A Mechanism for Water Splitting and Oxygen Production in Photosynthesis James Barber*

HYPOTHESIS PUBLISHED: 3 APRIL 2017 | VOLUME: 3 | ARTICLE NUMBER: 17041

A mechanism for water splitting and oxygen production in photosynthesis James Barber*

Sunlight is absorbed and converted to chemical energy by photosynthetic organisms. At the heart of this process is the most fundamental reaction on Earth, the light-driven splitting of water into its elemental constituents. In this way molecular oxygen is released, maintaining an aerobic atmosphere and creating the ozone layer. The hydrogen that is released is used to convert carbon dioxide into the organic molecules that constitute life and were the origin of fossil fuels. Oxidation of these organic molecules, either by respiration or combustion, leads to the recombination of the stored hydrogen with oxygen, releasing energy and reforming water. This water splitting is achieved by the enzyme photosystem II (PSII). Its appearance at least 3 billion years ago, and linkage through an electron transfer chain to photosystem I, directly led to the emergence of eukaryotic and multicellular organisms. Before this, biological organisms had been dependent on hydrogen/ 2+ electron donors, such as H2S, NH3, organic acids and Fe , that were in limited supply compared with the oceans of liquid water. However, it is likely that water was also used as a hydrogen source before the emergence of PSII, as found today in anaerobic prokaryotic organisms that use carbon monoxide as an energy source to split water. The enzyme that catalyses this reaction is carbon monoxide dehydrogenase (CODH). Similarities between PSII and the iron- and nickel-containing form of this enzyme (Fe-Ni CODH) suggest a possible mechanism for the photosynthetic O–O bond formation.

n photosystem II (PSII), the energy of four photons (4 hv) of light details of this final oxidation state are unknown because O–Obond is used by the oxygen-evolving complex (OEC) to drive the formation is very fast. The cycle is powered by the oxidation of a Isplitting of two water molecules to produce dioxygen and four chlorophyll, known as P680, generated by light-driven primary reducing equivalents (equation (1)): charge separation in the reaction centre of PSII coupled to a redox-active tyrosine (YZ), serving as an intermediate electron −4→hn + − + + + 7 2H2O O2 4e 4H (1) carrier between P680 and the Mn cluster (see Fig. 1) . Understanding the molecular mechanism of O–O bond In Fe-Ni CODH, the energy released by the oxidation of a CO formation in PSII during the S-state cycle is one of the most molecule is used to split one water molecule to produce carbon outstanding challenges of bioinorganic chemistry. The importance dioxide and two reducing equivalents (equation (2)): of this cannot be overstated since splitting water into its elemental constituents is thermodynamically and chemically demanding, + −→ + − + + CO H2O CO2 2e 2H (2) being accomplished by the relatively low energy content of four photons of long-wavelength visible light. Over the years, This chemistry is the well-known “water-gas shift reaction” there have been many postulates of the mechanism for O–O bond discovered in 1780 by the Italian physicist Felice Fontana1, and formation in PSII8–12. Here, I present support for a chemical today adopted as a large-scale industrial process to make pure mechanism which is consistent with the structure of the catalytic hydrogen gas from carbon monoxide. This commercial process centre of PSII and which shares striking similarities with the requires shifts in reaction temperature from high (approximately proposed mechanism of water splitting by Fe-Ni CODH13. 400 °C with an iron-chromium oxide catalyst) to low (approximately I, together with my colleagues at Imperial College London14, 225 °C with copper-based catalysts)2. concluded from X-ray diffraction analysis of PSII crystals at 3.5 Å However, in the case of Fe-Ni CODH, the generated reducing resolution, that the oxygen-generating catalytic centre of PSII 2+ “ ” equivalents are produced at ambient temperatures and are used consisted of a Mn3Ca O4 cubane with a fourth dangler Mn to drive the reductive chemistry of the organism rather than attached to the cubane via one of its bridging oxo bonds. Further fi 2+ 15 producing hydrogen gas, usually by electron transport involving re nement of this Mn4Ca O4 model at 1.9 Å by Umena et al. 3,4 fi nearby Fe4-S4 centres . con rmed this geometry but added one additional bridging oxo For PSII, it was elegantly demonstrated that it took four flashes of between the external dangler Mn and the cubane, to make a 2+ 2+ light to obtain the maximum yield of molecular oxygen which was Mn4Ca O5 cluster (see Fig. 2a). Recently, a Mn4Ca O4 cluster fi interpreted as a catalytic cycle consisting of ve main states (S0 to S4), has been synthesized as a pure inorganic molecule with no whereby oxidizing equivalents of about the same potential were protein ligands16 and is essentially identical to that originally sequentially accumulated at the catalytic site with each flash proposed by Ferreira et al.14. As far as it is known, no other 5 (Fig. 1) . The S4 state stores the four oxidizing equivalents needed manganese enzyme has a comparable structure but the catalytic to oxidize two water molecules and in so doing reverts back to the centre of Fe-Ni CODH has remarkably similar geometry (Fig. 2b). 13,17 S0 state. The oxidizing equivalents are stored by four Mn ions in X-ray crystallography at high resolution has shown that Fe-Ni “ ” the catalytic centre leading to four Mn(IV) ions in the S3 state CODH consists of an Fe3NiS4 cubane with a fourth dangler Fe 6 just before the last photochemical step to the S4 state . The precise attached to the cubane via a bridging S of the cubane and an

