Adrien Chauvet

Time-resolved X-ray absorption spectroscopy with a water-window high-harmonic source

An x-ray view of C–F and S–F bond breaks X-ray absorption spectroscopy is a useful probe of element-specific dynamics in molecular reactions. However, the required x-ray fluxes have rarely been available outside expensive dedicated facilities such as synchrotrons. Pertot et al. developed a tabletop laser-based high-harmonic source that extends far enough into the x-ray region to probe carbon K-edge and sulfur L-edge absorptions with femtosecond temporal resolution. They used this source to track the previously elusive dissociative dynamics of gaseous carbon tetrafluoride and sulfur hexafluoride after laser-induced ionization. Science, this issue p. 264 Ultrafast x-ray absorption spectroscopy at carbon and sulfur frequencies tracks dissociative dynamics of CF4+ and SF6+. Time-resolved x-ray absorption spectroscopy (TR-XAS) has so far practically been limited to large-scale facilities, to subpicosecond temporal resolution, and to the condensed phase. We report the realization of TR-XAS with a temporal resolution in the low femtosecond range by developing a tabletop high-harmonic source reaching up to 350 electron volts, thus partially covering the spectral region of 280 to 530 electron volts, where water is transmissive. We used this source to follow previously unexamined light-induced chemical reactions in the lowest electronic states of isolated CF4+ and SF6+ molecules in the gas phase. By probing element-specific core-to-valence transitions at the carbon K-edge or the sulfur L-edges, we characterized their reaction paths and observed the effect of symmetry breaking through the splitting of absorption bands and Rydberg-valence mixing induced by the geometry changes.

A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex.

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation–reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme–heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation–reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure–function parameter of membrane protein complexes.

Spectral Resolution of the Primary Electron Acceptor A0 in Photosystem I

The reduced state of the primary electron acceptor of Photosystem I, A(0), was resolved spectroscopically in its lowest energy Q(y) region for the first time without the addition of chemical reducing agents and without extensive data manipulation. To carry this out, we used the menB mutant of Synechocystis sp. PCC 6803 in which phylloquinone is replaced by plastoquinone-9 in the A(1) sites of Photosystem I. The presence of plastoquinone-9 slows electron transfer from A(0) to A(1), leading to a long-lived A(0)(-) state. This allows its spectral signature to be readily detected in a time-resolved optical pump-probe experiment. The maximum bleaching (A(0)(-) - A(0)) was found to occur at 684 nm with a corresponding extinction coefficient of 43 mM(-1) cm(-1). The data show evidence for an electrochromic shift of an accessory chlorophyll pigment, suggesting that the latter Q(y) absorption band is centered around 682 nm.

Set-up for broadband Fourier-transform multidimensional electronic spectroscopy

We present a compact passively phase-stabilized ultra-broadband 2D Fourier transform setup. A gas (argon)-filled hollow core fiber pumped by an amplified Ti:Al2O3 laser is used as a light source providing spectral range spanning from 420 to 900 nm. Sub-10-fs pulses were obtained using a deformable mirror-based pulse shaper. We probe the nonlinear response of Rhodamine 101 using 90 nm bandwidth and resolve vibrational coherences of 150 fs period in the ground state.

A microfluidic flow-cell for the study of the ultrafast dynamics of biological systems

The study of biochemical dynamics by ultrafast spectroscopic methods is often restricted by the limited amount of liquid sample available, while the high repetition rate of light sources can induce photodamage. In order to overcome these limitations, we designed a high flux, sub-ml, capillary flow-cell. While the 0.1 mm thin window of the 0.5 mm cross-section capillary ensures an optimal temporal resolution and a steady beam deviation, the cell-pump generates flows up to ∼0.35 ml/s that are suitable to pump laser repetition rates up to ∼14 kHz, assuming a focal spot-diameter of 100 μm. In addition, a decantation chamber efficiently removes bubbles and allows, via septum, for the addition of chemicals while preserving the closed atmosphere. The minimal useable amount of sample is ∼250 μl.

Does the singlet minus triplet spectrum with major photobleaching band near 680-682 nm represent an intact reaction center of Photosystem II?

We use both frequency- and time-domain low-temperature (5-20 K) spectroscopies to further elucidate the shape and spectral position of singlet minus triplet (triplet-bottleneck) spectra in the reaction centers (RCs) of Photosystem II (PSII) isolated from wild-type Chlamydomonas reinhardtii and spinach. It is shown that the shape of the nonresonant transient hole-burned spectrum in destabilized RCs from C. reinhardtii is very similar to that typically observed for spinach. This suggests that the previously observed difference in transient spectra between RCs from C. reinhardtii and spinach is not due to the sample origin but most likely due to a partial destabilization of the D1 and D2 polypeptides. This supports our previous assignments that destabilized RCs (referred to as RC680) (Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870), with a major photobleaching band near 680-682 nm and the absence of a photobleaching band near 673 nm, do not represent the intact RC residing within the PSII core complex. Time-resolved absorption difference spectra obtained for partially destabilized RCs of C. reinhardtii and for typical spinach RCs support the above conclusions. The absence of clear photobleaching bands near 673 and 684 nm (where the PD1 chlorophyll and the active pheophytin (PheoD1) contribute, respectively) in picosecond transient absorption spectra in both RCs studied in this work indicates that the cation can move from the primary electron donor (ChlD1) to PD1 (i.e., PD1ChlD1(+)PheoD1(-) → PD1(+)ChlD1PheoD1(-)). Therefore, we suggest that ChlD1 is the major electron donor in usually studied destabilized RCs (with a major photobleaching near 680-682 nm), although the PD1 path (where PD1 serves as the primary electron donor) is likely present in intact RCs, as discussed in Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870.

Note: Small anaerobic chamber for optical spectroscopy

The study of oxygen-sensitive biological samples requires an effective control of the atmosphere in which they are housed. In this aim however, no commercial anaerobic chamber is adequate to solely enclose the sample and small enough to fit in a compact spectroscopic system with which analysis can be performed. Furthermore, spectroscopic analysis requires the probe beam to pass through the whole chamber, introducing a requirement for adequate windows. In response to these challenges, we present a 1 l anaerobic chamber that is suitable for broad-band spectroscopic analysis. This chamber has the advantage of (1) providing access, via a septum, to the sample and (2) allows the sample position to be adjusted while keeping the chamber fixed and hermetic during the experiment.

Microfluidics for Ultrafast Spectroscopy

Ultrafast laser technologies became one of the essential tool in the characterization of mo‐ lecular compounds. Being comprised of spectroscopists, laser scientists, chemists and bi‐ ologists, the “ultrafast community” is often disconnected and consequently unaware of the developments in microfluidic systems. The challenges of studying limited amount of precious liquid sample by means of ultrafast spectroscopy remains silent and, while no commercial systems are available, each research group is developing its own “homemade” options. This chapter will therefore contribute in filling up the gap that exist be‐ tween the two communities, that of the ultrafast spectroscopy and that of microfluidics by revealing the importance of this analytical tool as well as the advantages of applying microfluidic technics to it. In this goal, the chapter will focus of the recently developed microfluidic flow-cell. With a minimal volume of about 250 μL, the flow-cell enables the study of precious protein complexes that are simply not available in larger quantities. The multiple advantages of the microfluidic flow-cell will be illustrated by the analysis of the cytochrome bc1. In particular, the study will describe how the capabilities of the mi‐ crofluidic flow-cell enabled the resolution of the ultrafast electronic and nuclear dynam‐ ics of specific embedded chromophores.