Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

A perfect X-ray beam splitter and its applications to time-domain interferometry and quantum optics exploiting free-electron lasers.

X-ray free-electron lasers (FELs) deliver ultrabright X-ray pulses, but not the sequences of phase-coherent pulses required for time-domain interferometry and control of quantum states. For conventional split-and-delay schemes to produce such sequences, the challenge stems from extreme stability requirements when splitting Ångstrom wavelength beams, where the tiniest path-length differences introduce phase jitter. We describe an FEL mode based on selective electron-bunch degradation and transverse beam shaping in the accelerator, combined with a self-seeded photon emission scheme. Instead of splitting the photon pulses after their generation by the FEL, we split the electron bunch in the accelerator, prior to photon generation, to obtain phase-locked X-ray pulses with subfemtosecond duration. Time-domain interferometry becomes possible, enabling the concomitant program of classical and quantum optics experiments with X-rays. The scheme leads to scientific benefits of cutting-edge FELs with attosecond and/or high-repetition rate capabilities, ranging from the X-ray analog of Fourier transform infrared spectroscopy to damage-free measurements.

Correlation of refractive index based and THz streaking arrival time tools for a hard X-ray free-electron laser.

To fully exploit ultra-short X-ray pulse durations routinely available at X-ray free-electron lasers to follow out-of-equilibrium dynamics, inherent arrival time fluctuations of the X-ray pulse with an external perturbing laser pulse need to be measured. In this work, two methods of arrival time measurement were compared to measure the arrival time jitter of hard X-ray pulses. The methods were photoelectron streaking by a THz field and a transient refractive index change of a semiconductor. The methods were validated by shot-to-shot correction of a pump-probe transient reflectivity measurement. An ultimate shot-to-shot full width at half-maximum error between the devices of 19.2 ± 0.1 fs was measured.

Spin cascade and doming in ferric hemes: Femtosecond X-ray absorption and X-ray emission studies.

The structure-function relationship is at the heart of biology, and major protein deformations are correlated to specific functions. For ferrous heme proteins, doming is associated with the respiratory function in hemoglobin and myoglobins. Cytochrome c (Cyt c) has evolved to become an important electron-transfer protein in humans. In its ferrous form, it undergoes ligand release and doming upon photoexcitation, but its ferric form does not release the distal ligand, while the return to the ground state has been attributed to thermal relaxation. Here, by combining femtosecond Fe Kα and Kß X-ray emission spectroscopy (XES) with Fe K-edge X-ray absorption near-edge structure (XANES), we demonstrate that the photocycle of ferric Cyt c is entirely due to a cascade among excited spin states of the iron ion, causing the ferric heme to undergo doming, which we identify. We also argue that this pattern is common to a wide diversity of ferric heme proteins, raising the question of the biological relevance of doming in such proteins.

Rovibrational investigation of a new high-lying 0u + state of Cu2 by using two-color resonant four-wave-mixing spectroscopy.

A highly excited electronic state of dicopper is observed and characterized for the first time. The [39.6]0u +-X1Σg +(0g +) system is measured at rotational resolution by using degenerate and two-color resonant four-wave-mixing, as well as laser induced fluorescence spectroscopy. Double-resonance experiments are performed by labeling selected rotational levels of the ground state by tuning the probe laser wavelength to transitions in the well-known (1-0) band of the B0u +-X1Σg +(0g +) electronic system. Spectra obtained by scans of the pump laser in the UV wavelength range were then assigned unambiguously by the stringent double-resonance selection rules. The absence of a Q-band suggests a parallel transition (ΔΩ = 0) and determines the term symbol of the state as 0u + in Hund's case (c) notation. The equilibrium constants for 63Cu2 are Te = 39 559.921(92) cm-1, ωe = 277.70(14) cm-1, Be = 0.104 942(66) cm-1, and re = 2.2595(11) Å. These findings are supported by high-level ab initio calculations at the MRCI+Q level, which clearly identifies this state as resulting from a 4p ← 3d transition. In addition, three dark perturber states are found in the v = 1 and v = 2 vibrational levels of the new state. A deperturbation analysis characterizes the interaction and rationalizes the anomalous dips in the excitation spectrum of the [39.6]0u +-X1Σg +(0g +) system.

Ultrafast Single-Particle Imaging with Intense X-Ray Pulses.

Ultrafast single-particle imaging with intense x-ray pulses from free-electron laser sources provides a new approach for visualizing structure and dynamics on the nanoscale. After a short introduction to the novel free-electron laser sources and methods, we highlight selected applications and discuss how ultrafast imaging flourishes from method development to early applications in physics and biology to opportunities for chemical sciences.

Femtosecond X-ray spectroscopy of haem proteins.

We discuss our recently reported femtosecond (fs) X-ray emission spectroscopy results on the ligand dissociation and recombination in nitrosylmyoglobin (MbNO) in the context of previous studies on ferrous haem proteins. We also present a preliminary account of femtosecond X-ray absorption studies on MbNO, pointing to the presence of more than one species formed upon photolysis.

