Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (... more Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side ...
Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at th... more Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes...
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by... more In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site o...
Photosystem II (PSII) is a membrane-spanning, multi-subunit pigment-protein complex responsible f... more Photosystem II (PSII) is a membrane-spanning, multi-subunit pigment-protein complex responsible for the oxidation of water and the reduction of plastoquinone in oxygenic photosynthesis. In the present review, the recent explosive increase in available structural information about the PSII core complex based on X-ray crystallography and cryo-electron microscopy is described at a level of detail that is suitable for a future structure-based analysis of light-harvesting processes. This description includes a proposal for a consistent numbering scheme of protein-bound pigment cofactors across species. The structural survey is complemented by an overview of the state of affairs in structure-based modeling of excitation energy transfer in the PSII core complex with emphasis on electrostatic computations, optical properties of the reaction center, the assignment of long-wavelength chlorophylls, and energy trapping mechanisms.
The binding of photosystem I (PS I) from to the native cytochrome (cyt) and cyt from horse heart ... more The binding of photosystem I (PS I) from to the native cytochrome (cyt) and cyt from horse heart (cyt ), is analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. While PS I has a higher affinity for cyt than for cyt , the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations reveal the existence of unspecific binding sites for cyt , besides one specific binding site close to P Binding to PS I is found to be the same for reduced and oxidized cyt Based on this information, suitable conditions for a co-crystallization of cyt with PS I have been found, resulting in crystals with a PS I:cyt ratio of 1:1. A crystal structure at 3.4 Å resolution has been obtained, but cyt cannot be identified in the electron density map because of unspecific binding sites and/or a high flexibility at the specific binding site. Modeling t...
Journal of the American Chemical Society, Nov 22, 2017
Artificial light-driven signal chains are particularly important for the development of systems c... more Artificial light-driven signal chains are particularly important for the development of systems converting light into a current, into chemicals or for light-induced sensing. Here, we report on the construction of an all-protein, light-triggered, catalytic circuit based on photosystem I, cytochrome c (cyt c) and human sulfite oxidase (hSOX). The defined assembly of all components using a modular design results in an artificial biohybrid electrode architecture, combining the photophysical features of PSI with the biocatalytic properties of hSOX for advanced light-controlled bioelectronics. The working principle is based on a competitive switch between electron supply from the electrode or by enzymatic substrate conversion.
In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of... more In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn 4 CaO 5 -cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from <italic>Thermosynechococcus elongatus</italic> without the Mn 4 CaO 5 -cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn 4 CaO 5 -cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depleti...
The structure of monomeric and trimeric photosystem I (PS I) of Thermosynechococcus elongatus BP1... more The structure of monomeric and trimeric photosystem I (PS I) of Thermosynechococcus elongatus BP1 (T. elongatus) was investigated using small angle X-ray scattering (SAXS). The scattering data reveal that the protein-detergent complexes possess a radius of gyration of 58 and 78 Å in the cases of monomeric and trimeric PS I, respectively. The results also show that the samples are monodisperse, virtually free of aggregation and contain free detergent micelles. The shape of the protein-detergent complexes can be well approximated by elliptical cylinders with heights of 78 Å. Monomeric PS I in buffer solution exhibits minor and major radii of 50 and 85 Å, respectively, while both radii are equal to about 110 Å in the case of trimeric PS I. The data have been analysed using a structure reconstitution by the xyz method. The measurements reveal, that monomeric and trimeric PS I are larger than their respective crystal structures. The reconstituted structures are larger by about 20 Å mainly in the region of the hydrophobic surfaces of the PS I complexes. This contradiction can be resolved by addition of a detergent belt constituted by a single layer of dodecylβ-D-maltoside molecules to the models of monomeric and trimeric PS I, containing 1540 and 480 molecules of the detergent, respectively.
X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macr... more 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...
Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has... more 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-fla...
The engineering of renewable and sustainable protein-based light-to-energy converting systems is ... more The engineering of renewable and sustainable protein-based light-to-energy converting systems is an emerging field of research. Here, we report on the development of supramolecular light-harvesting electrodes, consisting of the redox protein cytochrome c working as a molecular scaffold as well as a conductive wiring network and photosystem I as a photo-functional matrix element. Both proteins form complexes in solution, which in turn can be adsorbed on thiol-modified gold electrodes through a self-assembly mechanism. To overcome the limited stability of self-grown assemblies, DNA, a natural polyelectrolyte, is used as a further building block for the construction of a photo-active 3D architecture. DNA acts as a structural matrix element holding larger protein amounts and thus remarkably improving the maximum photocurrent and electrode stability. On investigating the photophysical properties, this system demonstrates that effective electron pathways have been created.
