Matthew J. Latimer

Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature.

One Protein, Two Probes A central challenge in the use of x-ray diffraction to characterize macromolecular structure is the propensity of the high-energy radiation to damage the sample during data collection. Recently, a powerful accelerator-based, ultrafast x-ray laser source has been used to determine the geometric structures of small protein crystals too fragile for conventional diffraction techniques. Kern et al. (p. 491, published online 14 February) now pair this method with concurrent x-ray emission spectroscopy to probe electronic structure, as well as geometry, and were able to characterize the metal oxidation states in the oxygen-evolving complex within photosystem II crystals, while simultaneously verifying the surrounding protein structure. A powerful x-ray laser source can extract the geometry and electronic structure of metalloenzymes prior to damaging them. Intense femtosecond x-ray pulses produced at the Linac Coherent Light Source (LCLS) were used for simultaneous x-ray diffraction (XRD) and x-ray emission spectroscopy (XES) of microcrystals of photosystem II (PS II) at room temperature. This method probes the overall protein structure and the electronic structure of the Mn4CaO5 cluster in the oxygen-evolving complex of PS II. XRD data are presented from both the dark state (S1) and the first illuminated state (S2) of PS II. Our simultaneous XRD-XES study shows that the PS II crystals are intact during our measurements at the LCLS, not only with respect to the structure of PS II, but also with regard to the electronic structure of the highly radiation-sensitive Mn4CaO5 cluster, opening new directions for future dynamics studies.

Room temperature femtosecond X-ray diffraction of photosystem II microcrystals

Most of the dioxygen on earth is generated by the oxidation of water by photosystem II (PS II) using light from the sun. This light-driven, four-photon reaction is catalyzed by the Mn4CaO5 cluster located at the lumenal side of PS II. Various X-ray studies have been carried out at cryogenic temperatures to understand the intermediate steps involved in the water oxidation mechanism. However, the necessity for collecting data at room temperature, especially for studying the transient steps during the O–O bond formation, requires the development of new methodologies. In this paper we report room temperature X-ray diffraction data of PS II microcrystals obtained using ultrashort (< 50 fs) 9 keV X-ray pulses from a hard X-ray free electron laser, namely the Linac Coherent Light Source. The results presented here demonstrate that the ”probe before destroy” approach using an X-ray free electron laser works even for the highly-sensitive Mn4CaO5 cluster in PS II at room temperature. We show that these data are comparable to those obtained in synchrotron radiation studies as seen by the similarities in the overall structure of the helices, the protein subunits and the location of the various cofactors. This work is, therefore, an important step toward future studies for resolving the structure of the Mn4CaO5 cluster without any damage at room temperature, and of the reaction intermediates of PS II during O–O bond formation.

Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers

X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode

The ultrabright femtosecond X-ray pulses provided by X-ray free-electron lasers open capabilities for studying the structure and dynamics of a wide variety of systems beyond what is possible with synchrotron sources. Recently, this “probe-before-destroy” approach has been demonstrated for atomic structure determination by serial X-ray diffraction of microcrystals. There has been the question whether a similar approach can be extended to probe the local electronic structure by X-ray spectroscopy. To address this, we have carried out femtosecond X-ray emission spectroscopy (XES) at the Linac Coherent Light Source using redox-active Mn complexes. XES probes the charge and spin states as well as the ligand environment, critical for understanding the functional role of redox-active metal sites. Kβ1,3 XES spectra of MnII and Mn2III,IV complexes at room temperature were collected using a wavelength dispersive spectrometer and femtosecond X-ray pulses with an individual dose of up to >100 MGy. The spectra were found in agreement with undamaged spectra collected at low dose using synchrotron radiation. Our results demonstrate that the intact electronic structure of redox active transition metal compounds in different oxidation states can be characterized with this shot-by-shot method. This opens the door for studying the chemical dynamics of metal catalytic sites by following reactions under functional conditions. The technique can be combined with X-ray diffraction to simultaneously obtain the geometric structure of the overall protein and the local chemistry of active metal sites and is expected to prove valuable for understanding the mechanism of important metalloproteins, such as photosystem II.

