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Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II: water here, water there, water everywhere
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0003-2975-8395
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few.

The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn4Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn4Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn4Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism.

In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S2-S3 state transition of the catalytic cycle.

Furthermore, through time-resolved membrane-inlet mass spectrometry (TR-MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water Ws is controlled by a dynamic equilibrium between conformations in the S2-state that give rise to either the low-spin multiline (LS-ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of Ws and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle.

To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry. With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S3-states fit best with D61 being in the vicinity of the fast exchanging water. With this information we found the data best explained in a scenario where the water insertion of the S2-S3 transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S2 state. In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S2 or the S3 states.

Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn4Ca-cluster to show that the briding oxygen O5  is exchanging with a near identical rate to Ws, further supporting the assignment that Ws=O5.

In conclusion, O-O bond formation most likely occurs between W2 (Wf) and O5 (Ws) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2020. , p. 104
Keywords [en]
Oxygenic Photosynthesis, Photosystem II, TR-MIMS, isotope exchange, EPR, EDNMR, water splitting, water oxidation
National Category
Physical Chemistry Biophysics Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-174116ISBN: 978-91-7855-343-3 (print)ISBN: 978-91-7855-344-0 (electronic)OAI: oai:DiVA.org:umu-174116DiVA, id: diva2:1458877
Public defence
2020-09-11, Glasburen, KBC Huset, Linnaeus väg 6, Umeå, 13:00 (English)
Opponent
Supervisors
Note

ISBN för den tryckta versionen saknas i fulltext och spikblad: 978-91-7855-343-3.

Available from: 2020-08-21 Created: 2020-08-18 Last updated: 2024-07-05Bibliographically approved
List of papers
1. Structure of photosystem II and substrate binding at room temperature
Open this publication in new window or tab >>Structure of photosystem II and substrate binding at room temperature
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2016 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 540, no 7633, p. 453-457Article in journal (Refereed) Published
Abstract [en]

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)1, in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution2,3. 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 site4–6. 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-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL7 provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10–13. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.

Place, publisher, year, edition, pages
Macmillan Publishers Ltd., 2016
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-128748 (URN)10.1038/nature20161 (DOI)000389716800046 ()27871088 (PubMedID)2-s2.0-84994458775 (Scopus ID)
Available from: 2016-12-14 Created: 2016-12-14 Last updated: 2023-03-24Bibliographically approved
2. Structures of the intermediates of Kok’s photosynthetic water oxidation clock
Open this publication in new window or tab >>Structures of the intermediates of Kok’s photosynthetic water oxidation clock
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2018 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 563, p. 421-425Article in journal, Letter (Refereed) Published
Abstract [en]

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 cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3–7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, 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 S2→S3 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 S3 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 O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-153920 (URN)10.1038/s41586-018-0681-2 (DOI)000450048400064 ()2-s2.0-85056636787 (Scopus ID)
Funder
NIH (National Institute of Health), GM055302NIH (National Institute of Health), GM110501NIH (National Institute of Health), GM126289NIH (National Institute of Health), GM117126NIH (National Institute of Health), GM124149NIH (National Institute of Health), GM124169Swedish Research Council, 2016-05183Knut and Alice Wallenberg Foundation, 2011.0055NIH (National Institute of Health), P41GM103393
Available from: 2018-12-07 Created: 2018-12-07 Last updated: 2023-03-24Bibliographically approved
3. Untangling the sequence of events during the S2 -> S3 transition in photosystem II and implications for the water oxidation mechanism
Open this publication in new window or tab >>Untangling the sequence of events during the S2 -> S3 transition in photosystem II and implications for the water oxidation mechanism
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2020 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, no 23, p. 12624-12635Article in journal (Refereed) Published
Abstract [en]

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 -> Stransition. 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.

