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Fuller, Franklin D.

Publications and source records attributed to Fuller, Franklin D..

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Structure of a ribonucleotide reductase R2 protein radical

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). Here we present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O–O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Revealing core-valence interactions in solution with femtosecond X-ray pump X-ray probe spectroscopy

Abstract Femtosecond pump-probe spectroscopy using ultrafast optical and infrared pulses has become an essential tool to discover and understand complex electronic and structural dynamics in solvated molecular, biological, and material systems. Here we report the experimental realization of an ultrafast two-color X-ray pump X-ray probe transient absorption experiment performed in solution. A 10 fs X-ray pump pulse creates a localized excitation by removing a 1 s electron from an Fe atom in solvated ferro- and ferricyanide complexes. Following the ensuing Auger–Meitner cascade, the second X-ray pulse probes the Fe 1 s → 3 p transitions in resultant novel core-excited electronic states. Careful comparison of the experimental spectra with theory, extracts +2 eV shifts in transition energies per valence hole, providing insight into correlated interactions of valence 3 d with 3 p and deeper-lying electrons. Such information is essential for accurate modeling and predictive synthesis of transition metal complexes relevant for applications ranging from catalysis to information storage technology. This study demonstrates the experimental realization of the scientific opportunities possible with the continued development of multicolor multi-pulse X-ray spectroscopy to study electronic correlations in complex condensed phase systems.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗
DOE OSTI2023

Structural evidence for intermediates during O 2 formation in photosystem II

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn 4 CaO 5 cluster first stores four oxidizing equivalents, the S 0 to S 4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S 3 →[S 4 ]→S 0 transition where O 2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn 4 CaO 5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom O x , which was introduced as a bridging ligand between Ca and Mn1 during the S 2 →S 3 transition disappears or relocates in parallel with Y z reduction starting at approximately 700 μs after the third flash. The onset of O 2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O 2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Generation of intense phase-stable femtosecond hard X-ray pulse pairs

Significance The generation of phase-stable femtosecond X-ray pulse pairs will advance nonlinear spectroscopies and imaging, providing direct insight into the coupled motions of electrons and nuclei with resolution on the electronic length scale and timescale. This paper presents the generation of such pulse pairs in the X-ray domain. The approach uses X-ray free-electron laser pulses to induce highly directional, intense, phase-stable pairs of superfluorescence and seeded stimulated emission at the 5.9 keV manganese Kα 1 line. The finding is evidenced by strong interference fringes in the superfluorescence and stimulated emission signals.

43 PARTICLE ACCELERATORS↗
DOE OSTI2020

High-Resolution XFEL Structure of the Soluble Methane Monooxygenase Hydroxylase Complex with its Regulatory Component at Ambient Temperature in Two Oxidation States

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O 2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. Furthermore, this study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Observation of Seeded Mn K β Stimulated X-Ray Emission Using Two-Color X-Ray Free-Electron Laser Pulses

Kβ x-ray emission spectroscopy is a powerful probe for electronic structure analysis of 3d transition metal systems and their ultrafast dynamics. Selectively enhancing specific spectral regions would increase this sensitivity and provide fundamentally new insights. Recently we reported the observation and analysis of Kα amplified spontaneous x-ray emission from Mn solutions using an x-ray free-electron laser to create the 1s core-hole population inversion [Kroll et al., Phys. Rev. Lett. 120, 133203 (2018)]. To apply this new approach to the chemically more sensitive but much weaker Kβ x-ray emission lines requires a mechanism to outcompete the dominant amplification of the Kα emission. Here we report the observation of seeded amplified Kβ x-ray emission from a NaMnO 4 solution using two colors of x-ray free-electron laser pulses, one to create the 1s core-hole population inversion and the other to seed the amplified Kβ emission. Comparing the observed seeded amplified Kβ emission signal with that from conventional Kβ emission into the same solid angle, we obtain a signal enhancement of more than 10 5 . Finally, our findings are the first important step of enhancing and controlling the emission of selected final states of the Kβ spectrum with applications in chemical and materials science.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2020

Artificial Iron Proteins: Modeling the Active Sites in Non-Heme Dioxygenases

An important class of non-heme dioxygenases contains a conserved Fe binding site that consists of a 2-His-1-carboxylate facial triad. Results from structural biology show that, in the resting state, these proteins are six-coordinate with aqua ligands occupying the remaining three coordination sites. Here, we have utilized biotin–streptavidin (Sav) technology to design new artificial Fe proteins (ArMs) that have many of the same structural features found within active sites of these non-heme dioxygenases. An Sav variant was isolated that contains the S 112 E mutation, which installed a carboxylate side chain in the appropriate position to bind to a synthetic Fe II complex confined within Sav. Structural studies using X-ray diffraction (XRD) methods revealed a facial triad binding site that is composed of two N donors from the biotinylated ligand and the monodentate coordination of the carboxylate from S 112 E. Two aqua ligands complete the primary coordination sphere of the Fe II center with both involved in hydrogen bond networks within Sav. The corresponding Fe III protein was also prepared and structurally characterized to show a six-coordinate complex with two exogenous acetato ligands. The Fe III protein was further shown to bind an exogenous azido ligand through replacement of one acetato ligand. Spectroscopic studies of the ArMs in solution support the results found by XRD.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

