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.

Ultrafast Excited State Relaxation of a Metalloporphyrin Revealed by Femtosecond X-ray Absorption Spectroscopy.

Photoexcited Nickel(II) tetramesitylporphyrin (NiTMP), like many open-shell metalloporphyrins, relaxes rapidly through multiple electronic states following an initial porphyrin-based excitation, some involving metal centered electronic configuration changes that could be harnessed catalytically before excited state relaxation. While a NiTMP excited state present at 100 ps was previously identified by X-ray transient absorption (XTA) spectroscopy at a synchrotron source as a relaxed (d,d) state, the lowest energy excited state (J. Am. Chem. Soc., 2007, 129, 9616 and Chem. Sci., 2010, 1, 642), structural dynamics before thermalization were not resolved due to the ∼100 ps duration of the available X-ray probe pulse. Using the femtosecond (fs) X-ray pulses of the Linac Coherent Light Source (LCLS), the Ni center electronic configuration from the initial excited state to the relaxed (d,d) state has been obtained via ultrafast Ni K-edge XANES (X-ray absorption near edge structure) on a time scale from hundreds of femtoseconds to 100 ps. This enabled the identification of a short-lived Ni(I) species aided by time-dependent density functional theory (TDDFT) methods. Computed electronic and nuclear structure for critical excited electronic states in the relaxation pathway characterize the dependence of the complex's geometry on the electron occupation of the 3d orbitals. Calculated XANES transitions for these excited states assign a short-lived transient signal to the spectroscopic signature of the Ni(I) species, resulting from intramolecular charge transfer on a time scale that has eluded previous synchrotron studies. These combined results enable us to examine the excited state structural dynamics of NiTMP prior to thermal relaxation and to capture intermediates of potential photocatalytic significance.

Imaging ultrafast excited state pathways in transition metal complexes by X-ray transient absorption and scattering using X-ray free electron laser source.

This report will describe our recent studies of transition metal complex structural dynamics on the fs and ps time scales using an X-ray free electron laser source, Linac Coherent Light Source (LCLS). Ultrafast XANES spectra at the Ni K-edge of nickel(ii) tetramesitylporphyrin (NiTMP) were measured for optically excited states at a timescale from 100 fs to 50 ps, providing insight into its sub-ps electronic and structural relaxation processes. Importantly, a transient reduced state Ni(i) (π, 3dx2-y2) electronic state is captured through the interpretation of a short-lived excited state absorption on the low-energy shoulder of the edge, which is aided by the computation of X-ray transitions for postulated excited electronic states. The observed and computed inner shell to valence orbital transition energies demonstrate and quantify the influence of the electronic configuration on specific metal orbital energies. A strong influence of the valence orbital occupation on the inner shell orbital energies indicates that one should not use the transition energy from 1s to other orbitals to draw conclusions about the d-orbital energies. For photocatalysis, a transient electronic configuration could influence d-orbital energies up to a few eV and any attempt to steer the reaction pathway should account for this to ensure that external energies can be used optimally in driving desirable processes. NiTMP structural evolution and the influence of the porphyrin macrocycle conformation on relaxation kinetics can be likewise inferred from this study.

Butterfly Deformation Modes in a Photoexcited Pyrazolate-Bridged Pt Complex Measured by Time-Resolved X-Ray Scattering in Solution.

Pyrazolate-bridged dinuclear Pt(II) complexes represent a series of molecules with tunable absorption and emission properties that can be directly modulated by structural factors, such as the Pt-Pt distance. However, direct experimental information regarding the structure of the emissive triplet excited state has remained scarce. Using time-resolved wide-angle X-ray scattering (WAXS), the excited triplet state molecular structure of [Pt(ppy)(µ-t-Bu2pz)]2 (ppy = 2-phenylpyridine; t-Bu2pz = 3,5-di-tert-butylpyrazolate), complex 1, was obtained in a dilute (0.5 mM) toluene solution utilizing the monochromatic X-ray pulses at Beamline 11IDD of the Advanced Photon Source. The excited-state structural analysis of 1 was performed based on the results from both transient WAXS measurements and density functional theory calculations to shed light on the primary structural changes in its triplet metal-metal-to-ligand charge-transfer (MMLCT) state, in particular, the Pt-Pt distance and ligand rotation. We found a pronounced Pt-Pt distance contraction accompanied by rotational motions of ppy ligands toward one another in the MMLCT state of 1. Our results suggest that the contraction is larger than what has previously been reported, but they are in good agreement with recent theoretical efforts and suggest the ppy moieties as targets for rational synthesis aimed at tuning the excited-state structure and properties.