Department of Life Sciences, Imperial College London, Sir Ernst Chain Building, South Kensington Campus, London SW7 2AZ, UK. *e-mail: [email protected]

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O a b c 2 Mn2 Fe3 hν 2H2O Ni Fe2 P680 Mn3 Ca Y S + z 4 1 ms H+ Fe4 Fe1 P680·+ H+ Mn4 Mn1 ·+ Yz S0 S + Figure 2 | Comparison of the Mn Ca2+O cluster of PSII with Fe Ni2+S 3 + Y 4 5 4 5 H z 2+ 30 µs P680 cluster of CODH. a,MnCa O cluster of PSII at 1.9 Å using PDB 3WU2 ·+ + 4 5 Y H 2+ z 350 µs (ref. 15). b,FeNi S cluster of CODH at 1.03 Å using PDB 4UDX (ref. 49). P680·+ 4 5 ·+ hν c, Overlay of the two structures with a RMSD of 0.65 between the metal Yz · Y S P680 + z S2+ 100 µs 1 atoms. Oxo bonds are in red, sulfur bonds in yellow, manganese ions in P680 purple, calcium and nickel ions in green as labelled and iron ions in orange. Y ·+ z Yz hν cluster of PSII derived from X-ray crystallography by myself and P680·+ P680 colleagues was consistent with this proposed mechanism and this was discussed in our paper14. Importantly, the dangler Mn4 was ν h found to be immediately adjacent to the Ca2+ and close to side chains of several key amino acids, including the redox-active Figure 1 | The S-state cycle showing how the absorption of four photons of tyrosine, Y , and therefore we suggested that Mn4 and Ca2+ form hv Z light ( ) by P680 drives the splitting of two water molecules and a ‘catalytic’ surface outside the cubane for binding the two substrate formation of O through a consecutive series of five intermediates (S ,S, 2 0 1 water molecules and their subsequent oxidation. The progression S ,S and S ). Protons (H+) are released during this cycle except for the S 2 3 4 1 through the S-state cycle would lead to deprotonation of these to S transition. Electron donation from the Mn Ca2+ cluster to P680•+ is 2 4 water molecules and the formation in S of a highly acidic electro- aided by the redox-active tyrosine Y . Each step involves a single oxidation 4 Z philic Mn(V)-oxo or Mn(IV)-oxyl radical, and to a nucleophilic of a Mn ion in the cluster, starting at S with 3 Mn(III) + Mn(IV) advancing 2+ 0 hydroxyl on the Ca . The formation of the O–O bond of dioxygen to S with 4 Mn(IV). The exact oxidation state of S is unknown but could 3 4 would result from the nucleophilic attack of hydroxyl on the electro- be 3 Mn(IV) + Mn(V) or 3 Mn(IV) + Mn(IV)-oxyl radical (see below). Also philic oxo of Mn4 with a possible peroxide intermediate. The elec- shown are half-times for the various steps of the cycle. trophilicity of Mn(V)-oxo would be enhanced by the high oxidation level of the cubane (3xMn(IV) plus a net positive charge generated additional S bridge, to make an Fe4NiS5 cluster as shown in Fig. 2b. during the S1 to S2 transition). Therefore, this mechanism of Despite the two catalytic centres being composed of different oxygen atom transfer is very similar to that proposed for Fe-Ni elements, the overlay of their structures, shown in Fig. 2c, is CODH13 and shown diagrammatically as a comparison in Fig. 3. remarkably good with a respectable root-mean-square deviation We also identified seven amino acids as possible ligands to the 14 (RMSD) of 0.65 between the metal atoms. In fact, they are the Mn4Ca cluster , six from the D1 reaction centre protein: only known examples of catalytic centres having heterocubanes D1Asp170, D1Glu189, D1His332, D1Ala344, D1Glu333 and with an additional metal in exo. D1Asp342 and CP43Glu354 of the inner antenna chlorophyll- It is known that, in CODH, the CO is activated by binding to the binding protein, CP43. That these seven amino acids are