Photoreduction of the active site of the metalloprotein putidaredoxin by synchrotron radiation

X-ray damage to protein crystals is often assessed on the basis of the degradation of diffraction intensity, yet this measure is not sensitive to the rapid changes that occur at photosensitive groups such as the active sites of metalloproteins. Here, X-ray absorption spectroscopy is used to study the X-ray dose-dependent photoreduction of crystals of the [Fe(2)S(2)]-containing metalloprotein putidaredoxin. A dramatic decrease in the rate of photoreduction is observed in crystals cryocooled with liquid helium at 40 K compared with those cooled with liquid nitrogen at 110 K. Whereas structural changes consistent with cluster reduction occur in the active site of the crystal measured at 110 K, no such changes occur in the crystal measured at 40 K, even after an eightfold increase in dose. When the structural results from extended X-ray absorption fine-structure measurements are compared with those obtained by crystallography on this and similar proteins, it is apparent that X-ray-induced photoreduction has had an impact on the crystallographic data and subsequent structure solutions. These results strongly indicate the importance of using liquid-helium-based cooling for metalloprotein crystallography in order to avoid the subtle yet important changes that can take place at the metalloprotein active sites when liquid-nitrogen-based cooling is used. The study also illustrates the need for direct measurement of the redox states of the metals, through X-ray absorption spectroscopy, simultaneously with the crystallographic measurements.

Efficient xylem transport and phloem remobilization of Zn in the hyperaccumulator plant species Sedum alfredii

Sedum alfredii is one of a few species known to hyperaccumulate zinc (Zn) and cadmium (Cd). Xylem transport and phloem remobilization of Zn in hyperaccumulating (HP) and nonhyperaccumulating (NHP) populations of S. alfredii were compared. Micro-X-ray fluorescence (μ-XRF) images of Zn in the roots of the two S. alfredii populations suggested an efficient xylem loading of Zn in HP S. alfredii, confirmed by the seven-fold higher Zn concentrations detected in the xylem sap collected from HP, when compared with NHP, populations. Zn was predominantly transported as aqueous Zn (> 55.9%), with the remaining proportion (36.7-42.3%) associated with the predominant organic acid, citric acid, in the xylem sap of HP S. alfredii. The stable isotope (68)Zn was used to trace Zn remobilization from mature leaves to new growing leaves for both populations. Remobilization of (68)Zn was seven-fold higher in HP than in NHP S. alfredii. Subsequent analysis by μ-XRF, combined with LA-ICPMS (laser ablation-inductively coupled plasma mass spectrometry), confirmed the enhanced ability of HP S. alfredii to remobilize Zn and to preferentially distribute the metal to mesophyll cells surrounding phloem in the new leaves. The results suggest that Zn hyperaccumulation by HP S. alfredii is largely associated with enhanced xylem transport and phloem remobilization of the metal. To our knowledge, this report is the first to reveal enhanced remobilization of metal by phloem transport in hyperaccumulators.

Refinement of the nickel site structure in Desulfovibrio gigas hydrogenase using range-extended EXAFS spectroscopy

We have reexamined the Ni EXAFS of oxidized, inactive (as-isolated) and H(2) reduced Desulfovibrio gigas hydrogenase. Better spatial resolution was achieved by analyzing the data over a 50% wider k-range than was previously available. A lower k(min) was obtained using the FEFF code for phase shifts and amplitudes. A higher k(max) was obtained by removing an interfering Cu signal from the raw spectra using multiple energy fluorescence detection. The larger k-range allowed us to better resolve the Ni-S bond lengths and to define more accurately the Ni-O and Ni-Fe bond lengths. We find that as-isolated, hydrogenase has two Ni-S bonds at approximately 2.2 A, but also 1-2 Ni-S bonds in the 2.35+/-0.05 A range. A Ni-O interaction is evident at 1.91 A. The as-isolated Ni-Fe distance cannot be unambiguously determined. Upon H(2) reduction, two short Ni-S bonds persist at approximately 2.2 A, but the remaining Ni-S bonds lengthen to 2.47+/-0.05 A. Good simulations are obtained with a Ni-Fe distance at 2.52 A, in agreement with crystal structures of the reduced enzyme. Although not evident in the crystal structures, an improvement in the fit is obtained by inclusion of one Ni-O interaction at 2.03 A. Implications of these distances for the spin-state of H(2) reduced H(2)ase are discussed.

