| 1. |
- Alonso-Mori, Roberto, et al.
(författare)
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Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:47, s. 19103-19107
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Tidskriftsartikel (refereegranskat)abstract
- 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. K beta(1,3) XES spectra of Mn-II and Mn-2(III,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.
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| 2. |
- Cheah, Mun Hon, et al.
(författare)
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Assessment of the manganese cluster’s oxidation state via photoactivation of photosystem II microcrystals
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:1, s. 141-145
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Tidskriftsartikel (refereegranskat)abstract
- Knowledge of the manganese oxidation states of the oxygen-evolving Mn4CaO5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S2 state of this cluster. This signal implies an overall oxidation state of either Mn(III)3Mn(IV) or Mn(III)Mn(IV)3 for the S2 state. These 2 competing assignments are commonly known as “low oxidation (LO)” and “high oxidation (HO)” models of the Mn4CaO5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn4CaO5 cluster starting from Mn(II) and Mn-free PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn4CaO5 cluster’s oxidation states.
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| 3. |
- Chrysina, Maria, et al.
(författare)
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Five-coordinate Mn-IV intermediate in the activation of nature's water splitting cofactor
- 2019
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:34, s. 16841-16846
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Tidskriftsartikel (refereegranskat)abstract
- Nature's water splitting cofactor passes through a series of catalytic intermediates (S-0-S-4) before O-O bond formation and O-2 release. In the second last transition (S-2 to S-3) cofactor oxidation is coupled to water molecule binding to Mn1. It is this activated, water-enriched all Mn-IV form of the cofactor that goes on to form the O-O bond, after the next light-induced oxidation to S-4. How cofactor activation proceeds remains an open question. Here, we report a so far not described intermediate (S-3') in which cofactor oxidation has occurred without water insertion. This intermediate can be trapped in a significant fraction of centers (> 50%) in (i) chemical-modified cofactors in which Ca2+ is exchanged with Sr2+; the Mn4O5Sr cofactor remains active, but the S-2-S-3 and S-3-S-0 transitions are slower than for the Mn4O5Ca cofactor; and (ii) upon addition of 3% vol/vol methanol; methanol is thought to act as a substrate water analog. The S-3' electron paramagnetic resonance (EPR) signal is significantly broader than the untreated S-3 signal (2.5 T vs. 1.5 T), indicating the cofactor still contains a 5-coordinate Mn ion, as seen in the preceding S-2 state. Magnetic double resonance data extend these findings revealing the electronic connectivity of the S-3' cofactor is similar to the high spin form of the preceding S-2 state, which contains a cuboidal Mn3O4Ca unit tethered to an external, 5-coordinate Mn ion (Mn-4). These results demonstrate that cofactor oxidation regulates water molecule insertion via binding to Mn-4. The interaction of ammonia with the cofactor is also discussed.
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| 4. |
- de Lichtenberg, Casper, et al.
(författare)
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Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor
- 2024
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 121:11
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Tidskriftsartikel (refereegranskat)abstract
- Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation.
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| 5. |
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| 6. |
- Ibrahim, Mohamed, et al.
(författare)
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Untangling the sequence of events during the S-2 -> S-3 transition in photosystem II and implications for the water oxidation mechanism
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 117:23, s. 12624-12635
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Tidskriftsartikel (refereegranskat)abstract
- 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, S-1, S-2, S-3, and S-0, showing that a water molecule is inserted during the S-2 -> S-3 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 O-2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S-2 -> S-3 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 mu s 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 of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (t of similar to 350 mu s) during the S-2 -> S-3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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| 7. |
- Ibrahim, Mohamed, et al.
(författare)
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Untangling the sequence of events during the S2 -> S3 transition in photosystem II and implications for the water oxidation mechanism
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:23, s. 12624-12635
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Tidskriftsartikel (refereegranskat)abstract
- 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 of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 μs) during the S2 -> S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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| 8. |
- Kern, Jan, et al.
(författare)
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Room temperature femtosecond X-ray diffraction of photosystem II microcrystals
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:25, s. 9721-9726
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Tidskriftsartikel (refereegranskat)abstract
- Most of the dioxygen on earth is generated by the oxidation of water by photosystem II (PS II) using light from the sun. This lightdriven, 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.
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| 9. |
- Koroidov, Sergey, et al.
(författare)
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Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation
- 2014
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 11:17, s. 6299-6304
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Tidskriftsartikel (refereegranskat)abstract
- Cyanobacteria, algae and plants oxidize water to the O2 we breathe, and consume CO2 during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO2/HCO3− in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that HCO3− acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water-oxidation out into the chloroplast's lumen, resulting in a light-driven production of O2 and CO2. Depletion of HCO3− from the media leads, in the absence of added buffers, to a reversible down-regulation of O2 production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis, and conclude the more than 50-y-long quest for the function of CO2/ HCO3− in photosynthetic water oxidation.
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| 10. |
- Kosourov, Sergey, et al.
(författare)
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Water oxidation by photosystem II is the primary source of electrons for sustained H-2 photoproduction in nutrient-replete green algae
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 117:47, s. 29629-29636
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Tidskriftsartikel (refereegranskat)abstract
- The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H-2 production. H-2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H(2)ase), the key enzyme responsible for H-2 metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H(2)ase by O-2 coevolved in photosynthesis. Recently, we achieved sustainable H-2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H(2)ase. Employing membrane-inlet mass spectrometry and (H2O)-O-18, we now present clear evidence that efficient H-2 photoproduction in pulseilluminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H(2)ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H-2 photoproduction ceases and instead a slow overall H-2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H-2 photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H-2 yield.
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