ligated 2+ fi Ni of the cubane which causes a shift in the coordination of the Ni to the Mn4Ca cluster has been con rmed in the 1.9 Å structure of atom from square-planar to a square-pyramidal geometry13 and Umena et al.15 and, importantly, the precise details of the ligation increases the electrophilicity of CO. Similarly, water is activated by pattern revealed. They found that the coordination requirements forming a complex with the dangler Fe42+. This induces a move- of the three Mn ions of the cubane are totally satisfied by amino ment of the cysteine Ni ligand leading to the possible formation acid ligands. However, Umena et al.15 assigned two water ligands for of a carboxyl bridge between the Ni2+ and Fe4 (ref. 13). Thus, the dangler Mn4 (W1 and W2) and for the Ca2+ (W3 and W4) which mechanism is a base-catalysed nucleophilic attack by the would be consistent with the position of the substrate waters for Fe4-bound water/hydroxyl group onto CO. A decarboxylation of the nucleophilic attack mechanism depicted in Figs 3a and 4. the intermediate leads to CO2 release and the reduction of the As shown in Fig. 1, as the catalytic cycle proceeds from S0 to S4, 13 metal cluster . The oxidation back to the active state involves protons are released at each S-state step except for the S1 to S2 electron transfer to nearby Fe4-S4 centres including ferredoxin or transition, where the metal cluster accumulates one positive to a closely bound acetyl-CoA synthase depending on species4. charge. The redox-active Yz is a neutral tyrosyl radical where its Given the striking geometrical similarities between the catalytic phenolic proton is donated to a nearby base, D1His190, to which centres of CODH and PSII, together with the fact they both catalyse it is hydrogen bonded. Therefore, the neutral Yz radical is redox the release of reducing equivalents from water, it would be reason- adjusted to be an ideal candidate for facilitating a proton-coupled able to conclude that there may be common features in their cataly- electron transfer from the catalytic centre, as originally suggested tic mechanisms. Indeed, the idea that O–O bond formation in PSII by Hoganson and Babcock20. An important study21 indicates the resulting from a base-catalysed nucleophilic attack of a hydroxyl coupling is sequential, with a strictly alternating removal of group onto an electrophilic oxo, derived from the deprotonation electrons and protons rather than hydrogen atom transfer as of the second substrate water molecule, was suggested from suggested by Babcock and colleagues20,22. The lack of proton 18 studies on synthesized Mn complexes by Pecoraro et al. . Despite release during the S1 to S2 transition has been explained by the 2+ there being no crystal structure of the Mn4Ca cluster at that formation of an additional intermediate by deprotonation before time they proposed that a “nucleophilic attack by a calcium- transfer to Yz, resulting in the storage of a positive charge. The ligated hydroxide on an electrophilic oxo group ligated to a high- nature of this intermediate is unknown. valent manganese to achieve the critical O–O bond formation”. As stated above, the dangler Mn4 and the Ca2+ each bind two The high valent state was taken to be Mn(V) in line with the sugges- water molecules according to the 1.9 Å structure of PSII15. This tion of Limburg et al.19 but could also be Mn(IV)-oxyl radical. The may indicate a ‘carousel mechanism’ to provide efficient delivery of 2+ 2+ Ca would act as a weak Lewis acid. The structure of the Mn4Ca substrate water molecules to the active site and allow maximum

