) and normalized resistivity difference (Lower) between cooling and warming as a function of sample thickness. Open squares are corrections from contact resistance (Fig. S4). Hysteresis widens and resistivity difference decreases in thinner samples, whereas average transition temperature remains constant. Resistivity change can be used to estimate the discommensuration density 1/d at low temperature. (C) Circuit model of discommensuration network.Although ∆T increases when sample thickness is reduced, the resistivity jump associated with the phase transition decreases with decreasing thickness. In the bottom panel of Fig. 3B, we plot the resistivity difference ∆ρ between cooling and warming at T = 180 K, normalized to ρ in the more conducting state as a function of flake thickness. The closed circles are extracted directly from the data in Fig. 3A, whereas the open squares are corrections due to the effects of contact resistance (Fig. S4). For the 20-nm device, resistivity changes by an order of magnitude. The change is smaller for thinner devices and disappears completely for the 2-nm device, which indicates that more conducting NC discommensurations persist at low temperatures for thinner samples, consistent with the larger energy barriers required to remove them. Also, the resistivity jump becomes less abrupt, which is a reflection that the phase transition has slowed, as larger energy barriers generally act also to impede the kinetics of a phase transition. A simple circuit model presented in Fig. 3C allows us to connect the measured resistance jump in the NC-C transition, ∆R, with the estimated density of discommensurations 1/d left in the low temperature phase. We assume that the device resistance at low temperature is dominated by conduction through a random network of discommensuration channels (shown as white lines), which is generally sensitive to the particular microstructure formed. However, for device sizes much larger than d, we find the resistance with discommensuration channels would be R ∼ ρ_DC d, where ρ_DC is the resistivity per unit length of each discommensuration channel. Similarly, in the high temperature NC phase with a well-defined discommensuration network, we have R_NC ∼ ρ_DC D_NC, where we assume D_NC ∼ 80 Å (13, 14). From this, we can use the resistivity change in Fig. 3B to determine d: (ΔR/R_NC) ～ (d/D_NC) ? 1. On the right axis, we plotted d extracted for the different sample thicknesses. For the 2-nm sample, d ∼ D_NC, whereas it grows to 70–160 nm for the 20-nm sample.Open in a separate windowFig. S4.Extracting contact resistivity. Two- and four-terminal resistivity vs. temperature for 4-nm-thick flakes. The difference is proportional to resistivity of edge contacts.We can further substantiate the microscopic picture presented above by providing atomic structural analysis based on TEM. As before, the CDW structure is preserved by environmentally controlled hBN encapsulation. In Fig. 4A, we show diffraction images taken from two 1T-TaS₂ flakes of different thicknesses (12 and 2 nm). To highlight their temperature dependence, we have overlaid the diffraction patterns for each flake at 295 K (red peaks) and 100 K (blue peaks), our lowest achievable temperature. Ta Bragg peaks are again connected by a dashed triangle. Multiple scattering from hBN creates additional discernable peaks. The CDW peaks have been circled for easy identification. Although the peaks circled in gray appear qualitatively similar for both flakes, only the thicker flake displays additional peaks (circled in blue) at 100 K, indicating that it makes the transition to the C phase (compare with blue panel in Fig. 1A), whereas the thinner flake remains in the NC phase. This observation is consistent with our transport data as larger energy barriers in thinner samples require lower temperatures to realize the C phase.The movement of the gray-circled peaks with cooling (denoted by arrows, Fig. 4A) can be understood more quantitatively with reference to the zoom-in schematic shown in Fig. 4B (Upper Right). The position of this CDW peak is related to the periodicity D_NC of the NC domains (Upper Left) by a simple geometric expression (14): DNC=a/(2πΔϕ/360°)2+(Δλ/λC)2, where ∆ϕ is the difference in degrees between ϕ and ϕ_C = 13.9°, and ∆λ is the difference between the apparent wavelength averaged over many domains and λ_C = 13a. Thus, as the domain size grows, the NC peaks move closer to the C phase positions. We explicitly measured the position and angle of the CDW wave vectors for these two samples at several different temperatures during cooling to determine the domain period D_NC using the expression above. The results are plotted in the lower panel of Fig. 4B. For comparison, we also reproduce STM results obtained by Thomson et al. on the surface of a bulk crystal (14). For bulk samples, D_NC grows steadily from 60 to 90 Å on cooling from 340 to 215 K and then jumps to an arbitrarily large value on transition into the C phase at ∼180 K. At the same time, the width of the discommensuration regions remains relatively constant (∼22 Å) in all of the NC phase (13). As with our transport results, we find that reducing sample thickness suppresses the NC to C phase transition to lower temperatures during cooling and slows the CDW domain growth rate during the transition. For both of the thin flakes, the initial domain size at room temperature is similar to that that of the bulk crystal (D_NC = 60–70 Å). D_NC increases slightly upon cooling in the NC phase. For the 12-nm flake, the C phase is formed between 100 and 150 K, whereas the 2-nm flake remains in the NC phase even at 100 K. Its domain size here is much larger (D_NC ∼ 500 Å), however, indicating that the phase transition has begun to take place. This result is in clear contrast to bulk samples where the transition is abrupt.Our transport and TEM measurements both indicate that reduced dimensionality increases the energy barrier separating the NC and C CDW phases and thus widens the metastable region of the phase transition. The transition into the C phase involves melting or removal of the NC discommensuration network. Microscopically, energy barriers to discommensuration motion have been attributed to the presence of defects or impurities in the material, which act to pin them locally (20). Even in nominally pure CDW samples, clusters of localized defects have been observed by STM (21, 22), where the distance between defects is on the order of ∼10 nm. In bulk 1T-TaS₂, the interlayer stacking of NC domains make the discommensuration walls extended planar objects (15, 23), which are generally more difficult to pin. In two dimensions, however, the discommensurations become lines, which may be more easily immobilized. We have constructed a model of discommensuration pinning for a 2D system of thickness t (Fig. S5). We find that in the ultrathin limit where t is smaller than the mean distance between impurities, the pinning energy for a discommensuration plane scales as E_pin ∼ t^−2/3, corresponding to a cross-over from collective weak pinning to strong individual pinning. These strong pinning centers stabilize the NC discommensuration network at low temperatures during cooling and will also hinder the nucleation and growth of discommensurations when warming from the C phase, thus increasing the temperature region of metastability for both CDW phases in accordance with our experimental observation.Open in a separate windowFig. S5.Schematic picture of a DC plane and important length scales. A shows 3D view and B shows 2D projection. Red dots denote the location of impurities inside a dc plane. The effective mean impurity distance is l for t > l, whereas it is l_1D for t < l.By using this microscopic understanding of the NC-C phase transition in 2D samples, we may further elucidate the role of dc current in the measurements of Fig. 2 B and C. When the sample is cooled in equilibrium starting in the NC phase, the activation barrier between the NC and C states is continuously lowered, and therefore discommensurations are driven away and domain size grows steadily. Near the transition temperature, the small barrier can then be overcome with sufficient current flow, which depins the discommensurations to form the C phase ground state (Fig. 2C). On the other hand, when the sample is cooled out of equilibrium in the presence of a large dc current, it is likely that the domain size does not grow—the activation barrier remains large and the small-domain NC state persists on cooling to the lowest temperatures (Fig. 2B). The dc current is thus effectively a way to control the activation barrier between the NC and C phases.Although a spatially resolved study is still needed to fully understand these effects, our results have both clarified the nature of the 2D state in 1T-TaS₂ and demonstrated clear electrical control over the NC-C phase transition in ultrathin samples, further establishing the material’s relevance for device applications. We also expect our environmentally controlled techniques to be applicable for the study of other 2D transition-metal dichalcogenides that may be unstable under ambient conditions (24).     

Crystallization kinetics of atomic crystals revealed by a single-shot and single-particle X-ray diffraction experiment

Crystallization is a fundamental natural phenomenon and the ubiquitous physical process in materials science for the design of new materials. So far, experimental observations of the structural dynamics in crystallization have been mostly restricted to slow dynamics. We present here an exclusive way to explore the dynamics of crystallization in highly controlled conditions (i.e., in the absence of impurities acting as seeds of the crystallites) as it occurs in vacuum. We have measured the early formation stage of solid Xe nanoparticles nucleated in an expanding supercooled Xe jet by means of an X-ray diffraction experiment with 10-fs X-ray free-electron laser (XFEL) pulses. We found that the structure of Xe nanoparticles is not pure face-centered cubic (fcc), the expected stable phase, but a mixture of fcc and randomly stacked hexagonal close-packed (rhcp) structures. Furthermore, we identified the instantaneous coexistence of the comparably sized fcc and rhcp domains in single Xe nanoparticles. The observations are explained by the scenario of structural aging, in which the nanoparticles initially crystallize in the highly stacking-disordered rhcp phase and the structure later forms the stable fcc phase. The results are reminiscent of analogous observations in hard-sphere systems, indicating the universal role of the stacking-disordered phase in nucleation.