Place, publisher, year, edition, pages
National Academy of Sciences, 2020
Keywords
photosynthesis, photosystem II, water oxidation, oxygen-evolving complex, X-ray free electron laser
National Category
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-173597 (URN)10.1073/pnas.2000529117 (DOI)000545947700028 ()32434915 (PubMedID)2-s2.0-85086146729 (Scopus ID)
Available from: 2020-07-22 Created: 2020-07-22 Last updated: 2023-03-24Bibliographically approved
4. Substrate water exchange in the S2 state of photosystem II is dependent on the conformation of the Mn4Ca cluster
Open this publication in new window or tab >>Substrate water exchange in the S2 state of photosystem II is dependent on the conformation of the Mn4Ca cluster
2020 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 22, no 23, p. 12894-12908Article in journal (Refereed) Published
Abstract [en]

In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn4Ca cluster. During catalysis, the OEC cycles through five redox states, S0 to S4. In the Sstate, the Mn4Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H218O/H216O exchange between bulk water and the two substrate waters bound at the Mn4Ca cluster in the SLS2, SHS2, and the S3 states in both Ca-PSII and Sr-PSII core complexes from T. elongatus. We found that the slowly exchanging substrate water exchanges 10 times faster in the SHS2 than in the SLS2 state, and that the SLS2 → SHS2 conversion has at physiological temperature an activation barrier of 17 ± 1 kcal mol−1. Of the presently suggested SHS2 models, our findings are best in agreement with a water exchange pathway involving a SHS2state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S3 state is governed by a different equilibrium than in S2, and that the exchange of the fast substrate water in the Sstate is unaffected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining possibilities for O–O bond formation in PSII are discussed.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2020
National Category
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-173575 (URN)10.1039/d0cp01380c (DOI)000543038500047 ()32373850 (PubMedID)2-s2.0-85086748140 (Scopus ID)
Available from: 2020-07-24 Created: 2020-07-24 Last updated: 2023-03-23Bibliographically approved
5. The D1-V185N mutation alters substrate water exchange by stabilizing alternative structures of the Mn4Ca-cluster in photosystem II
Open this publication in new window or tab >>The D1-V185N mutation alters substrate water exchange by stabilizing alternative structures of the Mn4Ca-cluster in photosystem II
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2021 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1862, no 1, article id 148319Article in journal (Refereed) Published
Abstract [en]

In photosynthesis, the oxygen-evolving complex (OEC) of the pigment-protein complex photosystem II (PSII) orchestrates the oxidation of water. Introduction of the V185N mutation into the D1 protein was previously reported to drastically slow O2-release and strongly perturb the water network surrounding the Mn4Ca cluster. Employing time-resolved membrane inlet mass spectrometry, we measured here the H218O/H216O-exchange kinetics of the fast (Wf) and slow (Ws) exchanging substrate waters bound in the S1, S2 and S3 states to the Mn4Ca cluster of PSII core complexes isolated from wild type and D1-V185N strains of Synechocystis sp. PCC 6803. We found that the rate of exchange for Ws was increased in the S1 and S2 states, while both Wf and Ws exchange rates were decreased in the S3 state. Additionally, we used EPR spectroscopy to characterize the Mn4Ca cluster and its interaction with the redox active D1-Tyr161 (YZ). In the S2 state, we observed a greatly diminished multiline signal in the V185N-PSII that could be recovered by addition of ammonia. The split signal in the S1 state was not affected, while the split signal in the S3 state was absent in the D1-V185N mutant. These findings are rationalized by the proposal that the N185 residue stabilizes the binding of an additional water-derived ligand at the Mn1 site of the Mn4Ca cluster via hydrogen bonding. Implications for the sites of substrate water binding are discussed.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
Photosystem II, Substrate water exchange, EPR, Manganese cluster, Water oxidation, O-O bond formation
National Category
Biophysics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-174108 (URN)10.1016/j.bbabio.2020.148319 (DOI)000601394200010 ()32979346 (PubMedID)2-s2.0-85092054718 (Scopus ID)
Note

Originally included in thesis in manuscript form.

Available from: 2020-08-18 Created: 2020-08-18 Last updated: 2023-03-24Bibliographically approved
6. Effects of the D61A and E189Q mutations on the exchange of substrate water in photosystem II
Open this publication in new window or tab >>Effects of the D61A and E189Q mutations on the exchange of substrate water in photosystem II
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:umu:diva-174110 (URN)
Available from: 2020-08-18 Created: 2020-08-18 Last updated: 2021-12-02
7. Assignment of the slow exchanging substrate water of Nature's water splitting cofactor
Open this publication in new window or tab >>Assignment of the slow exchanging substrate water of Nature's water splitting cofactor
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(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-174112 (URN)
Available from: 2020-08-18 Created: 2020-08-18 Last updated: 2021-12-02

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