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hν a Highly electrophilic oxo H O IV 2 H2O (or Mn oxyl radical) 2+ 2+ Nucleophilic attack H2O Ca H O Ca OH O 2 2+ H2O O Ca O H+/e– OH O III III Mn4 O MnIV Mn IV O O1 4 O Mn hν III MnIII IV O H2O O Mn H O O MnIII Mn 3 O 2 V IV O Mn O5 + Mn S S 4 1 0 1 – IV e IV Mn H2O O4 Mn 2 O 3 2 Ca2+ O2 2H O H2O H+ 2 O S2 OH − + O H2O+H2O O2 + 4e + 4H – III + IV 4e Mn4 O Mn IVMnIV H OOMn S3 2 O b Nucleophilic attack H2O S4 H O O C Ni2+ 2 OH Ca2+ S OH Ca2+ + – HO O O O H /e S O OH O hν Mn V O + IV IV + Fe S Fe 4 Mn Mn4 O MnIV 4 1 IV Fe O MnIVMn IV MnIV 2 H2O H+/e–H2O O Mn S Fe3 O O S − + hν CO + H2O CO2 + 2e + 2H

Figure 3 | A structurally based diagrammatic comparison of a base Figure 4 | Diagrammatic representation of a mechanistic scheme for water nucleophilic attack of a hydroxyl on to an electrophile leading to oxygen splitting and dioxygen formation in PSII based on the arguments and – atom transfer. a,O O formation in PSII in the S4 state. b, CO conversion to discussion presented in this communication. The substrate water molecules CO2 in Fe-Ni CODH. Note that the numbering of Fe ions in b are different to and products of the oxidation reactions for each S state are shown in red. in PDB 4UDX so as to compare with numbering of the Mn ions in a taken Intermediates that may exist between the S-state transitions21 are not from PDB 3WU2 (ref. 15). Also note that, unfortunately, the numbering of depicted, nor is the possible peroxide intermediate just before O–Obond 2+ the Mn4Ca O5 in PDB 3WU2 (ref. 15) differs from that of the earlier formation. Although the electrophilic oxo in the S4 state is shown attached 2+ Mn4Ca O4 in PDB 1S5L (ref. 14). As shown in the S4 state of the OEC, to Mn(V), it could equally be incorporated in a terminal Mn(IV)-oxyl radical − there is the storage of a net positive charge resulting from the S1 to S2 as indicated in Fig. 3. Note that the four electrons (4e ) generated as the – 2+ transition (see Figs 1 and 4). O O bond forms reduce the Mn4Ca cluster to its S0 state. turnover rates for the S-state cycle rather than being restricted by the of substrate water into the cubane to generate the S3 state. Whatever rate of water diffusion to the catalytic site just before formation of S0. turns out to be right, there are indications from electron spin resonance fi The idea of a carousel mechanism was rst proposed by Batista and spectroscopy that the S2 state consists of two inter-convertible colleagues, but for a different mechanism for O–Obondformation23. isoenergetic forms differing in their valence distribution and The hydroxyl/water nucleophilic attack mechanism for O–Obond multiplicity without there being any significant structural formation in PSII makes good chemical sense, as it does for change32. In the case of CODH, crystal structures obtained for the O–CO bond formation in CODH. Indeed, the former has been intermediate redox states of its catalytic cycle show that the fi demonstrated in Mn complexes synthesized to check this mechan- Fe3NiS4 cubane does not undergo any detectable modi cation in ism and the involvement of Mn(V)24,25. Moreover, the nucleophilic its structure during catalyses13. attack mechanism is a dominant reaction for water splitting and Figure 4 is a mechanistic scheme for water splitting and dioxygen oxygen formation by mononuclear Ru complexes oxidized by Ce4+, formation in PSII based on the arguments and discussion presented where the electrophile is Ru(V)=O and the nucleophile is solvent in this communication. This acid-base, hydroxyl/water nucleophilic water26,27. Some of these Ru complexes have very high turnover attack mechanism for O–O bond formation, first speculated by rates, often matching that of PSII28. The nucleophilic attack Wydrzynski and colleagues33, advocated by Pecoraro et al.18 and mechanism for Fe-Ni CODH has been concluded from X-ray presented on a structural basis by Ferreira et al.14, has been extensively diffraction analyses of the enzyme in different redox states13. championed by Brudvig and associates over several years11,34–36. An important common feature of the two enzymes is that the It must be said that, to date, there has been no overwhelming distances between Mn4 and Ca2+ and Fe4 and Ni2+ are essentially direct experimental evidence of the legitimacy of the mechanism identical, at 3.8 Å and 3.6 Å. Moreover, both reactions occur with shown in Figs 3a and 4 or of any alternative mechanisms. Mn(V) minimum overpotentials; just over 1 eV is available for each has not been detected experimentally in the OEC, possibly S-state transition, generating about 4 to 4.5 V to drive the oxidation because of its very short lifetime. Alternatively, the iso-energetic of two water molecules where the minimum thermodynamic energy Mn(IV) oxyl radical may be the active species on Mn4 as stated requirement is 1.23 eV. In the case of CODH, the energy available throughout the above discussions. Although water replacement 18 16 from CO oxidation to CO2 is about 0.56 eV, and indeed this using O /O isotope experiments has shown that both substrate enzyme is able to work in reverse under ambient and cellular waters are already bound by the S2 state37, they have not differen- conditions, although the rate of the back reaction is very slow4,13. tiated between the nucleophilic attack mechanism advocated in Because both enzymes operate on the ‘thermodynamic edge’ it is this article and other possibilities38. This approach has concluded, unlikely that their reaction mechanisms involve large energy-requiring however, that one of the substrate water molecules is bound to the structural changes such as breakage and reformation of the oxo or Ca2+ as required for the nucleophilic attack mechanism39. sulfur bonds of the cubane, as advocated in some suggested In principle, the new technique of femtosecond X-ray free- mechanisms for PSII water oxidation29,30. This therefore questions electron laser (XFEL) diffraction could provide evidence for or the involvement of oxyl radical formation in the cubane before against this proposed mechanism and a start in this direction is O–O bond formation as proposed by Siegbahn30. Initially, underway40–42. The recent XFEL study by Young et al.43 obtained Siegbahn favoured the nucleophilic attack mechanism involving a diffraction data at room temperature with a resolution of 2.0 Å for 31 terminal Mn(IV)-oxyl radical but according to his calculation, the S1 state and compared it with the XFEL structure of S1 obtained using density function theory, this mechanism has a higher at cryogenic temperature42. There were no large structural differences. fl energy barrier than the oxyl-oxo mechanism involving the insertion After giving two ashes of light to obtain a high population of the S3

