Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion

X ray induced sample damage at the Mn L edge a case study for soft X ray spectroscopy of transition metal complexes in solution

X ray induced sample damage can impede electronic and structural investigations of radiation sensitive samples studied with X rays. Here we quantify dose dependent sample damage to the prototypical MnIII acac 3 complex in solution and at room temperature for the soft X ray range, using X ray absorption spectroscopy at the Mn L edge. We observe the appearance of a reduced MnII species as the X ray dose is increased. We find a half damage dose of 1.6 MGy and quantify a spectroscopically tolerable dose on the order of 0.3 MGy 1 Gy 1 J kg 1 , where 90 of MnIII acac 3 are intact. Our dose limit is around one order of magnitude lower than the Henderson limit half damage dose of 20 MGy which is commonly employed for protein crystallography with hard X rays. It is comparable, however, to the dose limits obtained for collecting un damaged Mn K edge spectra of the photosystem II protein, using hard X rays. The dose dependent reduction of MnIII observed here for solution samples occurs at a dose limit that is two to four orders of magnitude smaller than the dose limits previously reported for soft X ray spectroscopy of iron samples in the solid phase. We compare our measured to calculated spectra from ab initio restricted active space RAS theory and discuss possible mechanisms for the observed dose dependent damage of MnIII acac 3 in solution. On the basis of our results, we assess the influence of sample damage in other experimental studies with soft X rays from storage ring synchrotron radiation sources and X ray free electron laser

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 measurement

How ultrafast X-ray pulses can reveal hidden secrets of photosynthesis

X-ray-based techniques are extremely versatile and can provide information regarding the atomic arrangement of atoms in a molecule and are often the method of choice for exploring the structures of proteins and their ligands. The photosynthetic splitting of water and evolution of molecular oxygen by plants and cyanobacteria is one of the key reactions in nature, which is catalysed by a metal site in a membrane-bound photosynthetic protein complex. In this article, we will describe how X-ray pulse lasers can simultaneously probe the overall atomic structure of the photosynthetic system and the electronic structure of a catalytic metal site under physiological conditions in real time

Electronic Structure and Oxidation State Changes in the Mn (4) Ca Cluster of Photosystem II

Oxygen-evolving complex (Mn{sub 4}Ca cluster) of Photosystem II cycles through five intermediate states (S{sub i}-states, i = 0-4) before a molecule of dioxygen is released. During the S-state transitions, electrons are extracted from the OEC, either from Mn or alternatively from a Mn ligand. The oxidation state of Mn is widely accepted as Mn{sub 4}(III{sub 2},IV{sub 2}) and Mn{sub 4}(III,IV{sub 3}) for S{sub 1} and S{sub 2} states, while it is still controversial for the S{sub 0} and S{sub 3} states. We used resonant inelastic X-ray scattering (RIXS) to study the electronic structure of Mn{sub 4}Ca complex in the OEC. The RIXS data yield two-dimensional plots that provide a significant advantage by obtaining both K-edge pre-edge and L-edge-like spectra (metal spin state) simultaneously. We have collected data from PSII samples in the each of the S-states and compared them with data from various inorganic Mn complexes. The spectral changes in the Mn 1s2p{sub 3/2} RIXS spectra between the S-states were compared to those of the oxides of Mn and coordination complexes. The results indicate strong covalency for the electronic configuration in the OEC, and we conclude that the electron is transferred from a strongly delocalized orbital, compared to those in Mn oxides or coordination complexes. The magnitude for the S{sub 0} to S{sub 1}, and S{sub 1} to S{sub 2} transitions is twice as large as that during the S{sub 2} to S{sub 3} transition, indicating that the electron for this transition is extracted from a highly delocalized orbital with little change in charge density at the Mn atoms

Structural changes in the Mn4Ca cluster and the mechanism of photosynthetic water splitting

Photosynthetic water oxidation, where water is oxidized to dioxygen, is a fundamental chemical reaction that sustains the biosphere. This reaction is catalyzed by a Mn4Ca complex in the photosystem II (PS II) oxygen-evolving complex (OEC): a multiprotein assembly embedded in the thylakoid membranes of green plants, cyanobacteria, and algae. The mechanism of photosynthetic water oxidation by the Mn4Ca cluster in photosystem II is the subject of much debate, although lacking structural characterization of the catalytic intermediates. Biosynthetically exchanged Ca/Sr-PS II preparations and x-ray spectroscopy, including extended x-ray absorption fine structure (EXAFS), allowed us to monitor Mn–Mn and Ca(Sr)–Mn distances in the four intermediate S states, S0 through S3, of the catalytic cycle that couples the one-electron photochemistry occurring at the PS II reaction center with the four-electron water-oxidation chemistry taking place at the Mn4Ca(Sr) cluster. We have detected significant changes in the structure of the complex, especially in the Mn–Mn and Ca(Sr)–Mn distances, on the S2-to-S3 and S3-to-S0 transitions. These results implicate the involvement of at least one common bridging oxygen atom between the Mn–Mn and Mn–Ca(Sr) atoms in the O–O bond formation. Because PS II cannot advance beyond the S2 state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical components in the mechanism of the enzyme. The results also show that Ca is not just a spectator atom involved in providing a structural framework, but is actively involved in the mechanism of water oxidation and represents a rare example of a catalytically active Ca cofactor