Fluoride substitution in the Mn cluster from Photosystem II: EPR and X-ray absorption spectroscopy studies

Abstract X-band electron paramagnetic resonance (EPR) and Mn K-edge X-ray fluorescence absorption were used to study the effects of fluoride inhibition on the Mn complex in Photosystem II. The tetrameric Mn complex, responsible for the light-induced oxidation of H2O to form molecular oxygen, is influenced by treatments in which the naturally occurring chloride salts are removed or replaced. Inhibition of the complex by fluoride is examined by parallel enzyme activity and EPR studies. It is found that, as a function of increasing fluoride concentration, the declining enzymatic activity is paralleled initially by an exchange of the S = 1 2 ‘multiline’ EPR signal for the S > 1 2 , ‘g = 4’ EPR signal in illuminated samples. High concentrations of fluoride induce a broad (≈ 200 G), featureless radical signal in samples which have not been illuminated; subsequent illumination of these samples also generates the g = 4 EPR signal. X-ray absorption studies (XAS) of fluoride-inhibited samples show subtle alterations of the conformation of the Mn complex that are consistent with the presence of two dissimilar pairs of Mn atoms. The halide studies are discussed in terms of structural models for the Mn complex.

Mo K- and L-edge X-ray absorption spectroscopic study of the ADP.AlF4--stabilized nitrogenase complex: comparison with MoFe protein in solution and single crystal

The utility of using X-ray absorption spectroscopy (XAS) to study metalloproteins and, specifically, the enzyme complex nitrogenase, is highlighted by this study comparing both the structural and Mo-localized electronic features of the iron-molybdenum cofactor (FeMoco) in isolated MoFe protein and in the ADP.AlF4--stabilized complex of the MoFe protein with the Fe protein. No major differences are found at Mo between the two protein forms. The excellent quality of the data at both the Mo K and L edges will provide a baseline for analysis of other intermediates in the nitrogenase cycle. A new capability to delineate various contributions in the resting state of FeMoco is being pursued through polarized single-crystal XAS. The initial results point to the feasibility of using this technique for the analysis of scattering from the as yet unidentified atom at the center of FeMoco.

Integrated instrumentation for combined polarized single-crystal XAS and diffraction data acquisition for biological applications

Single-crystal X-ray absorption spectroscopy (XAS) instrumentation, allowing sequential integrated XAS and crystallographic data acquisition during the same experiment and on the same beamline, has been developed for SSRL beamline 9-3, a wiggler side station dedicated to general user biological XAS. The implementation includes a Huber kappa goniometer, Canberra 30-element Ge detector for XAS data collection, open-flow LHe and LN2 crystal coolers, a microscope for crystal alignment in the beam, and a MarCCD crystallography detector. The kappa goniometer allows a large accessible angular range with an open geometry, affording access to detectors and open stream coolers, as well as future instrumentation. Applicable standard hardware on SSRL crystallography beamlines has been incorporated, with crystallographic data collection controlled via the Blu-Ice software developed by the SSRL SMB macromolecular crystallography group. XAS data collection is handled through the SSRL standard XAS-Collect software. Initial diffraction and XAS data from single crystals using an open-flow cryostat are presented. The instrument will be available to general users after the SPEAR3 upgrade in 2004, and future expansion for use in high-throughput structural genomics XAS is proposed.