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© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. HYPOTHESIS NATURE PLANTS state, the diffraction data did not provide any evidence of structural 14. Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J. & Iwata, S. Architecture changes indicative of substrate water insertion into the cubane as of the photosynthetic oxygen-evolving center. Science 303, 1831–1838 (2004). required by the Siegbahn mechanism29,44. The binding of NH 15. Umena, Y., Kawakami, K., Shen, J. R. & Kamiya, N. Crystal structure of oxygen- 3 evolving photosystem II at a resolution of 1.9 Å. Nature 473, 55–60 (2011). was used as a water analogue to investigate the mechanism of 16. Zhang, C. et al. A synthetic Mn4Ca-cluster mimicking the oxygen-evolving O–O bond formation. Unfortunately, the resolution of the diffraction center of photosynthesis. Science 348, 690–693 (2015). data (2.8 Å) was not good enough to identify a specific mechanism. 17. Dobbek, H., Svetlitchnyi, V., Gremer, L., Huber, R. & Meyer, O. Crystal structure However, one suggestion was a nucleophilic attack of a Ca2+ water of a carbon monoxide dehydrogenase reveals a [Ni-4Fe-5S] cluster. Science 293, – ligand onto the bridging oxo (O5) between the cubane and Mn4 1281 1285 (2001). 12 18. Pecoraro, V. L., Baldwin, M. J., Caudle, M. T., Hsieh, W. Y. & Law, N. A. A proposal which had previously been proposed by Yamanaka et al. .Tomy for water oxidation in photosystem II. Pure Appl. Chem. 70, 925–929 (1998). mind, this seems an unlikely alternative to the involvement of a 19. Limburg, J., Brudvig, G. W. & Crabtree, R. H. O2 evolution and permanganate Mn4-terminal oxo, which in its triple-bonded state is an extremely formation from high-valent manganese complexes. J. Am. Chem. Soc. 119, strong acid45, and also if it did occur it would result in the energe- 2761–2762 (1997). tically unfavourable opening up and reformation of the cubane 20. Hoganson, C. W. & Babcock, G. T. A metalloradical mechanism for the generation of oxygen from water in photosynthesis. Science 277, 1953–1956 (1997). during the S4 to S0 transition. Also, it is important to note that, 21. Dau, H. & Haumann, M. Eight steps preceding O–O bond formation in 46,47 unlike O4, O5 forms a stable oxo bond in synthesized mimics oxygenic photosynthesis—a basic reaction cycle of the photosystem II although it is lengthened in the OEC as emphasized by manganese complex. Biochim. Biophys. Acta 1767, 472–483 (2007). Umena et al.15. Despite my reservations, the most recent XFEL 22. Tommos, C. & Babcock, G. T. Oxygen production in nature: a light-driven 31, – studies at 2.35 Å48 indicate some structural changes in the vicinity metalloradical enzyme process. Acc. Chem. Res. 18 25 (1998). – 23. Askerka, M., Wang, J., Vinyard, D. J., Brudvig, G. W. & Batista, V. S. 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A molecular ruthenium catalyst with water-oxidation activity reducing equivalents from water. comparable to that of photosystem II. Nat. Chem. 4, 418–423 (2012). 29. Siegbahn, P. E. M. A structure-consistent mechanism for dioxygen formation in photosystem II. Chemistry 14, 8290–8302 (2008). Data availability. – No datasets were generated or analysed during the 30. Siegbahn, P. E. M. O O bond formation in the S4 state of the oxygen evolving current study. complex in photosystem II. Chem. Eur. J. 12, 9217–9237 (2006). 31. Siegbahn, P. E. & Crabtree, R. H. Manganese oxyl radical intermediates and Received 25 November 2016; accepted 3 March 2017; O–O bond formation in photosynthetic oxygen evolution and a proposed role 121, – published 3 April 2017 for the calcium cofactor in photosystem II. J. Am. Chem. Soc. 117 127 (1999). 32. Zimmermann, J. L. & Rutherford, A. W. Electron paramagnetic resonance