Electron Injection from Copper Diimine Sensitizers into TiO2: Structural Effects and Their Implications for Solar Energy Conversion Devices.

Copper(I) diimine complexes have emerged as low cost replacements for ruthenium complexes as light sensitizers and electron donors, but their shorter metal-to-ligand-charge-transfer (MLCT) states lifetimes and lability of transient Cu(II) species impede their intended functions. Two carboxylated Cu(I) bis-2,9-diphenylphenanthroline (dpp) complexes [Cu(I)(dpp-O(CH2CH2O)5)(dpp-(COOH)2)](+) and [Cu(I)(dpp-O(CH2CH2O)5)(dpp-(Φ-COOH)2)](+) (Φ = tolyl) with different linker lengths were synthesized in which the MLCT-state solvent quenching pathways are effectively blocked, the lifetime of the singlet MLCT state is prolonged, and the transient Cu(II) ligands are stabilized. Aiming at understanding the mechanisms of structural influence to the interfacial charge transfer in the dye-sensitized solar cell mimics, electronic and geometric structures as well as dynamics for the MLCT state of these complexes and their hybrid with TiO2 nanoparticles were investigated using optical transient spectroscopy, X-ray transient absorption spectroscopy, time-dependent density functional theory, and quantum dynamics simulations. The combined results show that these complexes exhibit strong absorption throughout the visible spectrum due to the severely flattened ground state, and a long-lived charge-separated Cu(II) has been achieved via ultrafast electron injection (<300 fs) from the (1)MLCT state into TiO2 nanoparticles. The results also indicate that the TiO2-phen distance in these systems does not have significant effect on the efficiency of the interfacial electron-transfer process. The mechanisms for electron transfer in these systems are discussed and used to develop new strategies in optimizing copper(I) diimine complexes in solar energy conversion devices.

Cobaloxime-based artificial hydrogenases.

Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions.

Ultrafast structural dynamics of Cu(I)-bicinchoninic acid and their implications for solar energy applications.

In this study, ultrafast optical transient absorption and X-ray transient absorption (XTA) spectroscopy are used to probe the excited-state dynamics and structural evolution of copper(I) bicinchoninic acid ([Cu(I)(BCA)2](+)), which has similar but less frequently studied biquinoline-based ligands compared to phenanthroline-based complexes. The optical transient absorption measurements performed on the complex in a series of polar protic solvents demonstrate a strong solvent dependency for the excited lifetime, which ranges from approximately 40 ps in water to over 300 ps in 2-methoxyethanol. The XTA experiments showed a reduction of the prominent 1s → 4pz edge peak in the excited-state X-ray absorption near-edge structure (XANES) spectrum, which is indicative of an interaction with a fifth ligand, most likely the solvent. Analysis of the extended X-ray absorption fine structure (EXAFS) spectrum shows a shortening of the metal-ligand bond in the excited state and an increase in the coordination number for the Cu(II) metal center. A flattened structure is supported by DFT calculations that show that the system relaxes into a flattened geometry with a lowest-energy triplet state that has a dipole-forbidden transition to the ground state. While the short excited-state lifetime relative to previously studied Cu(I) diimine complexes could be attributed to this dark triplet state, the strong solvent dependency and the reduction of the 1s → 4pz peak in the XTA data suggest that solvent interaction could also play a role. This detailed study of the dynamics in different solvents provides guidance for modulating excited-state pathways and lifetimes through structural factors such as solvent accessibility to fulfill the excited-state property requirements for efficient light harvesting and electron injection.

Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers.

Serial crystallography at large facilities, such as x-ray free-electron lasers and synchrotrons, evolved as a powerful method for the high-resolution structural investigation of proteins that are critical for human health, thus advancing drug discovery and novel therapies. However, a critical barrier to successful serial crystallography experiments lies in the efficient handling of the protein microcrystals and solutions at microscales. Microfluidics are the obvious approach for any high-throughput, nano-to-microliter sample handling, that also requires design flexibility and rapid prototyping to deal with the variable shapes, sizes, and density of crystals. Here, we discuss recent advances in polymer 3D printing for microfluidics-based serial crystallography research and present a demonstration of emerging, large-scale, nano-3D printing approaches leading into the future of 3D sample environment and delivery device fabrication from liquid jet gas-dynamic virtual nozzles devices to fixed-target sample environment technology.