Crystallization is one of the most ubiquitous physical phenomena in nature, yet its microscopic and mesoscopic mechanisms are not fully understood. Crystallization is generally assumed to start from nucleation, a nanometer-sized nucleus formation, which is followed by the nucleus growth. The initially grown phase structure has been a long-standing subject of controversy. Classical nucleation theory (CNT) (1, 2) assumes that nucleation occurs at a spherical crystallite having the stable lattice structure of the bulk. More than a century ago, Ostwald (3) pointed out the role of metastable phases in nucleation and advocated his step rule, which states that a metastable phase is initially formed and that it may change to the stable phase later. Alexander and McTague (4) argued that the initial phase would be body-centered cubic regardless of the stable phase structure. Meanwhile, a recent numerical simulation of ice nucleation (5) suggests that ice nucleation occurs in a stacking-disordered phase, thereby increasing the nucleation rate by several orders of magnitude as compared with the CNT prediction. Understanding the structural dynamics upon crystallization is crucial for the creation of various new materials, as the structures and properties of the final product are crucially influenced by the kinetic pathway of the phase transition. To date, the nonequilibrium dynamics upon crystallization have been extensively studied numerically, whereas experimental observations have been mostly restricted to slow dynamics, such as crystallization of colloids (6) and proteins (7), or more recently, to the crystallization of supercooled liquids (8).The crystallization dynamics in atomic systems is challenging to investigate experimentally. First, sufficiently high temporal resolution and atomic spatial resolutions are required to resolve transient structures emerging upon crystallization. Second, orientation and ensemble averaging of the structural information can lead to significant structural detail loss in individual crystallites; nevertheless, the averaged information is helpful in identifying the phase in the early stages of crystallization. Third, growth in the presence of a substrate would influence the nucleation and growth process, potentially causing disagreement with theories. Recently, ultrashort and intense X-ray pulses from X-ray free-electron lasers (XFELs) (9, 10) became available, providing novel opportunities for the structural determination of fragile samples, such as aerosol particles (11), superfluid nanodroplets (12), and live biospecimens (13). In single-shot diffraction experiments using XFELs, instantaneous structures of single free-flying nanoparticles can be captured with snapshot exposures of femtosecond X-ray pulses. Therefore, single-shot diffraction has the potential to overcome the above-mentioned technical challenges. Although the structural determination of single small nuclei at the critical stage (typically consisting of several hundred atoms) is not achievable with the current fluence of XFEL beams, the structure of the initially grown phase can be inferred by examining larger crystals immediately after the growth.In this study, we demonstrate the uniqueness of the single-shot diffraction to conclusively elucidate the structural dynamics upon crystallization. We investigate the structure of Xe nanoparticles crystallized in a supercooled Xe gas jet by single-shot and single-particle diffraction using XFEL pulses. Rare-gas systems are suitable atomic model systems for investigating the structural dynamics upon crystallization as the atomic interactions are well approximated by the simple Lennard–Jones interaction. In addition, the absence of interaction between the sample and a supporting substrate provided us with an ideal experimental environment for the investigation of the homogeneous nucleation process. The comparison of the experimental diffraction data with numerical simulation results revealed the coexistence of the face-centered cubic (fcc) and randomly stacked hexagonal close-packed (rhcp) phases in single Xe nanoparticles. Based on our observations, we discuss the crystallization kinetics of rare-gas nanoparticles in connection with the literature on crystallization of other systems, such as hard-sphere systems and water.     

Aligned hemozoin crystals in curved clusters in malarial red blood cells revealed by nanoprobe X-ray Fe fluorescence and diffraction

The human malaria parasite Plasmodium falciparum detoxifies the heme byproduct of hemoglobin digestion in infected red blood cells by sequestration into submicron-sized hemozoin crystals. The crystal is composed of heme units interlinked to form cyclic dimers via reciprocal Fe─O (propionate) bonds. Templated hemozoin nucleation was envisaged to explain a classic observation by electron microscopy of a cluster of aligned hemozoin crystals within the parasite digestive vacuole. This dovetails with evidence that acylglycerol lipids are involved in hemozoin nucleation in vivo, and nucleation of β-hematin, the synthetic analogue of hemozoin, was consistently induced at an acylglycerol-water interface via their {100} crystal faces. In order to ascertain the nature of hemozoin nucleation in vivo, we probed the mutual orientations of hemozoin crystals in situ within RBCs using synchrotron-based X-ray nanoprobe Fe fluorescence and diffraction. The X-ray patterns indicated the presence of hemozoin clusters, each comprising several crystals aligned along their needle c axes and exposing {100} side faces to an approximately cylindrical surface, suggestive of nucleation via a common lipid layer. This experimental finding, and the associated nucleation model, are difficult to reconcile with recent reports of hemozoin formation within lipid droplets in the digestive vacuole. The diffraction results are verified by a study of the nucleation process using emerging tools of three-dimensional cellular microscopy, described in the companion paper.     

From antenna to reaction center: Pathways of ultrafast energy and charge transfer in photosystem II

The photosystem II core complex (PSII-CC) is the smallest subunit of the oxygenic photosynthetic apparatus that contains core antennas and a reaction center, which together allow for rapid energy transfer and charge separation, ultimately leading to efficient solar energy conversion. However, there is a lack of consensus on the interplay between the energy transfer and charge separation dynamics of the core complex. Here, we report the application of two-dimensional electronic-vibrational (2DEV) spectroscopy to the spinach PSII-CC at 77 K. The simultaneous temporal and spectral resolution afforded by 2DEV spectroscopy facilitates the separation and direct assignment of coexisting dynamical processes. Our results show that the dominant dynamics of the PSII-CC are distinct in different excitation energy regions. By separating the excitation regions, we are able to distinguish the intraprotein dynamics and interprotein energy transfer. Additionally, with the improved resolution, we are able to identify the key pigments involved in the pathways, allowing for a direct connection between dynamical and structural information. Specifically, we show that C505 in CP43 and the peripheral chlorophyll Chlz_D1 in the reaction center are most likely responsible for energy transfer from CP43 to the reaction center.

Photosynthesis is the process through which solar energy is converted into chemical energy (1–3). Photosystem II (PSII), a pigment–protein complex found in cyanobacteria, algae, and land plants, is the site of water splitting and is therefore crucial for photosynthetic function (4–6). It is connected with a large light-harvesting antenna system that collects solar energy and transfers the energy to the reaction center (RC), where charge separation (CS) occurs. Unlike the antenna system of purple bacteria that has a clear energy funnel, the PSII antenna system has a more complicated composition and a very complex energy landscape (4–7). These features allow for regulation that responds to rapid environmental fluctuations and protect the organisms in, for example, excess light, while maintaining highly efficient electronic energy transfer (EET) under optimal conditions (8). To understand the intricate interactions between the subunits that allow for the robustness of this photosynthetic system, the first step is to understand how the antenna system is connected to the RC. The PSII core complex (PSII-CC) is the smallest unit in which the RC is connected to the antenna proteins. It is a dimeric pigment–protein complex in which each monomer contains an RC and two core antenna proteins, namely, CP43 and CP47 (1, 7). These core antennas not only harvest solar energy but also act as the crucial bridge between the peripheral light-harvesting antenna system and the RC. Fig. 1A shows the pigment arrangement of the PSII-CC. The RC, consisting of the D1 and D2 branches, binds the following pigments: 1) two special pair chlorophyll a (P_D1 and P_D2), 2) two accessory chlorophyll a (Chl_D1 and Chl_D2), 3) two pheophytin a (Pheo_D1 and Pheo_D2), and 4) two peripheral chlorophyll a (Chlz_D1 and Chlz_D2) (9, 10). Despite the similarity between the D1 and D2 branches, CS occurs only along the D1 branch (11, 12). CP43, one of the two core antenna proteins, contains 13 chlorophyll a (Chls) and is located closer to the D1 active branch. CP47 contains 16 Chls and is located closer to the D2 branch (10). Together, these proteins provide highly effective EET and CS, which are key to the high quantum yield of CS in the RC.Open in a separate windowFig. 1.(A) Pigment arrangement of monomeric PSII-CC (whereas it is typically found as a dimer) depicted based on the cryoelectron microscopy structure (3JCU) reported by Wei et al. (10). The pigments of CP43, RC, and CP47 are shown in green, blue, and red, respectively. (B) Corresponding excitonic energy levels of monomeric PSII-CC color coded to match pigments in A (55–57). The gray shaded regions in the background represent the three groups based on similar characteristic dynamics. Note that the boundaries between the groups provide only a rough separation region as the dynamical behaviors change gradually along ω_exc. The asterisk (for the RC state) indicates an optically dark state.Despite the importance of the PSII-CC, its early time dynamics is not fully understood—specifically the competition between EET and CS (5, 7). This is largely due to the highly congested excitonic manifold (Fig. 1B) and ultrafast EET timescales, which challenge ultrafast spectroscopic techniques. Two distinct models have been put forth to try to describe the function of the PSII-CC. These two models are the “exciton/radical pair equilibrium” (ERPE) model (13–17) and the “transfer-to-trap limited” (TTTL) model (18–22). An early fluorescence decay experiment (13, 14) suggested that rapid EET allows the excitonic states to reach an equilibrium between the core antennas and the RC before CS occurs (k_EET ≫ k_CS), which is the basis for the ERPE model. This model was later supported by improved time-resolved fluorescence (15) and transient absorption experiments (16). However, a major discrepancy in this model arose with the measurement of the X-ray crystal structure of the PSII-CC (18). It was suggested that the large distances (center-to-center distance, >20 Å) between antenna and RC pigments resolved in the crystal structure would mean that ultrafast EET between the antenna proteins and the RC is unlikely. A model was then put forth that instead suggested that the EET from the core antenna to the RC is slow compared to CS (k_EET ≪ k_CS), and therefore, the EET to the trap becomes a kinetic bottleneck (18). This TTTL model was later supported by transient infrared (IR) (19) and time-resolved fluorescence experiments (20, 21) as well as structure-based simulations (22). Additionally, Kaucikas et al. (23) performed a polarized transient IR experiment on an oriented single PSII-CC crystal. The decay of the polarization-dependent signature (50–100 ps) observed in their experiment suggests that equilibration between different subunits is slow, consistent with the TTTL model. However, it has been pointed out that satisfactory fitting of the spectral evolution to this model does not necessarily imply that it is correct (24, 25), especially as others have shown that the EET dynamics cannot be adequately described by a single hopping scheme (26, 27). A recent two-dimensional electronic spectroscopy (2DES) experiment (28) with improved time resolution has also revealed the existence of ultrafast EET (<100 fs) that was not predicted by theoretical calculations. In their work, Pan et al. (28) attributed the origin of this unexpectedly fast EET pathway to polaron formation. Vibronic effects on the ultrafast EET and CS dynamics of other photosynthetic proteins have also been discussed (29–38).The lack of detailed understanding of the PSII-CC early time dynamics, in particular the EET between the core antennas and the RC, highlights the need for further experimental input with the ability to make specific assignments of the dynamical pathways. This, however, requires simultaneous high temporal and spectral resolution, which remains a challenge for ultrafast spectroscopic techniques. Here, we describe the application of two-dimensional electronic-vibrational (2DEV) spectroscopy (39–41) to the PSII-CC. The combination of both spectral dimensions provides an improved resolution that allows us to obtain much more detailed dynamical information in complex systems. The excitonic energy landscapes generated by electronic coupling in photosynthetic complexes, combined with site-dependent and charge state–dependent vibrational spectra, allow the resolution along both axes of 2DEV spectra to provide a direct connection between energetic space (via visible excitation) and physical space (via IR detection). This advantage has proven to be useful for the studies of dynamics in photosynthetic pigment–protein complexes (33, 40–45). Specifically, the resolution along the electronic excitation axis allows for the separation of the contributions from different pathways, while the resolution along the vibrational detection axis provides a way to identify the protein subunits or even specific states involved in the dynamics. As we will show, this unique feature of 2DEV spectroscopy provides insight into the complex dynamics of the PSII-CC.In the following text, we will show that the sub-100-ps dynamics of the PSII-CC extracted from spinach are highly dependent on the excitation frequency range. The resolution along the detection axis allows different dominant dynamics to be identified. In addition, we will demonstrate how 2DEV spectroscopy allows us to connect the observed dynamics to specific excitonic states. This connection allows us to obtain a more specific pigment assignment for the EET pathways and therefore provides a more detailed understanding of the finely tuned interactions between the RC and the core antennas (specifically CP43, which is closer to the active D1 branch). We will conclude with a comparison between our results and the existing models in order to provide a path forward in the understanding of this critical photosynthetic component.     