Optical second harmonic generation in LiB3O5 modulated by intense femtosecond X-ray pulses.

Many of the scientific applications for X-ray free-electron lasers seek to exploit the ultrashort pulse durations of intense X-rays to obtain femtosecond time resolution of various processes in a "pump-probe" scheme. One of the limiting factors for such experiments is the timing jitter between the X-rays and ultrashort pulses from more conventional lasers operating at near-optical wavelengths. In this work, we investigate the potential of using X-ray-induced changes in the optical second harmonic generation efficiency of a nonlinear crystal to retrieve single-shot arrival times of X-ray pulses with respect to optical laser pulses. Our experimental results and simulations show changes to the efficiency of the second harmonic generation of 12%, approximately three times larger than the measured changes in the transmission of the 800 nm center-wavelength fundamental pulse. Further experiments showing even larger changes in the transmission of 400 nm center-wavelength pulses show that the mechanism of the second harmonic generation efficiency modulation is mainly the result of X-ray-induced changes in the linear absorption coefficients near 400 nm. We demonstrate and characterize a cross-correlation tool based on this effect in reference to a previously demonstrated method of X-ray/optical cross-correlation.

How the methyl group position influences the ultrafast deactivation in aromatic radicals.

Excited xylyl (methyl-benzyl) radical isomers have been studied by femtosecond time-resolved photoelectron spectroscopy and mass spectrometry. Depending on the substitution we find different deactivation channels after excitation into the D3(2A'') state (310 nm, 4 eV). While the ortho and para isomer exhibit deactivation rates similar to the benzyl radical, meta-xylyl sticks out and depletes twice as fast into the vibrationally hot ground state. We found that a ring deformation mode rather than the methyl pseudorotation enables access to a conical intersection, which is responsible for the faster deactivation. Transitions in the photoelectron spectrum can be assigned to several Rydberg series with mostly d angular momentum components. Absorption of two 4 eV photons triggers hydrogen loss reactions on a femtosecond timescale.

Integrated Evaluation Concept to Assess the Efficacy of Advanced Wastewater Treatment Processes for the Elimination of Micropollutants and Pathogens.

A multidisciplinary concept has been developed to compare advanced wastewater treatment processes for their efficacy of eliminating micropollutants and pathogens. The concept is based on (i) the removal/formation of selected indicator substances and their transformation products (TPs), (ii) the assessment of ecotoxicity via in vitro tests, and (iii) the removal of pathogens and antibiotic resistant bacteria. It includes substances passing biological wastewater treatment plants regulated or proposed to be regulated in the European Water Framework Directive, TPs formed in biological processes or during ozonation, agonistic/antagonistic endocrine activities, mutagenic/genotoxic activities, cytotoxic activities, further activities like neurotoxicity as well as antibiotics resistance genes, and taxonomic gene markers for pathogens. At a pilot plant, ozonation of conventionally treated wastewater resulted in the removal of micropollutants and pathogens and the reduction of estrogenic effects, whereas the in vitro mutagenicity increased. Subsequent post-treatment of the ozonated water by granular activated carbon (GAC) significantly reduced the mutagenic effects as well as the concentrations of remaining micropollutants, whereas this was not the case for biofiltration. The results demonstrate the suitability of the evaluation concept to assess processes of advanced wastewater treatment including ozonation and GAC by considering chemical, ecotoxicological, and microbiological parameters.

Opportunities for Chemistry at the SwissFEL X-ray Free Electron Laser.

X-ray techniques have long been applied to chemical research, ranging from powder diffraction tools to analyse material structure to X-ray fluorescence measurements for sample composition. The development of high-brightness, accelerator-based X-ray sources has allowed chemists to use similar techniques but on more demanding samples and using more photon-hungry methods. X-ray Free Electron Lasers (XFELs) are the latest in the development of these large-scale user facilities, opening up new avenues of research and the possibility of more advanced applications for a range of research. The SwissFEL XFEL project at the Paul Scherrer Institute will begin user operation in the hard X-ray (2.1-12.4 keV) photon energy range in 2018 with soft X-ray (240-1930 eV) user operation to follow and here we will present the details of this project, it's operating capabilities, and some aspects of the experimental stations that will be particularly attractive for chemistry research. SwissFEL is a revolutionary new machine that will complement and extend the time-resolved chemistry efforts in the Swiss research community.

Visualizing competing intersystem crossing and internal conversion with a complementary measurement.

A complementary measurement method based on a home-built double-sided velocity map imaging setup is introduced. This method can simultaneously obtain time-resolved photoelectron imaging and fragment ion imaging. It has been successfully applied to investigate the ultrafast dynamics of the second singlet electronically excited state (S2) in m-xylene. Time-resolved photoelectron and ion signals derived from the initial populated S2 state are tracked following two-photon absorption of a pump pulse. Time-of-flight mass spectra (TOFMS) show that there are dominant parent ions and one fragment ions with methyl loss during such a process. According to the measured photoelectron images and fragment ions images, transient kinetic energy distributions and angular distributions of the generated photoelectrons and fragments are obtained and analyzed. Compared to stand-alone photoelectron imaging, the obtained fragment ion imaging is powerful for further understanding the mechanisms especially when the dissociation occurs during the pump-probe ionization. Two competing channels intersystem crossing T3←S2 and internal conversion S1←S2 are attributed to the deactivation of the S2 state. A lifetime of ∼50 fs for the initially excited S2 state, of ∼276 fs for the secondary populated S1 state, and of 5.76 ps for the T3 state is inferred.