All polymer microfluidic chips-A fixed target sample delivery workhorse for serial crystallography.

The development of x-ray free electron laser (XFEL) light sources and serial crystallography methodologies has led to a revolution in protein crystallography, enabling the determination of previously unobtainable protein structures and near-atomic resolution of otherwise poorly diffracting protein crystals. However, to utilize XFEL sources efficiently demands the continuous, rapid delivery of a large number of difficult-to-handle microcrystals to the x-ray beam. A recently developed fixed-target system, in which crystals of interest are enclosed within a sample holder, which is rastered through the x-ray beam, is discussed in detail in this Perspective. The fixed target is easy to use, maintains sample hydration, and can be readily modified to allow a broad range of sample types and different beamline requirements. Recent innovations demonstrate the potential of such microfluidic-based fixed targets to be an all-around "workhorse" for serial crystallography measurements. This Perspective will summarize recent advancements in microfluidic fixed targets for serial crystallography, examine needs for future development, and guide users in designing, choosing, and utilizing a fixed-target sample delivery device for their system.

A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Co-flow injection for serial crystallography at X-ray free-electron lasers.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

Plug-and-play polymer microfluidic chips for hydrated, room temperature, fixed-target serial crystallography.

The practice of serial X-ray crystallography (SX) depends on efficient, continuous delivery of hydrated protein crystals while minimizing background scattering. Of the two major types of sample delivery devices, fixed-target devices offer several advantages over widely adopted jet injectors, including: lower sample consumption, clog-free delivery, and the ability to control on-chip crystal density to improve hit rates. Here we present our development of versatile, inexpensive, and robust polymer microfluidic chips for routine and reliable room temperature serial measurements at both synchrotrons and X-ray free electron lasers (XFELs). Our design includes highly X-ray-transparent enclosing thin film layers tuned to minimize scatter background, adaptable sample flow layers tuned to match crystal size, and a large sample area compatible with both raster scanning and rotation based serial data collection. The optically transparent chips can be used both for in situ protein crystallization (to eliminate crystal handling) or crystal slurry loading, with prepared samples stable for weeks in a humidified environment and for several hours in ambient conditions. Serial oscillation crystallography, using a multi-crystal rotational data collection approach, at a microfocus synchrotron beamline (SSRL, beamline 12-1) was used to benchmark the performance of the chips. High-resolution structures (1.3-2.7 Å) were collected from five different proteins - hen egg white lysozyme, thaumatin, bovine liver catalase, concanavalin-A (type VI), and SARS-CoV-2 nonstructural protein NSP5. Overall, our modular fabrication approach enables precise control over the cross-section of materials in the X-ray beam path and facilitates chip adaption to different sample and beamline requirements for user-friendly, straightforward diffraction measurements at room temperature.

Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins.

Membranes proteins make up more than 60% of current drug targets and account for approximately 30% or more of the cellular proteome. Access to this important class of proteins has been difficult due to their inherent insolubility and tendency to aggregate in aqueous solutions. Understanding membrane protein structure and function demands novel means of membrane protein production that preserve both their native conformational state as well as function. Over the last decade, cell-free expression systems have emerged as an important complement to cell-based expression of membrane proteins due to their simple and customizable experimental parameters. One approach to overcome the solubility and stability limitations of purified membrane proteins is to support them in stable, native-like states within nanolipoprotein particles (NLPs), aka nanodiscs. This has become common practice to facilitate biochemical and biophysical characterization of proteins of interest. NLP technology can be easily coupled with cell-free systems to achieve functional membrane protein production for this purpose. Our approach involves utilizing cell-free expression systems in the presence of NLPs or using co-translation techniques to perform one-pot expression and self-assembly of membrane protein/NLP complexes. We describe how cell-free reactions can be modified to render control over nanoparticle size and monodispersity in support of membrane protein production. These modifications have been exploited to facilitate co-expression of full-length functional membrane proteins such as G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In particular, we summarize the state of the art in NLP-assisted cell-free coexpression of these important classes of membrane proteins as well as evaluate the advances in and prospects for this technology that will drive drug discovery against these targets. We conclude with a prospective on the use of NLPs to produce as well as deliver functional mammalian membrane-bound proteins for a range of applications.