Pressure Evolution of Ultrafast Photocarrier Dynamics and Electron–Phonon Coupling in FeTe0.5Se0.5

Understanding the coupling between electrons and phonons in iron chalcogenides FeTexSe1−x has remained a critical but arduous project in recent decades. The direct observation of the electron–phonon coupling effect through electron dynamics and vibrational properties has been lacking. Here, we report the first pressure-dependent ultrafast photocarrier dynamics and Raman scattering studies on an iron chalcogenide FeTe0.5Se0.5 to explore the interaction between electrons and phonons in this unconventional superconductor. The lifetime of the excited electrons evidently decreases as the pressure increases from 0 to 2.2 GPa, and then increases with further compression. The vibrational properties of the A1g phonon mode exhibit similar behavior, with a pronounced frequency reduction appearing at approximately 2.3 GPa. The dual evidence reveals the enhanced electron–phonon coupling strength with pressure in FeTe0.5Se0.5. Our results give an insight into the role of the electron–phonon coupling effect in iron-based superconductors.     

Efficient UV-induced charge separation and recombination in an 8-oxoguanine-containing dinucleotide

During the early evolution of life, 8-oxo-7,8-dihydro-2′-deoxyguanosine (O) may have functioned as a proto-flavin capable of repairing cyclobutane pyrimidine dimers in DNA or RNA by photoinduced electron transfer using longer wavelength UVB radiation. To investigate the ability of O to act as an excited-state electron donor, a dinucleotide mimic of the FADH₂ cofactor containing O at the 5′-end and 2′-deoxyadenosine at the 3′-end was studied by femtosecond transient absorption spectroscopy in aqueous solution. Following excitation with a UV pulse, a broadband mid-IR pulse probed vibrational modes of ground-state and electronically excited molecules in the double-bond stretching region. Global analysis of time- and frequency-resolved transient absorption data coupled with ab initio quantum mechanical calculations reveal vibrational marker bands of nucleobase radical ions formed by electron transfer from O to 2′-deoxyadenosine. The quantum yield of charge separation is 0.4 at 265 nm, but decreases to 0.1 at 295 nm. Charge recombination occurs in 60 ps before the O radical cation can lose a deuteron to water. Kinetic and thermodynamic considerations strongly suggest that all nucleobases can undergo ultrafast charge separation when π-stacked in DNA or RNA. Interbase charge transfer is proposed to be a major decay pathway for UV excited states of nucleic acids of great importance for photostability as well as photoredox activity.The RNA world hypothesis suggests that ancient life originated from RNA-based oligomers due to their ability to both store genetic information and catalyze reactions in a manner similar to protein-based enzymes (1). In proteins, the 20 canonical amino acids are not versatile enough in their redox activity for many purposes, and special redox cofactors, such as the dinucleotides FADH₂ and NAD(P)H, are often recruited to facilitate a desired transformation. Similarly, facile oxidation or reduction reactions involving the canonical nucleobases could put the integrity of the genome at risk. Recent work has shown that 8-oxo-7,8-dihydroguanine (8-oxo-G), an oxidatively damaged form of guanine (G), is a redox-active base capable of photoinduced reversal of thymine dimers in DNA oligomers (2, 3). In particular, continuous irradiation of substrates containing a thymine dimer and a nearby 8-oxo-G using a UVB lamp decreases the amount of dimers over time (2, 3), but direct evidence of photoinduced electron transfer (ET) has been lacking.The ability of 8-oxo-G to act as an excited-state electron donor is plausible, but by no means assured in light of conflicting indications. On the one hand, 8-oxo-G is easier to oxidize in its electronic ground state by ∼30 kJ⋅mol^–1 compared with G (4), the most easily oxidized of the canonical bases, but this advantage may be lost for excited-state oxidation due to the lower energy of the first excited singlet state of 8-oxo-G. On the other hand, the 2′-deoxynucleoside of 8-oxo-G (8-oxo-dGuo, or O) has an excited-state lifetime of just 0.9 ± 0.1 ps at physiological pH (5)—a value that is similar to the subpicosecond lifetimes of the undamaged bases (6). Rapid nonradiative decay could thus frustrate ET despite favorable thermodynamics. Time-resolved spectroscopy can resolve this puzzle by detecting any short-lived radicals produced by photoinduced ET. Here, we use femtosecond time-resolved infrared (TRIR) spectroscopy to definitively show that UV excitation of the dinucleotide d(OA) (Fig. 1) transfers an electron from O to 2′-deoxyadenosine (A) on a subpicosecond timescale to form a contact radical ion pair (exciplex) that can be unambiguously identified by comparison with density functional theory (DFT) calculations.Open in a separate windowFig. 1.UV-visible (A) and FTIR spectra (B) for d(OA) at neutral pH. The spectra of monomeric 8-oxo-dGuo (red dashed curves) and AMP (green dotted curves) are shown for comparison. The excitation wavelengths used in the pump-probe experiments are indicated in A by arrows.The dinucleotide d(OA) was chosen as a crude mimic of FADH₂ in which 8-oxo-G replaces the dihydroflavin moiety of the cofactor that serves as the electron source for photoinitiated ET to a cyclobutane pyrimidine dimer (CPD) (7). Combining O with A offers the further advantage that the steady-state UV-visible and IR absorption spectra have several nonoverlapping transitions that arise from just one of the two chromophores (Fig. 1 and Fig. S1). These spectral characteristics permit selective excitation of O and selective detection of the localization site of an excited state via mid-IR probing of either O or A vibrations. A vibrational spectrum with its comparatively narrow resonances is frequently easier to assign than overlapping electronic absorption spectra. Consequently, TRIR spectroscopy can often differentiate between charge transfer (CT) states and other excited states that may have similar electronic absorption spectra (8, 9).Besides its interest as a mimic of a redox cofactor, the d(OA) dinucleotide also provides valuable insights into the role of CT states in DNA. As shown below, the CT state of d(OA) decays in ∼60 ps by charge recombination (CR). This decay is much longer than the excited-state lifetimes of either A or O as monomers. Similarly long-lived excited states are formed in high yields whenever DNA bases are stacked with one another both in single- and double-stranded forms (10–14). The identity of these long-lived states has been one of the most debated issues in the photophysics and photochemistry of nucleic acids during the past decade. The results from this study suggest that a primary decay channel for excited states of stacked nucleobases in DNA, whether modified or not, is ultrafast interbase ET.     

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

The flavin cofactor in photoenzyme photolyase and photoreceptor cryptochrome may exist in an oxidized state and should be converted into reduced state(s) for biological functions. Such redox changes can be efficiently achieved by photoinduced electron transfer (ET) through a series of aromatic residues in the enzyme. Here, we report our complete characterization of photoreduction dynamics of photolyase with femtosecond resolution. With various site-directed mutations, we identified all possible electron donors in the enzyme and determined their ET timescales. The excited cofactor behaves as an electron sink to draw electron flow from a series of encircling aromatic molecules in three distinct layers from the active site in the center to the protein surface. The dominant electron flow follows the conserved tryptophan triad in a hopping pathway across the layers with multiple tunneling steps. These ET dynamics occur ultrafast in less than 150 ps and are strongly coupled with local protein and solvent relaxations. The reverse electron flow from the flavin is slow and in the nanosecond range to ensure high reduction efficiency. With 12 experimentally determined elementary ET steps and 6 ET reaction pairs, the enzyme exhibits a distinct reduction–potential gradient along the same aromatic residues with favorable reorganization energies to drive a highly unidirectional electron flow toward the active-site center from the protein surface.     

