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Substrate water binding to the oxygen-evolving complex in photosystem II
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Johannes Messinger)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxygenic photosynthesis in plants, algae and cyanobacteria converts sunlight into chemical energy. In this process electrons are transferred from water molecules to CO2 leading to the assembly of carbohydrates, the building blocks of life. A cluster of four manganese ions and one calcium ion, linked together by five oxygen bridges, constitutes the catalyst for water oxidation in photosystem II (Mn4CaO5 cluster). This cluster stores up to four oxidizing equivalents (S0,..,S4 states), which are then used in a concerted reaction to convert two substrate water molecules into molecular oxygen. The reaction mechanism of this four-electron four-proton reaction is not settled yet and several hypotheses have been put forward. The work presented in this thesis aims at clarifying several aspects of the water oxidation reaction by analyzing the mode of substrate water binding to the Mn4CaO5 cluster.

Time-resolved membrane-inlet mass spectrometric detection of flash-induced O2 production after fast H218O labelling was employed to study the exchange rates between substrate waters bound to the Mn4CaO5 cluster and the surrounding bulk water. By employing this approach to dimeric photosystem II core complexes of the red alga Cyanidoschyzon merolae it was demonstrated that both substrate water molecules are already bound in the S2 state of the Mn4CaO5 cluster. This was confirmed with samples from the thermophilic cyanobacterium Thermosynechococcus elongatus. Addition of the water analogue ammonia, that is shown to bind to the Mn4CaO5 cluster by replacing the crystallographic water W1, did not significantly affect the exchange rates of the two substrate waters. Thus, these experiments exclude that W1 is a substrate water molecule.

The mechanism of O-O bond formation was studied by characterizing the substrate exchange in the S3YZ● state. For this the half-life time of this transient state into S0 was extended from 1.1 ms to 45 ms by replacing the native cofactors Ca2+ and Cl- by Sr2+ and I-. The data show that both substrate waters exchange significantly slower in the S3YZ● state than in the S3 state. A detailed discussion of this finding lead to the conclusions that (i) the calcium ion in the Mn4CaO5 cluster is not a substrate binding site and (ii) O-O bond formation occurs via the direct coupling between two Mn-bound water-derived oxygens, which were assigned to be the terminal water/hydroxy ligand W2 and the central oxo-bridging O5.

The driving force for the O2 producing S4→S0 transition was studied by comparing the effects of N2 and O2 pressures of about 20 bar on the flash-induced O2 production of photosystem II samples containing either the native cofactors Ca2+ and Cl- or the surrogates Sr2+ and Br-. While for the Ca/Cl-PSII samples no product inhibition was observed, a kinetic limitation of O2 production was found for the Sr/Br-PSII samples under O2 pressure. This was tentatively assigned to a significant slowdown of the O2 release in the Sr/Br-PSII samples. In addition, the equilibrium between the S0 state and the early intermediates of the S4 state family was studied under 18O2 atmosphere in photosystem II centers devoid of tyrosine YD. Water-exchange in the transiently formed early S4 states would have led to 16,18O2 release, but none was observed during a three day incubation time. Both experiments thus indicate that the S4→S0 transition has a large driving force. Thus, photosynthesis is not limited by the O2 partial pressure in the atmosphere.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2014. , 51 p.
Keyword [en]
Photosynthesis, Photosystem II, water oxidation, oxygen evolution, substrate water exchange, membrane-inlet mass spectrometry
National Category
Chemical Engineering
Identifiers
URN: urn:nbn:se:umu:diva-86500ISBN: 978-91-7459-802-5 (print)OAI: oai:DiVA.org:umu-86500DiVA: diva2:699578
Public defence
2014-03-20, KBC huset, Stora Hörsalen, KB3B1, Umeå universitet, Linnaeus väg 10, SE-901 87 Umeå, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2014-03-06 Created: 2014-02-27 Last updated: 2014-08-05Bibliographically approved
List of papers
1. Substrate water exchange in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae
Open this publication in new window or tab >>Substrate water exchange in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae
2014 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1837, no 8, 1257-1262 p.Article in journal (Other academic) Published
Abstract [en]