Real-time observation of ultrafast internal conversion in ethylbenzene by femtosecond time-resolved photoelectron imaging.

The ultrafast dynamics of the second singlet electronically excited state (S2) in ethylbenzene has been studied by femtosecond time-resolved photoelectron imaging. The time evolution of the photoelectron signal can be well described by a biexponential decay: a rapid relaxation pathway with a time constant of 60 ( ± 9) fs and a longer-lived channel on a timescale of 2.58 ( ± 0.22) ps. The rapid relaxation is ascribed to the ultrafast internal conversion from the S2 state to the vibrationally hot S1 state. This internal conversion process has been observed in real time. The slow photoelectron signal reflects the depopulation of secondarily populated high vibronic S1 state.

Ultrafast imaging of electronic relaxation in o-xylene: a new competing intersystem crossing channel.

The ultrafast dynamics of the second singlet electronically excited state (S2) in o-xylene was investigated by femtosecond time-resolved photoelectron imaging. A new competing relaxation channel of the S2 state was observed and assigned to the T3 ← S2 intersystem crossing. Interestingly, it is found that the relaxation via this channel occurs on a comparable femtosecond timescale as the S1 ← S2 internal conversion. A lifetime of ~60 fs for the initially excited S2 state, of 540 (±17) fs for the secondary populated S1 state, and of 7.23 (±0.21) ps for the T3 state could be inferred.

Ultrafast Energy Transfer from Photoexcited Tryptophan to the Haem in Cytochrome c.

We report femtosecond Fe K-edge absorption (XAS) and nonresonant X-ray emission (XES) spectra of ferric cytochrome C (Cyt c) upon excitation of the haem (>300 nm) or mixed excitation of the haem and tryptophan (<300 nm). The XAS and XES transients obtained in both excitation energy ranges show no evidence for electron transfer processes between photoexcited tryptophan (Trp) and the haem, but rather an ultrafast energy transfer, in agreement with previous ultrafast optical fluorescence and transient absorption studies. The reported (J. Phys. Chem. B 2011, 115 (46), 13723-13730) decay times of Trp fluorescence in ferrous (∼350 fs) and ferric (∼700 fs) Cyt c are among the shortest ever reported for Trp in a protein. The observed time scales cannot be rationalized in terms of Förster or Dexter energy transfer mechanisms and call for a more thorough theoretical investigation.

An X-ray free-electron laser with a highly configurable undulator and integrated chicanes for tailored pulse properties.

X-ray free-electron lasers (FELs) are state-of-the-art scientific tools capable to study matter on the scale of atomic processes. Since the initial operation of X-ray FELs more than a decade ago, several facilities with upgraded performance have been put in operation. Here we present the first lasing results of Athos, the soft X-ray FEL beamline of SwissFEL at the Paul Scherrer Institute in Switzerland. Athos features an undulator layout based on short APPLE-X modules providing full polarisation control, interleaved with small magnetic chicanes. This versatile configuration allows for many operational modes, giving control over many FEL properties. We show, for example, a 35% reduction of the required undulator length to achieve FEL saturation with respect to standard undulator configurations. We also demonstrate the generation of more powerful pulses than the ones obtained in typical undulators. Athos represents a fundamental step forward in the design of FEL facilities, creating opportunities in FEL-based sciences.

Disentangling the evolution of electrons and holes in photoexcited ZnO nanoparticles.

The evolution of charge carriers in photoexcited room temperature ZnO nanoparticles in solution is investigated using ultrafast ultraviolet photoluminescence spectroscopy, ultrafast Zn K-edge absorption spectroscopy, and ab initio molecular dynamics (MD) simulations. The photoluminescence is excited at 4.66 eV, well above the band edge, and shows that electron cooling in the conduction band and exciton formation occur in <500 fs, in excellent agreement with theoretical predictions. The x-ray absorption measurements, obtained upon excitation close to the band edge at 3.49 eV, are sensitive to the migration and trapping of holes. They reveal that the 2 ps transient largely reproduces the previously reported transient obtained at 100 ps time delay in synchrotron studies. In addition, the x-ray absorption signal is found to rise in ∼1.4 ps, which we attribute to the diffusion of holes through the lattice prior to their trapping at singly charged oxygen vacancies. Indeed, the MD simulations show that impulsive trapping of holes induces an ultrafast expansion of the cage of Zn atoms in <200 fs, followed by an oscillatory response at a frequency of ∼100 cm-1, which corresponds to a phonon mode of the system involving the Zn sub-lattice.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.