Unveiling the spatial distribution of molecular coherences at conical intersections by covariance X-ray diffraction signals

The outcomes and timescales of molecular nonadiabatic dynamics are decisively impacted by the quantum coherences generated at localized molecular regions. In time-resolved X-ray diffraction imaging, these coherences create distinct signatures via inelastic photon scattering, but they are buried under much stronger background elastic features. Here, we exploit the rich dynamical information encoded in the inelastic patterns, which we reveal by frequency-dispersed covariance ultrafast powder X-ray diffraction of stochastic X-ray free-electron laser pulses. This is demonstrated for the photoisomerization of azobenzene involving the passage through a conical intersection, where the nuclear wave packet branches and explores different quantum pathways. Snapshots of the coherence dynamics are obtained at high frequency shifts, not accessible with conventional diffraction measurements. These provide access to the timing and to the confined spatial distribution of the valence electrons directly involved in the conical intersection passage. This study can be extended to full three-dimensional imaging of conical intersections with ultrafast X-ray and electron diffraction.

The transient pathways of virtually all photophysical and photochemical processes in molecules are dictated by conical intersections (CoIns). These are degeneracy regions in electronic potential energy surfaces where electrons and nuclei move on comparable timescales and become strongly coupled (1, 2). Despite CoIns being ubiquitous in molecules, accessing them experimentally has been especially challenging (3, 4). Recent advances in extreme-ultraviolet (XUV) and X-ray light sources have significantly increased the experimental capability to target different properties of these elusive nonadiabatic passages. It is now possible to record the precise timings of CoIns with attosecond transient absorption spectroscopy (5, 6) or XUV photoelectron spectroscopy (7), measure the transport of electronic coherences through CoIns (8), and distinguish between coupled nuclear and electronic dynamics with ultrafast electron diffraction (9, 10). Techniques directly accessing the quantum coherences generated at CoIns have also been put forward (11–13).Real-space imaging of the evolution of the electronic charge densities at CoIns is a long-standing dream. With (sub)femtosecond hard X-ray pulses from free-electron lasers (FELs) (14) and laser-driven plasmas (15), X-ray diffraction, a technique that provides spatial information for structure determination, has recently entered the ultrafast regime (16–21). While diffraction experiments at third-generation synchrotron sources are typically performed in crystals, high-brilliance FEL sources are now enabling ultrafast X-ray diffraction (UXD) from samples of reduced size, such as nanocrystals (22), gases of aligned molecules (23), and macromolecules (24), with prospects for single-molecule experiments (25).In UXD, most X-ray photons are scattered elastically off electronic state densities, with contributions from all electrons in the molecule. This represents a challenge when monitoring CoIns, where most core and valence electrons are inactive, and only a few valence electrons are directly involved. Key temporal and spatial information about CoIn dynamics is encoded in the additional UXD inelastic patterns (19, 26, 27). These signatures directly stem from the localized quantum coherences generated at CoIns. They are scattered off transition charge densities, which map the few electrons involved in the CoIn passage without background contributions from the remaining inactive ones. However, observing these distinctive coherence signatures in the UXD signal has not been possible thus far. Frequency dispersing signals from existing stochastic broadband FEL pulses does not allow an effective separation of elastic and inelastic scattering. As a result, the coherence contributions are buried beneath the much stronger elastic scattering off state densities.Here, by frequency dispersion of time-resolved X-ray diffraction signals, and further employing a covariance-based protocol (28–36) with state-of-the-art stochastic FEL pulses, we separate the inelastic signatures due to CoIn coherences from the dominant, less distinctive elastic features. This is exemplified for powder diffraction off randomly oriented nanocrystals of azobenzene molecules undergoing photoisomerization through a CoIn (27). The signal allows us to monitor the molecular coherences emerging during the nonadiabatic dynamics, revealing a confined distribution of transition charge densities representing small distances in the molecule. This reflects the nπ* character of the excitation, with contributing orbitals localized around the nitrogen atoms. Implemented in a crystal, covariance UXD can offer background-free, full three-dimensional reconstruction of transition charge densities at CoIns. Our approach, based on energy dispersing the diffraction signal to isolate inelastic coherence contributions, can be analogously applied to ultrafast electron diffraction (9, 10, 37).The effectiveness of the covariance UXD signal is demonstrated for the photoisomerization of azobenzene, a textbook photochemical switch with cis and trans isomers, as shown in Fig. 1A. Azobenzene can be switched selectively with high quantum yield between both geometries on a femtosecond timescale (39–41). With its derivates, azobenzene has found application in photopharmacology and optogenetics, to control the activity of pharmaceutical compounds (42) or neurons (43). Understanding the primary events of its transformation is thus of broad relevance.Open in a separate windowFig. 1.Time- and frequency-resolved CUPXD signal of azobenzene molecules. (A) CUPXD measurement. (Left) An ensemble of molecules is excited by an off-resonant X-ray pulse with variable central time T and wavevector kX. The pulse encounters azobenzene molecules undergoing photoisomerization from a cis to a trans geometry. The signal scattered along different ks directions is recorded by a pixel array detector placed far from the sample. The time- and frequency-dependent X-ray diffraction signal is obtained by varying the X-ray pulse arrival time and spectrally dispersing the signal at every pixel point. The resulting rotationally averaged CUPXD signal (Eq. 5) is shown in arbitrary units for a pulse duration of 22 fs, time delay T = 50 fs, and (Bottom Right) ωR=0 eV on the qx–qy plane or (Top Right) frequency dispersed at q=3.3 Å−1. (B) Loop diagrams (38) for UXD off a nonstationary state of azobenzene molecules with the two electronic states g (ground) and e (excited). Population and coherence contributions are shown.To monitor the evolution of the electronic charge densities σ^(r) during the CoIn passage, we assume the powder diffraction setup shown in Fig. 1A. Stochastic X-ray pulses, with variable central time T and momentum kX, are scattered off randomly oriented nanocrystals of azobenzene molecules undergoing cis → trans photoisomerization. The spontaneously emitted photons are assumed to be dispersed in momentum ks and frequency ωs (44) by an array of frequency spectrometers far from the sample. Within each nanocrystal, owing to long-range structural order, the signal arises from the interference of X-ray photons scattered at pairs of molecular sites (19). Scanning the pulse arrival time results in the momentum-, frequency-, and time-resolved X-ray diffraction signalS(q,ωs′,T)∝|ϵ^s*⋅ϵ^X|2 α3ωs4π2 ×∫dt AX(t−T) eiωs′(t−T) [σ~pop(q,t)+σ~coh(q,t)]2=|ϵ^s*⋅ϵ^X|2 α3ωs4π2 ×∫dω2π ÃX(ωs′−ω) e−iωT [σ~~pop(q,ω)+σ~~coh(q,ω)]2,[1]with the diffraction momentum transfer q=ks−kX, the frequency difference ωs′=ωs−ωX, and the fine-structure constant α (see Materials and Methods for details). Atomic units are used throughout unless otherwise stated. AX(t) and ÃX(ω)=∫dt AX(t) eiωt are the complex time and frequency envelopes of the pulse vector potential 𝒜X(r,t)=ϵ^XAX(t−T) eikX⋅r e−iωX(t−T) with central frequency ωX, while ϵ^X and ϵ^s are the polarization unit vectors of the incident and scattered X-ray photon, respectively. σ~pop and σ~coh are later defined as the population and coherence contributions to the charge density in reciprocal space (19), σ~^(q)=∫d3r σ^(r) e−iq⋅r. We assume a powder diffraction setup, where X-ray pulses scatter off an ensemble of randomly oriented crystallites of dimensions between several hundreds of nanometers and a few micrometers (15). The charge densities σ~^(q)=∫dΩq/(4π) σ~^(q) accessed by the signal are thus rotationally averaged over the momentum space solid angle Ωq, and only depend on the modulus q=|q|.The charge density contributions from populations and coherences are exemplified in Fig. 1B for the two relevant electronic states in azobenzene photoisomerization. This is highlighted by expanding the wavefunction Ψ(t)=∑ici(t) χi(t) ϕi in the basis {ϕi} of adiabatic electronic states, with time-dependent normalized nuclear wavepackets χi(t) and state amplitudes ci(t). The resulting charge densities σ~^(q,t)=Ψ(t)|σ~^(q)|Ψ(t)=σ~pop(q,t)+σ~coh(q,t), with Fourier transform σ~~pop(q,ω)+σ~~coh(q,ω), are then given by the sum of diagonal charge densities stemming from states’ populations (Fig. 1 B, i and ii),σ~pop(q,t)=∑iσ~ii(q,t)=∑i|ci(t)|2 ⟨χi(t)|σ~^ii(q)|χi(t)⟩,[2]and transition charge densities associated with the quantum coherences (Fig. 1 B, iii and iv),σ~coh(q,t)=∑i,j≠iσ~ji(q,t)=∑i,j≠icj*(t) ci(t) ⟨χj(t)|σ~^ji(q)|χi(t)⟩.[3]The modulus square in Eq. 1 results in the sum of three contributions, S(q,ωs′,T)=Spop(q,ωs′,T)+Scoh(q,ωs′,T)+Shet(q,ωs′,T), which contain σ~pop(q,t)σ~pop(q,t′), σ~coh(q,t)σ~coh(q,t′), and their interference σ~pop(q,t)σ~coh(q,t′), respectively. The interference contribution Shet(q,ωs′,T) can be viewed as a heterodyne-detected coherence term, with the populations acting as a local oscillator. This is analogous to the setup employed in ref. 45, where the scattering off unexcited molecules served as local oscillator for the dynamical signal.The advantage of directly accessing coherence dynamics without population background contributions is apparent in Fig. 2A, where we display the evolution of the charge densities in real and in momentum space. Ab initio nuclear wavepacket simulations (see Materials and Methods) of ultrafast azobenzene photoisomerization involving the CoIn passage (27), with the full inclusion of coupled nuclear and electronic degrees of freedom, were performed on the basis of accurate two-dimensional potential energy surfaces (46) containing the ground state g and the electronically excited nπ* state e. The system is prepared at t = 0 fs in the e state by a vertical excitation, and the passage through the CoIn, taking place at approximately 100 fs, moves part of the populations to the g state, thereby generating a coherence between g and e. While our two-mode effective Hamiltonian captures the reactive pathway through the CoIn, additional effects like vibrational relaxation to other modes might come into play in a multimode treatment. It was shown recently that the coherence emerging at the CoIn can be appreciably large and long lived even in full-dimensional simulations of a much larger molecule with more decoherence pathways (47). We note that the diffraction signal in Eq. 1 assumes long-range order within each randomly oriented nanocrystal (22), such that the contribution from pairs of molecular sites dominates over the single-molecule one. From the molecular perspective, our azobenzene Hamiltonian is set up for the gas phase, and isomerization within a crystal will likely be different (48). From the signal perspective, the dynamics will be analogously captured by frequency-resolved UXD.Open in a separate windowFig. 2.Photoisomerization dynamics of azobenzene. (A) (Top Left) Representative molecular structure of azobenzene at the reactive CoIn, with real-space transition charge density Re{σcoh(r,t)} at t = 190 fs; and evolution of the rotationally averaged, momentum-space charge densities, with contributions from the populations (Top Right) |σ~ee(q,t)| and (Bottom Left) |σ~gg(q,t)| (Eq. 2), and (Bottom Right) the coherence |σ~coh(q,t)| (Eq. 3), all in atomic units. The system is initially prepared in the e state, with coherences and populations in the g state emerging during the passage through the CoIn starting at approximately 100 fs. The spatial localization of the transition charge densities at the nitrogen atoms is reflected by the similar magnitude of σ~coh(q,t) at different values of q. (B) Cuts along q=3.3 Å−1 of (Left) |σ~pop(q,t)| and (Right) |σ~coh(q,t)| in A (yellow, continuous), compared to the average temporal envelope of the stochastic pulse of duration τ=22 fs and centered at T = 140 fs (blue, dashed). The pulse duration is short compared to the quasi-static populations, so that inelastic coherence scattering can be resolved in the CUPXD signal at large frequency shifts.Fig. 2 A, Top Left shows the real-space transition charge density at t = 190 fs, with a clear localization at the nitrogen atoms stemming from the nπ* character of the e state. By highlighting the role of the valence electrons directly involved in the CoIn passage, transition charge densities carry more distinctive and localized information than state charge densities, which are spread through the entire molecule with strong contributions from inactive electrons. This is reflected in the rotationally averaged, momentum-space charge densities σ~pop(q,t) and σ~coh(q,t), also shown in Fig. 2A. The population contributions are stronger at small q, in contrast to the coherences which, albeit weaker overall, have similar magnitudes at varying momentum transfers and become more prominent at large q.When an incident X-ray photon interacts with the nonstationary superposition state of Fig. 2A, it can exchange energy with the molecule and be inelastically scattered. The faster the molecular dynamics, the larger the X-ray inelastic frequency shift is. Although population terms are much stronger than coherences, their evolution is, in general, much slower (Fig. 2A), and they predominantly lead to elastic photon scattering. If the pulse provides the high spectral resolution needed to separate elastic