XANES and EXAFS of dilute solutions of transition metals at XFELs

This work has demonstrated that X-ray absorption spectroscopy (XAS), both Mn XANES and EXAFS, of solutions with millimolar concentrations of metal is possible using the femtosecond X-ray pulses from XFELs. Mn XAS data were collected using two different sample delivery methods, a Rayleigh jet and a drop-on-demand setup, with varying concentrations of Mn. Here, a new method for normalization of XAS spectra based on solvent scattering that is compatible with data collection from a highly variable pulsed source is described. The measured XANES and EXAFS spectra of such dilute solution samples are in good agreement with data collected at synchrotron sources using traditional scanning protocols. The procedures described here will enable XFEL-based XAS on dilute biological samples, especially metalloproteins, with low sample consumption. Details of the experimental setup and data analysis methods used in this XANES and EXAFS study are presented. This method will also benefit XAS performed at high-repetition-rate XFELs such as the European XFEL, LCLS-II and LCLS-II-HE.

Chatterjee, Ruchira (ORCID:000000020865061X)↗
DOE OSTI2019

Structural isomers of the S 2 state in photosystem II: do they exist at room temperature and are they important for function?

In nature, an oxo‐bridged Mn 4 CaO 5 cluster embedded in photosystem II (PSII), a membrane‐bound multi‐subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light‐induced charge separations in the reaction center of PSII. The Mn 4 CaO 5 cluster accumulates four oxidizing equivalents to enable the four‐electron four‐proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S‐states, S 0 – S 4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen‐evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S‐states and if such equilibria are essential for the mechanism of the O‐O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S 2 state of PSII with structural data collected of the S 1 , S 2 and S 3 states by serial crystallography at neutral pH (∼6.5) using an X‐ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S 2 state, the room temperature crystallography data can be well‐described by just one S 2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.

Chatterjee, Ruchira↗
DOE OSTI2019

The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source

The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world's first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗
DOE OSTI2018

Structures of the intermediates of Kok’s photosynthetic water oxidation clock

Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle. The model comprises four (meta)stable intermediates (S 0 , S 1 , S 2 and S 3 ) and one transient S 4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn 4 CaO 5 ) cluster in the oxygen-evolving complex. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here in this paper, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S 2 →S 3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S 3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O 2 release. Thus, our results exclude peroxo-bond formation in the S 3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

X-ray Emission Spectroscopy as an in Situ Diagnostic Tool for X-ray Crystallography of Metalloproteins Using an X-ray Free-Electron Laser

Serial femtosecond crystallography (SFX) using the ultrashort X-ray pulses from a X-ray free-electron laser (XFEL) provides a new way of collecting structural data at room temperature that allows for following the reaction in real time after initiation. XFEL experiments are conducted in a shot-by-shot mode as the sample is destroyed and replenished after each X-ray pulse, and therefore, monitoring and controlling the data quality by using in situ diagnostic tools is critical. To study metalloenzymes, we developed the use of simultaneous collection of X-ray diffraction of crystals along with X-ray emission spectroscopy (XES) data that is used as a diagnostic tool for crystallography, by monitoring the chemical state of the metal catalytic center. We have optimized data analysis methods and sample delivery techniques for fast and active feedback to ensure the quality of each batch of samples and the turnover of the catalytic reaction caused by reaction triggering methods. Here, we describe this active in situ feedback system using Photosystem II as an example that catalyzes the oxidation of H2O to O2at the Mn4CaO5active site. We used the first moments of the Mn Kβ1,3emission spectra, which are sensitive to the oxidation state of Mn, as the primary diagnostics. This approach is applicable to different metalloproteins to determine the integrity of samples and follow changes in the chemical states of the reaction that can be initiated by light or activated by substrates and offers a metric for determining the diffraction images that are used for the final data sets.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Stimulated x-ray emission spectroscopy in transition metal complexes

Here, 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 four 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 three orders of magnitude, pointing to the build-up of transform limited pulses of ~1 fs duration. Driving the amplification into saturation leads to broadening and 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.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗
DOE OSTI2017

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.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