The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open cap... more The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ra...
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular ... more X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
One major vital element of the oxygenic photosynthesis is Photosystem I (PSI). We report on the c... more One major vital element of the oxygenic photosynthesis is Photosystem I (PSI). We report on the construction of graphene-based nanophotohybrid light-harvesting architectures consisting of PSI supercomplexes adsorbed onto π-system-modified graphene interfaces. The light-driven nanophotobioelectrochemical architectures have been designed on a modified carbon surface, based on π-π-stacking interactions between polycyclic aromatic compounds and graphene. As a result of the remarkable features of graphene and the feasibility of purposeful surface property adjustment, well-defined photoelectrochemical responses have been displayed by the nanophotohybrid electrodes. In particular, the PSI-graphene electrodes utilizing naphthalene derivatives provided a suitable surface for the adsorption of PSI and display already at the open circuit potential (OCP) a high cathodic photocurrent output of 4.5±0.1 µA/cm2. By applying an overpotential and addition of a soluble electron acceptor (methyl viologen) the photocurrent density can be further magnified to 20±0.5 µA/cm2. On the contrary, the investigated anthracene-based PSI graphene electrodes exhibit considerably smaller and not very directed photoelectrochemical responses. This study grants insights into the influences of different polycyclic aromatic compounds acting as interface between the very large protein supercomplex PSI and graphene while supporting the electrochemical communication of the biomolecule with the electrode. It needs to be emphasized that solely the naphthalene-based photoelectrodes reveal unidirectional cathodic photocurrents, establishing the feasibility of utilizing this advanced approach for the construction of next-generation photovoltaic devices.
In the version of this article initially published, the Hendrickson formula in the Figure 2 legen... more In the version of this article initially published, the Hendrickson formula in the Figure 2 legend incorrectly had (2N A /N P) 1/2 divided by (f''/Z eff); these terms should have been multiplied. The error has been corrected in the HTML and PDF versions of the article.
The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides under conditi... more The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides under conditions of membrane protein crystallization is investigated.
Proceedings of the National Academy of Sciences of the United States of America, Jan 5, 2001
Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyr... more Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS I...
Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (... more Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side ...
Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at th... more Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes...
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by... more In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site o...
Photosystem II (PSII) is a membrane-spanning, multi-subunit pigment-protein complex responsible f... more Photosystem II (PSII) is a membrane-spanning, multi-subunit pigment-protein complex responsible for the oxidation of water and the reduction of plastoquinone in oxygenic photosynthesis. In the present review, the recent explosive increase in available structural information about the PSII core complex based on X-ray crystallography and cryo-electron microscopy is described at a level of detail that is suitable for a future structure-based analysis of light-harvesting processes. This description includes a proposal for a consistent numbering scheme of protein-bound pigment cofactors across species. The structural survey is complemented by an overview of the state of affairs in structure-based modeling of excitation energy transfer in the PSII core complex with emphasis on electrostatic computations, optical properties of the reaction center, the assignment of long-wavelength chlorophylls, and energy trapping mechanisms.
The binding of photosystem I (PS I) from to the native cytochrome (cyt) and cyt from horse heart ... more The binding of photosystem I (PS I) from to the native cytochrome (cyt) and cyt from horse heart (cyt ), is analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. While PS I has a higher affinity for cyt than for cyt , the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations reveal the existence of unspecific binding sites for cyt , besides one specific binding site close to P Binding to PS I is found to be the same for reduced and oxidized cyt Based on this information, suitable conditions for a co-crystallization of cyt with PS I have been found, resulting in crystals with a PS I:cyt ratio of 1:1. A crystal structure at 3.4 Å resolution has been obtained, but cyt cannot be identified in the electron density map because of unspecific binding sites and/or a high flexibility at the specific binding site. Modeling t...
Journal of the American Chemical Society, Nov 22, 2017
Artificial light-driven signal chains are particularly important for the development of systems c... more Artificial light-driven signal chains are particularly important for the development of systems converting light into a current, into chemicals or for light-induced sensing. Here, we report on the construction of an all-protein, light-triggered, catalytic circuit based on photosystem I, cytochrome c (cyt c) and human sulfite oxidase (hSOX). The defined assembly of all components using a modular design results in an artificial biohybrid electrode architecture, combining the photophysical features of PSI with the biocatalytic properties of hSOX for advanced light-controlled bioelectronics. The working principle is based on a competitive switch between electron supply from the electrode or by enzymatic substrate conversion.