and inelastic scattering, coherence dynamics can then be isolated. For coherent pulses, the spectral and temporal resolutions of the X-ray diffraction signal in Eq. 1 would be simultaneously determined by the pulse envelope AX(t−T) and limited by Fourier uncertainty. To effectively detect inelastic coherence scattering, the pulse should cover an adequate range of coherence dynamics within its duration τ, as illustrated in Fig. 2B. If the populations remain approximately constant within this time window, their contribution to inelastic scattering at large frequency shifts is minor, and the coherences are better resolved. However, existing FEL sources based on the self-amplified spontaneous emission (SASE) mechanism produce stochastic pulses with spiky intensity profiles. This generates ensembles of stochastic signals for each pulse realization which, upon averaging, do not possess the spectral resolution necessary to separate elastic and inelastic scattering.To overcome this obstacle with existing X-ray FEL sources, we introduce the covariance ultrafast powder X-ray diffraction (CUPXD) signalC(q,ωs1′,ωs2′,T)=⟨|ÃX(ωs1′)|2 S(q,ωs2′,T)⟩−⟨|ÃX(ωs1′)|2⟩⟨S(q,ωs2′,T)⟩,[4]obtained by averaging the product of each stochastic signal and the spectral intensity of the specific pulse generating it over independent pulse realizations. Covariance signals do not require additional measurements and can be obtained experimentally at the data processing stage. To simulate the performance of CUPXD, we model the X-ray pulse envelope AX(t)=2πf(t)u(t) as the product of a broadband stochastic function f(t) (see Materials and Methods) and a long (narrow-band) temporal gating envelope u(t)=e−t2/(2τ2)/2π of duration τ. The model employed here was shown to reproduce the stochastic temporal and spectral spikes displayed by experimental SASE FEL pulses, with the average pulse duration τ and spectral bandwidth σ independently set by u(t) and f(t), respectively (36, 49).The average signal ⟨S(q,ωs′,T)⟩ calculated with the above model does not provide any frequency information (see Materials and Methods), and cannot spectrally separate elastic and inelastic contributions. In contrast, the CUPXD signal,C(q,ωR,T)∝∫dt e−(t−T)2τ2 eiωR(t−T) [σ~pop(q,t)+σ~coh(q,t)]2=∫dω2π πτ e−(ωR−ω)2τ24 e−iωT [σ~~pop(q,ω)+σ~~coh(q,ω)]2,[5]recovers the same expressions as for a single coherent X-ray pulse, with AX(t) now replaced by the average temporal envelope |u(t)|2=e−t2/τ2/(2π), and the frequency difference ωs′ by the Raman frequency ωR=ωs2−ωs1. The average pulse duration thus acts as a control parameter providing the spectral resolution needed to separate elastic and inelastic contributions and, at the same time, the temporal resolution necessary to follow ultrafast molecular dynamics.A representative section of the CUPXD signal in azobenzene is shown in Fig. 1A for an average pulse duration of τ=22 fs; others are presented in SI Appendix, Fig. S1. To highlight the information encoded in the diffraction patterns, we display, in Fig. 3, the time- and frequency-resolved CUPXD signal for a fixed, sufficiently large value of q=3.3 Å−1. In analogy with the three components already identified for S(q,ωs′,T) in Eq. 1, the covariance signal in Eq. 5 can be recast as the sum of three contributions, C(q,ωR,T)=Cpop(q,ωR,T)+Ccoh(q,ωR,T)+Chet(q,ωR,T). The total signal C(q,ωR,T) exhibits strong elastic scattering within a narrow frequency region around ωR=0 eV, mostly determined by contributions from quasi-stationary populations. These reflect the slow evolution of σ~pop(q,t) displayed in Fig. 2B, which leads to narrow elastic spectral features in σ~~pop(q,ω). These are convolved with the broader Fourier transform e−ω2τ2/4 of the average pulse intensity, so that the spectral width of the population term Cpop(q,ωR,T) is set by 1/τ. The faster coherence dynamics σ~coh(q,t), also shown in Fig. 2B, are encoded in the inelastic scattering patterns at larger Raman frequencies ωR. These features are broader than the pulse correlation bandwidth 1/τ, and can thus be distinguished in the coherence contribution Ccoh(q,ωR,T). The heterodyne term Chet(q,ωR,T) carries signatures of the coherences, magnified by interference with the larger population contribution. However, this amplification is limited to elastic scattering within a narrow region around ωR=0 eV, due to the narrow spectral width of σ~~pop(q,ω). We note that this heterodyne term Chet(q,ωR,T) is equal to the real part of the product between ∫dt e−(t−T)2/τ2 eiωR(t−T) σ~pop(q,t) and [∫dt e−(t−T)2/τ2 eiωR(t−T) σ~coh(q,t)]*. For fixed q and T, the ωR profile of Chet(q,ωR,T) and its positive or negative sign thus encode the phase of the molecular superposition state encountered by the X-ray pulse at a given time delay.Open in a separate windowFig. 3.Temporally and spectrally resolved CUPXD signal in azobenzene. The signal C(q,ωR,T) (Eq. 5) and its three components, Cpop(q,ωR,T), Chet(q,ωR,T), and Ccoh(q,ωR,T), are displayed in arbitrary units for q=3.3 Å−1 and τ=22 fs as a function of time delay T and Raman frequency ωR. Elastic scattering off quasi-stationary populations dominates the narrow spectral region around ωR=0 eV. At higher Raman frequencies, due to the drop in Cpop(q,ωR,T), the inelastic signal allows access to the coherence dynamics with significantly reduced background.At large Raman frequencies, elastic scattering from slowly varying populations is significantly inhibited, and the signal carries distinguishable, temporally resolved coherence signatures from Chet(q,ωR,T) and Ccoh(q,ωR,T). This is highlighted by the sections of the signal displayed in Fig. 4A. Up to ωR=0.1 eV, the signal is dominated by the strong elastic scattering off populations. At the intermediate Raman frequency ωR=0.15 eV, the role of the coherences starts to emerge, magnified in Chet(q,ωR,T) by the populations acting as local oscillator. At higher Raman frequency, ωR=0.2 eV, where scattering off populations is highly suppressed, Chet(q,ωR,T) and Ccoh(q,ωR,T) become comparable, and additional information can be extracted from the signal. For these high Raman frequencies, the inelastic coherence terms are strongest around 170 and 240 fs, providing direct access to the timing of the CoIn passage, and reflecting the faster coherence dynamics—hence stronger inelastic scattering—taking place during these time intervals (see, e.g., Fig. 2B). In Fig. 4B, analogous information is obtained by integrating C(q,ωR,T) over frequency after having filtered out a central spectral region of width 2ΔωR. In an experiment, a proper choice of ΔωR will thus ensure that population contributions are removed, and the coherence dynamics can be clearly viewed.Open in a separate windowFig. 4.Coherence contributions to the CUPXD signals in azobenzene. (A) Sections of the CUPXD signal (Eq. 5) of azobenzene molecules at q=3.3 Å−1 and for selected values of the Raman frequency ωR. The total signal C(q,ωR,T) (blue, continuous) and the three contributions, Cpop(q,ωR,T) (yellow, dashed), Chet(q,ωR,T) (green, dotted), and Ccoh(q,ωR,T) (red, dot-dashed), are compared. (B) Filtered CUPXD signal at q=3.3 Å−1, obtained by integrating the CUPXD signal after having filtered out the spectral region [−ΔωR, ΔωR] of indicated width ΔωR. Filtered signals from C(q,ωR,T) and their three contributions are displayed, with the same line styles used in A. For large Raman frequencies ωR, lying outside the spectral width of elastic population terms, the population contribution Cpop(q,ωR,T) is suppressed, and the coherence contributions become clearly visible.By suppressing the elastic (population) background via frequency dispersion, the CUPXD signal offers a direct access to the evolution of the transition charge densities, with distinctive spatial information on the localized molecular events determining the CoIn passage. The elastic (ωR=0 eV) CUPXD signal is shown in Fig. 5A. At each momentum transfer q, the signal is dominated by the elastic population scattering, whereas coherence contributions, both population heterodyne and homodyne, are significantly weaker. The signal is strongest around small momentum transfers, which reflects the underlying behavior of the populations in q space (see, e.g., σ~ee(q,t) and σ~gg(q,t) in Fig. 2A) and the spread of the state densities throughout the entire molecule. In contrast, the inelastic CUPXD signal, displayed in Fig. 5B at ωR=0.2 eV, carries clear signatures of the temporal evolution of the transition charge densities. For time delays between 150 and 250 fs, additional features appear at large q, reflecting the homogeneous distribution of σ~coh(q,t) in momentum space. As already seen in Fig. 4, these attributes are most intense at intervals of faster coherence dynamics, since they lead to stronger inelastic scattering. This appears by comparing the signal in Fig. 5B and the evolution of σ~coh(q,t) in Fig. 2A.Open in a separate windowFig. 5.CUPXD signals revealing reciprocal-space and real-space information on coherence dynamics in azobenzene. (A and B) The (A) elastic (ωR=0 eV) and (B) inelastic (at ωR=0.2 eV) CUPXD signals C(q,ωR,T) (Eq. 5), along with their three components Cpop(q,ωR,T), Chet(q,ωR,T), and Ccoh(q,ωR,T), are displayed in arbitrary units as a function of time delay T and momentum transfer amplitude q. The elastic signal is dominated by population contributions at small momentum transfers. In the inelastic signal at ωR=0.2 eV, coherence signatures at large momentum transfers appear between 150 and 250 fs, as indicated by the white arrow. (C and D) Spatial information encoded in |G(r,ωR,T)| and |Gpop(r,ωR,T)| (Eq. 6) for (C) elastic and (D) inelastic scattering. G(r,ωR,T) contains contributions from the total charge density ⟨σ~^(q,t)⟩=σ~pop(q,t)+σ~coh(q,t) (Eqs. 2 and 3), while Gpop(r,ωR,T) is calculated from population-only terms. Elastic scattering is dominated by the populations, whereas coherence features appear at small values of r for inelastic scattering, as highlighted by the white arrows. This reflects the underlying coherence dynamics within confined molecular regions at the nitrogen atoms (see also Fig. 2A).After inverting the CUPXD signal from momentum to real space, the inelastic contributions offer clear insight into the spatial distribution of the transition charge densities, highlighting the valence electrons directly involved in the CoIn dynamics. Fig. 5 C and D depicts the modulus of the spatial Fourier transformG(r,ωR,T)=∫dq2π eiqr∫dt e−(t−T)2τ2 eiωR(t−T) ⟨σ~^(q,t)⟩,[6]obtained from the square root of C(q,ωR,T). For powder diffraction, the variable r conjugated to q spans the distribution of real-space distances within the molecule. For comparison, we also display Gpop(r,ωR,T), calculated by including only scattering population amplitudes in Eq. 6, with the substitution ⟨σ~^(q,t)⟩→σ~pop(q,t). Fig. 5C depicts G(r,ωR,T) for the elastic scattering case of ωR=0 eV. In this case, the role and localization of the transition charge densities do not appear, as they are hidden by the much stronger state densities, stemming from all electrons in the molecule. In contrast, by frequency dispersing G(r,ωR,T) and accessing inelastic scattering, the spatial spread of the coherences and its variation with time are apparent. For ωR=0.2 eV, Fig. 5D exhibits new features at small molecular distances r, which reflect the localization of the transition charge densities at the nitrogen atoms due to the nπ* character of the excitation (Fig. 2A). This allows direct access to the primary events involved in the CoIn passage, revealing the evolution and localized nature of the molecular coherences.To summarize, we have shown how covariance UXD of existing, stochastic FEL pulses can provide direct access to electron densities around CoIns. This is achieved by separating the inelastic and elastic scattering, and thus revealing the distinct coherence signature from the dominating population background. We model the correlation properties of existing stochastic X-ray FEL pulses generated by the SASE mechanism, and employ a covariance-based analysis to recover the joint spectral and temporal resolutions needed for CoIn detection and hidden by the stochasticity of the pulses. The coherence contribution appears more spread in q space than the populations, reflecting the localized nature of the transition charge densities at the nitrogen atoms. The CUPXD signal shows that coherence contributions originate at small distances within the molecule, but does not identify where the coherences are located. In crystals, the covariance diffraction signal will feature Bragg peaks and can yield full three-dimensional information on the localization and dynamics of the transition charge densities.While the stochastic model used here aimed at reproducing the spiky temporal and spectral profiles of SASE FEL pulses, other models could be utilized to simulate, for example, shaped X-ray pulses available at FEL sources (50). By engineering suitable stochastic X-ray pulses, it may be possible to highlight desired molecular dynamics features. A high temporal and spectral resolution could be alternatively obtained by postprocessing time-resolved diffraction snapshots. In ref. 20, the Fourier transform of the time-dependent X-ray diffraction signal was employed to separate contributions from different molecular modes. For nonadiabatic dynamics, Wigner or frequency-resolved optical gating–like spectrograms of the time-resolved diffraction signal could be used instead (12), as shown in SI Appendix, Fig. S2, requiring, however, attosecond FEL pulses (51, 52). This differs from the covariance UXD signals presented here, where the frequency information is directly obtained in the experiment through frequency dispersion. Our protocol for the separation of elastic and inelastic scattering can be straightforwardly extended to ultrafast electron diffraction (9, 10). Similar to UXD, the electron diffraction signal contains most valuable contributions from mixed elastic and inelastic scattering (37). In contrast to UXD, however, electrons are diffracted from the total (electron and nuclear) charge density in the molecule. Dispersing the electron diffraction signal as a function of energy, and thereby isolating the inelastic scattering contribution, will provide additional imaging information on CoIns.     

