Franklin Fuller

Structure of photosystem II and substrate binding at room temperature.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution. A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

X-ray absorption spectroscopy using a self-seeded soft X-ray free-electron laser

X-ray free electron lasers (XFELs) enable unprecedented new ways to study the electronic structure and dynamics of transition metal systems. L-edge absorption spectroscopy is a powerful technique for such studies and the feasibility of this method at XFELs for solutions and solids has been demonstrated. However, the required x-ray bandwidth is an order of magnitude narrower than that of self-amplified spontaneous emission (SASE), and additional monochromatization is needed. Here we compare L-edge x-ray absorption spectroscopy (XAS) of a prototypical transition metal system based on monochromatizing the SASE radiation of the linac coherent light source (LCLS) with a new technique based on self-seeding of LCLS. We demonstrate how L-edge XAS can be performed using the self-seeding scheme without the need of an additional beam line monochromator. We show how the spectral shape and pulse energy depend on the undulator setup and how this affects the x-ray spectroscopy measurements.

Stimulated x-ray emission spectroscopy in transition metal complexes

We report the observation and analysis of the gain curve of amplified Kα x-ray emission from solutions of Mn(II) and Mn(VII) complexes using an x-ray free electron laser to create the 1s core-hole population inversion. We find spectra at amplification levels extending over 4 orders of magnitude until saturation. We observe bandwidths below the Mn 1s core-hole lifetime broadening in the onset of the stimulated emission. In the exponential amplification regime the resolution corrected spectral width of ∼1.7  eV FWHM is constant over 3 orders of magnitude, pointing to the buildup of transform limited pulses of ∼1  fs duration. Driving the amplification into saturation leads to broadening and a shift of the line. Importantly, the chemical sensitivity of the stimulated x-ray emission to the Mn oxidation state is preserved at power densities of ∼10^{20}  W/cm^{2} for the incoming x-ray pulses. Differences in signal sensitivity and spectral information compared to conventional (spontaneous) x-ray emission spectroscopy are discussed. Our findings build a baseline for nonlinear x-ray spectroscopy for a wide range of transition metal complexes in inorganic chemistry, catalysis, and materials science.

Soft x-ray absorption spectroscopy of metalloproteins and high-valent metal-complexes at room temperature using free-electron lasers

X-ray absorption spectroscopy at the L-edge of 3d transition metals provides unique information on the local metal charge and spin states by directly probing 3d-derived molecular orbitals through 2p-3d transitions. However, this soft x-ray technique has been rarely used at synchrotron facilities for mechanistic studies of metalloenzymes due to the difficulties of x-ray-induced sample damage and strong background signals from light elements that can dominate the low metal signal. Here, we combine femtosecond soft x-ray pulses from a free-electron laser with a novel x-ray fluorescence-yield spectrometer to overcome these difficulties. We present L-edge absorption spectra of inorganic high-valent Mn complexes (Mn ∼ 6–15 mmol/l) with no visible effects of radiation damage. We also present the first L-edge absorption spectra of the oxygen evolving complex (Mn4CaO5) in Photosystem II (Mn < 1 mmol/l) at room temperature, measured under similar conditions. Our approach opens new ways to study metalloenzymes under functional conditions.

Exploring the dynamic of PSII at room temperature by simultaneous femtosecond X-ray spectroscopy and dffraction

dioxygen, one of nature’s most fascinating and important reactions. The water-splitting reaction takes place at the oxygen evolving complex (OEC), through five intermediate Sstates (S0 to S4), where S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. The structure of PSII in the dark state has been solved by X-ray diffraction and X-ray free electron laser (XFEL) providing information of the structural geometry of the Mn4CaO5 cluster in OEC. However, to fully understand the O-O bond formation mechanism, elucidating the structures of the OEC in the different Sstates is essential. In our recently published study, we report high-resolution structures of PSII at room temperature using XFEL coupling with X-ray spectroscopy under different illumination conditions. This enables us to gain new insights about the dynamic changes in the structure of the Mn4CaO5 cluster as well as the ligands and the bound water molecules. We further use ammonia as water analogue to investigate the water-binding site(s) and discriminate between mechanisms proposed in literature. We discuss the precise role of the water bound to OEC in electron transfer and the water-splitting reaction.

Insights into the oxygen-evolving mechanism of photosynthesis using XFELs

X-ray free electron lasers (XFELs) provide a unique opportunity for time resolved, damage-free studies of dynamic systems due to the ability of femtosecond-long laser pulses of extreme intensity to diffract on a much shorter time scale than the onset of radiation damage. We have applied this technique to the study of water oxidation in photosystem II (PSII), the transmembrane protein where water is converted to oxygen in plants and photosynthetic cyanobacteria. A nanoflow liquid jet1 or acoustic droplet ejection2 is used to deliver microcrystals suspended in buffer to the XFEL beam. Placement of visible lasers in the path of the jet or droplets allows timed illumination for selection of any stable or transient state of the catalytic center in the water-splitting cycle. In recent XFEL experiments, we obtained the first high-resolution room-temperature diffraction data for the dark and twiceilluminated (2F) states of PSII, which allowed us to exclude several proposed mechanisms for water oxidation.3

Time-resolved structural biology benefits from complementary methods

Allen Milster Orville1, Franklin Fuller2, Sheraz Gul2, Jan Kern2, Aaron Brewster2, Nicholas Sauter2, Uwe Bergmann3, Roberto AlonsoMori4, Vittal Yachandra2, Junko Yano2 1Life Sciences, Diamond Light Source, Didcot, United Kingdom, 2Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States, 3Stanford PULSE Institute, SLAC National Accelerator Laboratory, Menlo Park, United States, 4LCLS, SLAC National Accelerator Laboratory, Menlo Park, United States E-mail: allen.orville@diamond.ac.uk

On-demand acoustic methods for time-resolved structural biology

Allen Milster Orville1, Franklin D. Fuller2, Sheraz Gul2, Jan Kern2, Pierre Aller1, Babak Andi3, Aaron Brewster2, Nicholas Sauter2, Vittal Yachandra2, Junko Yano2 1Life Sciences, Diamond Light Source, Didcot, United Kingdom, 2Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States, 3NSLS-II, Brookhaven National Lab, Upton, United States E-mail: allen.orville@diamond.ac.uk