In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of... more In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn 4 CaO 5 -cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from <italic>Thermosynechococcus elongatus</italic> without the Mn 4 CaO 5 -cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn 4 CaO 5 -cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depleti...
The structure of monomeric and trimeric photosystem I (PS I) of Thermosynechococcus elongatus BP1... more The structure of monomeric and trimeric photosystem I (PS I) of Thermosynechococcus elongatus BP1 (T. elongatus) was investigated using small angle X-ray scattering (SAXS). The scattering data reveal that the protein-detergent complexes possess a radius of gyration of 58 and 78 Å in the cases of monomeric and trimeric PS I, respectively. The results also show that the samples are monodisperse, virtually free of aggregation and contain free detergent micelles. The shape of the protein-detergent complexes can be well approximated by elliptical cylinders with heights of 78 Å. Monomeric PS I in buffer solution exhibits minor and major radii of 50 and 85 Å, respectively, while both radii are equal to about 110 Å in the case of trimeric PS I. The data have been analysed using a structure reconstitution by the xyz method. The measurements reveal, that monomeric and trimeric PS I are larger than their respective crystal structures. The reconstituted structures are larger by about 20 Å mainly in the region of the hydrophobic surfaces of the PS I complexes. This contradiction can be resolved by addition of a detergent belt constituted by a single layer of dodecylβ-D-maltoside molecules to the models of monomeric and trimeric PS I, containing 1540 and 480 molecules of the detergent, respectively.
X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macr... more 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...
Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has... more 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-fla...
The engineering of renewable and sustainable protein-based light-to-energy converting systems is ... more The engineering of renewable and sustainable protein-based light-to-energy converting systems is an emerging field of research. Here, we report on the development of supramolecular light-harvesting electrodes, consisting of the redox protein cytochrome c working as a molecular scaffold as well as a conductive wiring network and photosystem I as a photo-functional matrix element. Both proteins form complexes in solution, which in turn can be adsorbed on thiol-modified gold electrodes through a self-assembly mechanism. To overcome the limited stability of self-grown assemblies, DNA, a natural polyelectrolyte, is used as a further building block for the construction of a photo-active 3D architecture. DNA acts as a structural matrix element holding larger protein amounts and thus remarkably improving the maximum photocurrent and electrode stability. On investigating the photophysical properties, this system demonstrates that effective electron pathways have been created.
The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open cap... more The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ra...
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular ... more X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
One major vital element of the oxygenic photosynthesis is Photosystem I (PSI). We report on the c... more One major vital element of the oxygenic photosynthesis is Photosystem I (PSI). We report on the construction of graphene-based nanophotohybrid light-harvesting architectures consisting of PSI supercomplexes adsorbed onto π-system-modified graphene interfaces. The light-driven nanophotobioelectrochemical architectures have been designed on a modified carbon surface, based on π-π-stacking interactions between polycyclic aromatic compounds and graphene. As a result of the remarkable features of graphene and the feasibility of purposeful surface property adjustment, well-defined photoelectrochemical responses have been displayed by the nanophotohybrid electrodes. In particular, the PSI-graphene electrodes utilizing naphthalene derivatives provided a suitable surface for the adsorption of PSI and display already at the open circuit potential (OCP) a high cathodic photocurrent output of 4.5±0.1 µA/cm2. By applying an overpotential and addition of a soluble electron acceptor (methyl viologen) the photocurrent density can be further magnified to 20±0.5 µA/cm2. On the contrary, the investigated anthracene-based PSI graphene electrodes exhibit considerably smaller and not very directed photoelectrochemical responses. This study grants insights into the influences of different polycyclic aromatic compounds acting as interface between the very large protein supercomplex PSI and graphene while supporting the electrochemical communication of the biomolecule with the electrode. It needs to be emphasized that solely the naphthalene-based photoelectrodes reveal unidirectional cathodic photocurrents, establishing the feasibility of utilizing this advanced approach for the construction of next-generation photovoltaic devices.
In the version of this article initially published, the Hendrickson formula in the Figure 2 legen... more In the version of this article initially published, the Hendrickson formula in the Figure 2 legend incorrectly had (2N A /N P) 1/2 divided by (f''/Z eff); these terms should have been multiplied. The error has been corrected in the HTML and PDF versions of the article.
The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides under conditi... more The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides under conditions of membrane protein crystallization is investigated.
Proceedings of the National Academy of Sciences of the United States of America, Jan 5, 2001
Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyr... more Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS I...
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Papers by Athina Zouni