Dynamic determination of the functional state in photolyase and the implication for cryptochrome

The flavin adenine dinucleotide cofactor has an unusual bent configuration in photolyase and cryptochrome, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.     

Structural basis of mouse cytomegalovirus m152/gp40 interaction with RAE1γ reveals a paradigm for MHC/MHC interaction in immune evasion

Natural killer (NK) cells are activated by engagement of the NKG2D receptor with ligands on target cells stressed by infection or tumorigenesis. Several human and rodent cytomegalovirus (CMV) immunoevasins down-regulate surface expression of NKG2D ligands. The mouse CMV MHC class I (MHC-I)–like m152/gp40 glycoprotein down-regulates retinoic acid early inducible-1 (RAE1) NKG2D ligands as well as host MHC-I. Here we describe the crystal structure of an m152/RAE1γ complex and confirm the intermolecular contacts by mutagenesis. m152 interacts in a pincer-like manner with two sites on the α1 and α2 helices of RAE1 reminiscent of the NKG2D interaction with RAE1. This structure of an MHC-I–like immunoevasin/MHC-I–like ligand complex explains the binding specificity of m152 for RAE1 and allows modeling of the interaction of m152 with classical MHC-I and of related viral immunoevasins.     

Near-Infrared Femtosecond Laser Ablation of Au-Coated Ni: Effect of Organic Fluids and Water on Crater Morphology,Ablation Efficiency and Hydrodynamic Properties of NiAu Nanoparticles

Scanning electron microscopy (SEM) and profilometry of the crater morphology and ablation efficiency upon femtosecond laser ablation of Au-coated Ni targets in various fluids revealed a pronounced dependence on the ablation medium. For ethanol, a sufficient ablation efficiency was obtained, whereas for 2-butanol a higher efficiency indicated stronger laser–target interaction. Hierarchical features in the crater periphery pointed to asymmetrical energy deposition or a residual effect of the Coulomb-explosion-initiating ablation. Significant beam deviation in 2-butanol caused maximum multiple scattering at the crater bottom. The highest values of microstrain and increased grain size, obtained from Williamson–Hall plots, indicated the superposition of mechanical stress, defect formation and propagation of fatigue cracks in the crater circumference. For n-hexane, deposition of frozen droplets in the outer crater region suggested a femtosecond-laser-induced phase explosion. A maximum ablation depth occurred in water, likely due to its high cooling efficiency. Grazing incidence micro X-ray diffraction (GIXRD) of the used target showed residual carbon and partial surface oxidation. The produced nanoparticle colloids were examined by multiangle dynamic light scattering (DLS), employing larger scattering angles for higher sensitivity toward smaller nanoparticles. The smallest nanoparticles were obtained in 2-butanol and ethanol. In n-hexane, floating carbon flakes originated from femtosecond-laser-induced solvent decomposition.     