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45. Winkler, J. R. & Gray, H. B. in Molecular Electronic Structures of Transition R. Huber, who in 2007 told me about Fe-Ni CODH after a lecture I gave on PSII at the Metal Complexes I (eds Mingos, D. M. P., Day, P. & Dahl, J. P.) 17–28 University of Cardiff, UK. Finally, I most sincerely thank J. Murray, who helped me with the (Springer, 2011). construction of Fig. 2, and R. Malkin, who encouraged me to write this paper after ’ 46. Mukherjee, S. et al. Synthetic model of the asymmetric [Mn3CaO4] cubane core discussing my ideas with him in the quiet and peaceful ambience of Lago d Orta, Italy in of the oxygen-evolving complex of photosystem II. Proc. Natl Acad. Sci. USA September 2016. 109, 2257–2262 (2012). 47. Kanady, S., Tsui, E., Day, M. & Agapie, T. A synthetic model of the Mn3Ca subsite Additional information of the oxygen-evolving complex in photosystem II. Science 333, 733–736 (2011). Reprints and permissions information is available at www.nature.com/reprints. 48. Suga, M. et al. Light-induced structural changes and the site of O=O bond Correspondence and requests for materials should be addressed to J.B. formation in PSII caught by XFEL. Nature 543, 131–135 (2017). How to cite this article: Barber, J. A mechanism for water splitting and oxygen production in 49. Fesseler, J., Jeoung, J.-H. & Dobbek, H. How the [NiFe4S4] cluster of CO 54, photosynthesis. Nat. Plants 3, 17041 (2017). dehydrogenase activates CO2 and NCO. Angew. Chem. Int. Ed. 8560–8564 (2015). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements I want to dedicate this paper to the late G. Babcock, who influenced much of my thinking Competing interests about the mechanism of water oxidation in PSII over the years. I would also like to thank The authors declare no competing financial interests.

NATURE PLANTS 3, 17041 (2017) | DOI: 10.1038/nplants.2017.41 | www.nature.com/natureplants 5