The Nature of the Long-Lived Excited State in a NiII Phthalocyanine Complex Investigated by X-Ray Transient Absorption Spectroscopy.

The nature of the photoexcited state of octabutoxy nickel(II) phthalocyanine (NiPcOBu8 ) with a 500 ps lifetime was investigated by X-ray transient absorption (XTA) spectroscopy. Previous optical, vibrational, and computational studies have suggested that this photoexcited state has a ligand-to-metal charge transfer (LMCT) nature. By using XTA, which provides unambiguous information on the local electronic and nuclear configuration around the Ni center, the nature of the excited state of NiPcOBu8 was reassessed. Using X-ray probe pulses from a synchrotron source, the ground- and excited-state X-ray absorption spectra of NiPcOBu8 were measured. Based on the results, we identified that the excited state exhibits spectral features that are characteristic of a Ni1, 3 (3dz2 ,3dx2-y2 ) state rather than a LMCT state with a transiently reduced Ni center. This state resembles the (d,d) state of nickel(II) tetramesitylphorphyrin. The XTA features are rationalized based on the inherent cavity sizes of the macrocycles. These results may provide useful guidance for the design of photocatalysts in the future.

Synthesis, structure, ultrafast kinetics, and light-induced dynamics of CuHETPHEN chromophores.

Five heteroleptic Cu(i)bis(phenanthroline) chromophores with distinct variation in the steric bulk at the 2,9-phenanthroline position were synthesized using the HETPHEN method, and their ground and excited state properties are described. Analysis of the crystal structures reveals a significant distortion from tetrahedral geometry around the Cu(i) centre which is attributed to favourable aromatic interactions between the two phenanthroline ligands. Ultrafast and nanosecond transient optical spectroscopies reveal that the excited state lifetime can be tuned across two orders of magnitude up to 74 nanoseconds in acetonitrile by changing the 2,9-substituent from hydrogen to sec-butyl. X-ray transient absorption spectroscopy at the Cu K-edge confirmed Cu(i) oxidation to Cu(ii) and revealed a decrease of the Cu-N bond lengths in the excited state. The ground and excited state characterization presented here will guide the integration of CuHETPHEN chromophores into complex electron donor-acceptor architectures.

Removal of Ca(2+) from the Oxygen-Evolving Complex in Photosystem II Has Minimal Effect on the Mn4O5 Core Structure: A Polarized Mn X-ray Absorption Spectroscopy Study.

Ca(2+)-depleted and Ca(2+)-reconstituted spinach photosystem II was studied using polarized X-ray absorption spectroscopy of oriented PS II preparations to investigate the structural and functional role of the Ca(2+) ion in the Mn4O5Ca cluster of the oxygen-evolving complex (OEC). Samples were prepared by low pH/citrate treatment as one-dimensionally ordered membrane layers and poised in the Ca(2+)-depleted S1 (S1') and S2 (S2') states, the S2'YZ(•) state, at which point the catalytic cycle of water oxidation is inhibited, and the Ca(2+)-reconstituted S1 state. Polarized Mn K-edge XANES and EXAFS spectra exhibit pronounced dichroism. Polarized EXAFS data of all states of Ca(2+)-depleted PS II investigated show only minor changes in distances and orientations of the Mn-Mn vectors compared to the Ca(2+)-containing OEC, which may be attributed to some loss of rigidity of the core structure. Thus, removal of the Ca(2+) ion does not lead to fundamental distortion or rearrangement of the tetranuclear Mn cluster, which indicates that the Ca(2+) ion in the OEC is not critical for structural maintenance of the cluster, at least in the S1 and S2 states, but fulfills a crucial catalytic function in the mechanism of the water oxidation reaction. On the basis of this structural information, reasons for the inhibitory effect of Ca(2+) removal are discussed, attributing to the Ca(2+) ion a fundamental role in organizing the surrounding (substrate) water framework and in proton-coupled electron transfer to YZ(•) (D1-Tyr161).

Water-exchange rates of lanthanide ions in an ionic liquid.

The first ligand-exchange rate measurements of lanthanide ions in an ionic liquid are reported here. The trend of water-exchange rates in the ionic liquid is the opposite of the trend in water.