Independent initiation of primary electron transfer in the two branches of the photosystem I reaction center

Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with ∼100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P₇₀₀ (a pair of chlorophylls), and the tertiary acceptor, F_X (a Fe₄S₄ cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of F_X, it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 13¹-keto oxygen of either ec3_A (PsaA-Tyr696) or ec3_B (PsaB-Tyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2⁺ec3^- radical pair, followed by rapid reduction by P₇₀₀ in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.     

Fragile charge order in the nonsuperconducting ground state of the underdoped high-temperature superconductors

The normal state in the hole underdoped copper oxide superconductors has proven to be a source of mystery for decades. The measurement of a small Fermi surface by quantum oscillations on suppression of superconductivity by high applied magnetic fields, together with complementary spectroscopic measurements in the hole underdoped copper oxide superconductors, point to a nodal electron pocket from charge order in YBa₂Cu₃O6+δ. Here, we report quantum oscillation measurements in the closely related stoichiometric material YBa₂Cu₄O₈, which reveals similar Fermi surface properties to YBa₂Cu₃O6+δ, despite the nonobservation of charge order signatures in the same spectroscopic techniques, such as X-ray diffraction, that revealed signatures of charge order in YBa₂Cu₃O6+δ. Fermi surface reconstruction in YBa₂Cu₄O₈ is suggested to occur from magnetic field enhancement of charge order that is rendered fragile in zero magnetic fields because of its potential unconventional nature and/or its occurrence as a subsidiary to more robust underlying electronic correlations.The normal state of the underdoped copper oxide superconductors has proven to be even more perplexing than the d-wave superconducting state in these materials. At high temperatures in zero magnetic fields, the normal state of the underdoped cuprates comprises an unconventional Fermi surface of truncated “Fermi arcs” in momentum space, which is referred to as the pseudogap state (1). At low temperatures in high magnetic fields, quantum oscillations reveal the nonsuperconducting ground state in various families of underdoped hole-doped copper oxide superconductors to comprise small Fermi surface pockets (2–15). These small Fermi pockets in YBa₂Cu₃O6+δ have been identified as nodal electron pockets (2, 3, 11, 16, 17) originating from Fermi surface reconstruction associated with charge order measured by X-ray diffraction (18–20), ultrasound (21), nuclear magnetic resonance (22), and optical reflectometry (23). However, various aspects of the underlying charge order and the associated Fermi surface reconstruction remain obscure. A central question pertains to the origin of this charge order, curious features of which include a short correlation length in zero magnetic field that grows with increasing magnetic field and decreasing temperature (20). It is crucial to understand the nature of this ground-state order that is related to the high-temperature pseudogap state and delicately balanced with the superconducting ground state. Here, we shed light on the nature of this state by performing extended magnetic field, temperature, and tilt angle-resolved quantum oscillation experiments in the stoichiometric copper oxide superconductor YBa₂Cu₄O₈ (24). This material with double CuO chains has fixed oxygen stoichiometry, making it a model system to study. YBa₂Cu₄O₈ avoids disorder associated with the fractional oxygen stoichiometry in the YBa₂Cu₃O6+δ chains, which has been shown by microwave conductivity to be the dominant source of weak-limit (Born) scattering (25).Intriguingly, we find magnetic field- and angle-dependent signatures of quantum oscillations in YBa₂Cu₄O₈ (13, 14) that are very similar to those in YBa₂Cu₃O6+δ, indicating a similar nodal Fermi surface that arises from Fermi surface reconstruction by charge order with orthogonal wave vectors (16). However, the same X-ray diffraction measurements that show a Bragg peak characteristic of charge order in YBa₂Cu₃O6+δ for a range of hole dopings from 0.084≤p≤0.164 (19, 20, 26) have, thus far, not revealed a Bragg peak in the case of YBa₂Cu₄O₈ (19). We suggest that charge order enhanced by applied magnetic fields reconstructs the Fermi surface in YBa₂Cu₄O₈, whereas charge order is revealed even in zero magnetic fields in YBa₂Cu₃O6+δ because of pinning by increased disorder from oxygen vacancies.     

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations

Ultrafast structural dynamics with different spatial and temporal scales were investigated during photodissociation of carbon monoxide (CO) from iron(II)–heme in bovine myoglobin during the first 3 ps following laser excitation. We used simultaneous X-ray transient absorption (XTA) spectroscopy and X-ray transient solution scattering (XSS) at an X-ray free electron laser source with a time resolution of 80 fs. Kinetic traces at different characteristic X-ray energies were collected to give a global picture of the multistep pathway in the photodissociation of CO from heme. In order to extract the reaction coordinates along different directions of the CO departure, XTA data were collected with parallel and perpendicular relative polarizations of the laser pump and X-ray probe pulse to isolate the contributions of electronic spin state transition, bond breaking, and heme macrocycle nuclear relaxation. The time evolution of the iron K-edge X-ray absorption near edge structure (XANES) features along the two major photochemical reaction coordinates, i.e., the iron(II)–CO bond elongation and the heme macrocycle doming relaxation were modeled by time-dependent density functional theory calculations. Combined results from the experiments and computations reveal insight into interplays between the nuclear and electronic structural dynamics along the CO photodissociation trajectory. Time-resolved small-angle X-ray scattering data during the same process are also simultaneously collected, which show that the local CO dissociation causes a protein quake propagating on different spatial and temporal scales. These studies are important for understanding gas transport and protein deligation processes and shed light on the interplay of active site conformational changes and large-scale protein reorganization.

Enzymatic functions frequently involve local motions at the active site as well as large-amplitude motions of the protein, and the two are often strongly correlated. Many chemical processes at the active sites take place as a result of the interplay between atomic movement and electronic structural changes in response to external stimuli such as light, ligand binding, heat or electric field. While reaction kinetics can be predicted from thermodynamic properties, the intrinsic time scales for fundamental chemical events, such as bond breakage and formation, are often unresolved due to challenges in examining rapid electronic and atomic movements in real time. Advanced X-ray sources, especially those with intense photon bursts within the time scale of fundamental chemical events (i.e., femtoseconds), enable structural characterization in terms of the electronic and atomic motions. Combining such ultrashort X-ray pulses with laser excitation, we are able to detect the interplay of ultrafast electron and nuclear motions in the photodissociation of an axial CO ligand from the iron center in the heme site of myoglobin (Mb) (Fig. 1). The same process has been extensively studied due to numerous functions of heme or other iron porphyrins in hemoproteins, including electron transfer, catalytic oxidation or reduction of metabolites, neutralization of damaging reactive species, and famously the binding of diatomics such as dioxygen, carbon monoxide, and nitric oxide for transportation and sensing (1–6). Because the dissociation of diatomic ligands, such as CO and NO, can be synchronized through optical excitation of the porphyrin, diatomic ligand binding in hemoproteins is amenable to scrutiny by dynamic structural and electronic spectroscopies (1, 7–14). Several X-ray diffraction, solution scattering, and X-ray spectroscopy (including X-ray absorption and emission) studies have been carried out using intense X-ray pulses from synchrotron and X-ray free electron laser sources (11, 14–19). In this report, we focus on the correlations between the electronic structural change of the iron center and these nuclear motions. To investigate these correlations, we used X-ray transient absorption spectroscopy/scattering and theoretical calculations to detect and project detailed trajectories for the CO departure from Fe(II) in the heme site of bovine Mb.Open in a separate windowFig. 1.(A) Mb structure with heme in pink surrounded by helices of the protein. (B) Mb active site structural changes following CO photolysis. Upon photoexcitation, ground state MbCO (green) loses its bond to CO and adopts a square pyramidal structure with His93 (pink), resulting in the doming of the porphyrin where the Fe (red) comes out of the plane of the macrocycle. Structures are from photolysed MbCO trapped at low temperature (12) and ground state (9) MbCO, where their crystal structures are aligned by their respective porphyrin carbons.In carbonmonoxymyoglobin (MbCO), the low-spin (LS) Fe(II) center has a pseudo-octahedral coordination geometry, ligated with four nitrogens (N_p) from the heme, the nitrogen of an axial histidine (N_His, His93), and CO, a strong field ligand. Previous studies have pointed out that upon excitation of the heme Soret or Q band, photolysis occurs within ∼50 fs, although there is an ongoing debate about the mechanism of CO photodissociation and the subsequent relaxation of the heme, as well as the possible role of intermediate spin states, similar to those observed in photoexcited iron Tris(bipyridine) (20) and ferrous cytochrome c (14, 15, 21). With the loss of CO, the LS state of Fe(II) transforms to a high-spin (HS) state and adopts square-pyramidal pentacoordination with the axial histidine His93 moving ∼0.3 Å out of the porphyrin plane, perturbing the position of the alpha helix in which it sits (Fig. 1B) (4, 8, 22, 23). Protein control of this movement is critical, both because it is the first step of the mechanism of cooperativity in hemoglobin O₂ binding and because it may lead to a conformational rearrangement of the heme pocket that allows CO to escape and avoid geminate recombination (24).This CO photodissociation from MbCO, as well as the photolysis of other diatomics such as NO, and the subsequent recombination dynamics have been assessed using X-ray transient absorption (XTA) spectroscopy (Fig. 2) at synchrotron sources with ∼100-ps time resolution (19, 25), using Fe K-edge X-ray absorption near edge structure (XANES) spectral features shown in Fig. 2. The main differences between the spectral features of MbCO and Mb are an edge shift to a lower energy and a preedge conversion from two sharp peaks to one broader and weaker peak. These changes are consistent with loss of CO and a conversion of Fe(II) from LS to HS in Mb, as supported by optical and vibrational spectroscopic studies (8, 26).Open in a separate windowFig. 2.Fe K-edge XANES and difference spectra measured after CO photodissociation with 100 ps time resolution (19). Energies selected for measurement of polarization-dependent dynamics are marked in dashed vertical cyan lines: line 1, 7.112 keV, the depletion of the ground state transition of 1s → 3d_z2, 3d_x2-y2 character; line 2, 7.115 keV, the disappearance of the preedge peak associated with the CO back bonding antibonding orbital; line 3, 7.118 keV, the rising edge shoulder that appears in MbFe(II); line 4, 7.123 keV, the edge shift; and line 5, 7.172 keV, an EXAFS energy where changes are purely based on changes in the local geometry.While heme vibrational cooling was observed by time-resolved Raman techniques on the time scales of a few to tens of ps (26, 27), and optical transient absorption spectroscopy shows the development of broad excited state absorption features with lifetimes of ∼300 fs and 3 ps, there is an active discussion in the literature as to whether these features can be assigned to an excited-state evolution through a series of electronic intermediate states/species (28–31) or to an exclusively vibrational relaxation pathway (31–33). Because Fe K-edge XTA is sensitive to both the heme iron electronic configuration and the local structural geometry during heme relaxation, measurements of the XANES should distinguish between these mechanisms but only if very fast time scales are resolvable. In this regard, XTA at Linac Coherent Light Source (LCLS) provides a rare opportunity to investigate these relaxation processes with a technique with both high temporal and structural resolution.Although the photodissociation of CO from heme and the concomitant LS to HS transition and heme doming motion are well-known phenomena, many fundamental transformations in terms of electronic and nuclear motions that result in the CO departure are not well understood. The recent works on cytochtome c and NO-bound myoglobin have made progress in the timing of the spin state transitions on the femtosecond time scale and the identification of the intermediate spin state (14, 15). However, it is far from clear how the spin state change was induced electronically, how the iron spin state is related to the Fe–CO distance, and when the heme doming takes place as the Fe–CO distance elongates in dissociation processes. Understanding these correlations has important implications for other chemical and enzymatic processes involving ligand dissociation. It is therefore of great interest to link dynamic structural and electronic changes at the heme during ligand dissociation to more large-scale conformational changes, especially on the time scale of ligand departure and heme doming, which is expected to take place on tens of femtoseconds to a few picoseconds time scales.In order to address the dynamic interplay between electronic and nuclear motions that are beyond the Born–Oppenheimer approximation, we carried out combined XTA and X-ray transient scattering measurements during CO photodissociation with sub–100-fs time resolution at the X-ray Pump-Probe (XPP) facility of the LCLS. The kinetics of the spin state transition and the nuclear motion associated with doming, as well as the global motions of the protein matrix, have revealed a series of correlated events as CO departs from the heme. Although the exact trajectory of CO departure in terms of Fe–CO distance and other structural parameters is still difficult to resolve, such processes can be simulated via quantum mechanical calculations to model this process for the Fe(II) center and the ligands directly bound to the metal. The results provide the energetics of different excited states as well as their trajectories as functions of local structural changes, such as Fe–CO distance and heme relaxation, to distinguish the effect of different structural factors on the overall structural dynamics. These calculations also allow us to predict XTA features without and with the structural relaxation of the heme, providing insight into the interplays between the electronic spin state/configuration and corresponding nuclear motions as a function of the Fe–CO distance.     