The binding affinity of the two substrate–water molecules to the water-oxidizing Mn4CaO5 catalyst in photosystem II core complexes of the extremophilic red alga Cyanidioschyzon merolae was studied in the S2and S3 states by the exchange of bound 16O-substrate against 18O-labeled water. The rate of this exchange was detected via the membrane-inlet mass spectrometric analysis of flash-induced oxygen evolution. For both redox states a fast and slow phase of water-exchange was resolved at the mixed labeled m/z 34 mass peak: kf = 52 ± 8 s− 1 and ks = 1.9 ± 0.3 s− 1 in the S2 state, and kf = 42 ± 2 s− 1 and kslow = 1.2 ± 0.3 s− 1 in S3, respectively. Overall these exchange rates are similar to those observed previously with preparations of other organisms. The most remarkable finding is a significantly slower exchange at the fast substrate–water site in the S2 state, which confirms beyond doubt that both substrate–water molecules are already bound in the S2 state. This leads to a very small change of the affinity for both the fast and the slowly exchanging substrates during the S2 → S3 transition. Implications for recent models for water-oxidation are briefly discussed.

Keyword
Cyanidioschyzon merolae, photosystem II, Water oxidation, oxygen evolution, substrate–water exchange, membrane-inlet mass spectrometry
National Category
Chemical Engineering Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-86497 (URN)10.1016/j.bbabio.2014.04.001 (DOI)000339133800004 ()
Note

This paper is dedicated to the memory of Warwick Hillier (18.10.1967-10.01.2014). Using membrane-inlet mass spectrometry and FTIR spectroscopy Warwick made many important discoveries regarding substrate-water binding to the OEC and the mechanism of water-oxidation. He was a very good scientist and friend that was highly appreciated throughout the photosynthesis community. In 2007 he was awarded the Robin-Hill award of the International Society for Photosynthesis Research (ISPR).

Available from: 2014-02-27 Created: 2014-02-27 Last updated: 2017-12-05Bibliographically approved
2. Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (µ-oxo) of the manganese tetramer
Open this publication in new window or tab >>Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (µ-oxo) of the manganese tetramer
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2013 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 39, 15561-15566 p.Article in journal (Refereed) Published
Abstract [en]

The assignment of the two substrate water sites of the tetramanganese penta-oxygen calcium (Mn4O5Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (mu-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn4O5Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522-3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable mu-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable mu-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55-60]}; (ii) ammonia displaces a water ligand to the outer manganese (Mn-A4-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the Mn-A4-O5 bond, leading to the perturbation of the mu-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.

Keyword
PSII, OEC, water oxidizing complex, water-oxidation, Mn cluster
National Category
Chemical Engineering
Identifiers
urn:nbn:se:umu:diva-82288 (URN)10.1073/pnas.1304334110 (DOI)000324765100024 ()
Available from: 2014-02-18 Created: 2013-10-29 Last updated: 2017-12-06Bibliographically approved
3. Substrate-water exchange in photosystem II is arrested before dioxygen formation
Open this publication in new window or tab >>Substrate-water exchange in photosystem II is arrested before dioxygen formation
2014 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, 4305- p.Article in journal (Refereed) Published
Abstract [en]

Light-driven oxidation of water into dioxygen, catalysed by the oxygen-evolving complex (OEC) in photosystem II, is essential for life on Earth and provides the blueprint for devices for producing fuel from sunlight. Although the structure of the OEC is known at atomic level for its dark-stable state, the mechanism by which water is oxidized remains unsettled. Important mechanistic information was gained in the past two decades by mass spectrometric studies of the H2 18O/H2 16O substrate-water exchange in the four (semi) stable redox states of the OEC. However, until now such data were not attainable in the transient states formed immediately before the O-O bond formation. Using modified photosystem II complexes displaying up to 40-fold slower O2 production rates, we show here that in the transient state the substrate-water exchange is dramatically slowed as compared with the earlier S states. This further constrains the possible sites for substrate-water binding in photosystem II.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-91401 (URN)10.1038/ncomms5305 (DOI)000340613800013 ()
Available from: 2014-08-04 Created: 2014-08-04 Last updated: 2017-12-05Bibliographically approved
4. Estimation of the equilibrium constant of the molecular oxygen generating S4→S0 (S3+YZ→S0YZ) transition in photosystem II
Open this publication in new window or tab >>Estimation of the equilibrium constant of the molecular oxygen generating S4→S0 (S3+YZ→S0YZ) transition in photosystem II
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(English)Manuscript (preprint) (Other academic)
National Category
Chemical Engineering
Identifiers
urn:nbn:se:umu:diva-86499 (URN)
Available from: 2014-02-27 Created: 2014-02-27 Last updated: 2014-03-05

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