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease–related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme b_H of cytochrome bc₁, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme b_H caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

Hemes are common redox-active cofactors in biological electron transfer systems. Their major function is to transfer electrons within the cofactor chains or as part of the catalytic sites. The direction and rate of electron transfer are secured by the specific properties of hemes, among which the redox midpoint potential and the spin state are considered to be of crucial importance. Several studies have been carried out to understand how specific molecular elements contribute in adjusting the redox potential values to the levels required for efficient electron transfer rate (1–5). These studies recognized the importance of the heme-iron ligands, the degree of exposure of the heme to the solvent, and specific interactions, including hydrogen bonding, within the amino acids of heme-binding pocket. However, the overall structural complexity of proteins often makes an experimental extraction of individual elements of control extremely challenging, in particular those associated within the hydrogen bonding networks. It is indeed remarkable that the fluctuation of the hydrogen bond network was identified as the factor modulating the efficiency of long-range biological electron transfer (6, 7).This work focused on exploring the hydrogen bond-related elements of control of the redox properties of one of the hemes of cytochrome b subunit of cytochrome bc₁ (mitochondrial complex III). Cytochrome b is the only subunit of this complex encoded by mitochondrial DNA, thus is a subject of high susceptibility for spontaneous mutations in comparison to other subunits (encoded by nuclear DNA). Such mutations often lead to systemic disorders, mitochondrial diseases, manifesting in symptoms such as exercise intolerance, myopathies, or neuropathies. On the other hand, their mimic in the bacterial or yeast model systems not only provides insights into the possible molecular basis of the disease but also reveal molecular aspects of protein design (8, 9). With this in mind, we targeted a mutation G34S (human cytochrome b numbering), found in one 52-y-old female patient suffering from exercise intolerance (10). The mutation was present in mitochondria from muscle tissue and caused mild myopathy, lactic acidosis, and defect in mitochondrial complex III activity. The mutated glycine 34 is located on transmembrane helix A of cytochrome b subunit in close distance (∼8 Å) to heme b_H (SI Appendix, Fig. S1 A and B). This location suggests the possible effect of mutation on the properties of heme cofactor, in particular if one considers that a change of small and nonpolar glycine to a larger and polar serine may affect the local hydrogen bonding environment.Indeed, the presence of glycine at position 34 was previously noticed as important for heme packing in the cytochrome b subunit (11). Interestingly, the importance of G34 was emphasized by the remarkable evolutionary conservation in the equivalents of cytochrome bc₁ complex in distant organisms, such as bacteria, insects, fish, mammals and even plants (12). In a bacterial model (Rhodobacter sphaeroides cytochrome bc₁), the equivalent G48 residue was mutated to valine and aspartic acid, both rendering the bacteria photosynthetically incompetent (13). The equivalent of this mutation was also studied in Saccharomyces cerevisiae (8). Yeast with G33S (S. cerevisiae numbering) mutation were not able to grow aerobically, whereas the isolated bc₁ complex had lower enzymatic activity and disrupted subunit composition (lower level of the iron-sulfur protein assessed by Western blotting and lower cytochrome b content, measured optically to 55% of wild type [WT]). The same G33 position was found to be mutated to aspartic acid in a respiratory-deficient yeast (14). Among the 85 tested G33D revertants, 82 were D33G and 3 were D33A, suggesting that only a small amino acid without a charge can be tolerated at this position.Given the implicated importance of glycine at 34 position of mitochondrial cytochrome b, particularly in the context of hydrogen bonding network within the heme-binding pocket, we combined the experimental and computational methods to investigate the effects of introducing serine to the homologous position in purple photosynthetic bacterium Rhodobacter capsulatus (mutant G48S). Notably, the bacterial cytochrome b shares reasonable sequence similarity with human and yeast mitochondrial cytochrome b, close to 57%. We found that G48S perturbs the redox properties of heme b_H and causes structural and hydration changes in the vicinity of heme.The mutation also severely affects the spectral properties of heme b_H, which are best explained by a model assuming a reversible change from low to high spin state when the heme is oxidized. The latter comes as a rather unusual and unexpected molecular effect, considering all so-far reported effects of disease-related mitochondrial mutations (8, 9, 15). At the same time, it offers interesting insights into the role of hydrogen bonding and protein packing in maintaining the low spin state of the oxidized heme fostering electron-transfer relay function.     

肝素对腹膜透析大鼠腹膜电荷屏障及蛋白质丢失的影响

目的:观察尿毒症及腹膜透析(PD)两种状态对对大鼠腹膜电荷屏障的影响,研究电荷屏障与PD液白蛋白丢失的相关性,探讨肝素对PD大鼠腹膜电荷屏障与PD液白蛋白丢失的作用. 方法:选取SD大鼠40只,随机分为对照组(假手术组,n=10)、尿毒症组(5/6肾切除,n=10)、PD组(n=10)和肝素组(n=10);所有大鼠PD 4周后收集血清及PD液,采用清除法测定胰淀粉酶清除率(Cpam)及唾液淀粉酶的清除率(Csam)的比值(Cpam/Csam)用以评价腹膜电荷屏障,同时测定PD液白蛋白的丢失量. 结果:(1)对照组、尿毒症组、PD组及肝素组的腹膜Cpam/Csam结果分别为1.91 ±0.89,2.32 ±0.74、3.11 ±0.76和2.24±0.59.尿毒症组、PD组与对照组比较均有显著差异(P均＜0.01),但尿毒症组和PD组间无显著差异;肝素组与PD组比较也有显著差异(P =0.008).(2)尿毒症组、PD组及肝素组PD液白蛋白量显著高于对照组(41.3 ±8.34 mg,49.2 ±3.61 mg、39.9±3.73 mg vs 27.1±5.66 mg,P均＜0.01);同时,PD组还显著高于尿毒症组(P=0.04),肝素组与PD组相比,亦有显著差异(P=0.001).(3)所有大鼠Cpam/Csam与PD液白蛋白丢失量显著相关(r=-0.469,P=0.002).结论:尿毒症会损害腹膜的电荷特性,PD会进一步损害电荷屏障,增加PD液白蛋白的丢失.PD液中增加肝素可改善电荷屏障,减少白蛋白丢失.