Water oxidation in photosystem II
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Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55–60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.
KeywordsPhotosystem II Oxygen-evolving complex Water binding Triplet oxygen formation EPR spectroscopy Quantum chemical calculations
More than three billion years ago, the cyanobacteria evolved a light-driven enzyme that was able to split water into molecular oxygen and hydrogen. Biology couples this to the reduction of carbon dioxide (CO2) to carbohydrates and in so doing stores the energy of the sun in energy-rich compounds with water acting as the electron source. We call this whole process photosynthesis and it represents the central metabolic pathway of the biosphere. While there exists a wide variety of photosynthetic organisms from cyanobacteria to algae and higher plants, the cellular machinery responsible for water splitting is unique and uses the same mechanism across all species.
We benefit from photosynthesis in many ways. It is our only source of food and supplies us with much of our energy in the form of fossilized photosynthetic material—oil, coal and natural gas. Furthermore, it provides us with valuable natural materials like wood, paper, cotton, peat and biomass in general. Plants also contain many important bioactive materials that are used as drugs, in cosmetics, as natural dyes etc.
A basic understanding of the way how Nature stores energy and, in particular, uses sunlight to split water, could open future pathways to harvest and store the sun’s abundant energy, satisfying the energy demands of society in a sustainable fashion i.e., by an “artificial photosynthesis” (acatech et al. 2018; Chabi et al. 2017; Collings and Critchley 2005; Cox and Lubitz 2013; Cox et al. 2015; Faunce et al. 2013; Gratzel 2005; McKone et al. 2016; Messinger et al. 2014; Nocera 2012, 2017; Wydrzynski and Hillier 2012). In the short review below the current knowledge of the principles of photosynthetic water oxidation in photosystem II of oxygenic organisms are described with special emphasis on the recent X-ray crystallographic data and the results obtained on the electronic structure of the water oxidizing cluster and the water binding and processing derived from advanced EPR techniques in combination with quantum chemical calculations. For a more extensive and detailed description of photosynthetic water oxidation in PS II the reader is referred to some recent reviews (Junge 2019; Pantazis 2018; Shen 2015; Vinyard and Brudvig 2017; Yano and Yachandra 2014).
The structure of photosystem II and the oxygen-evolving complex
In oxygenic photosynthesis, sunlight is collected by the light-harvesting complexes (LHCs), specialized chlorophyll (and carotenoid) containing proteins, which show a large variation depending on the type of organism (Croce and van Amerongen 2014). The light energy is funneled to a reaction center (RC) where charge separation across the photosynthetic membrane takes place. Two RCs—photosystem I and II (PS I, PS II)—work in tandem to create the redox potential necessary to drive the coupled reactions (Blankenship 2014). This process provides electrons with sufficiently negative potential needed to reduce NADP+ to NADPH, which can be considered “bound biological hydrogen”. The electrons for this process originate from water oxidation that takes place in PS II. This process, and subsequent reactions, also generate a proton gradient across the photosynthetic membrane, which is used by the enzyme ATPase to produce ATP from ADP (Junge and Nelson 2015). In a different cell compartment, ATP and NADPH are then used to reduce CO2 to carbohydrates in the so-called dark reactions (Calvin–Benson cycle) (Calvin 1962).
Much of our current understanding of the photosynthetic machinery has come from high-resolution crystal structures of its constituents, particular photosystem I and II. The first crystal structure of a bacterial photosynthetic reaction center (BRC) was reported by Deisenhofer, Michel and Huber in the early eighties (Deisenhofer et al. 1985; Michel 1982, 1983)—who subsequently received the Nobel Prize in 1988 for this work. The structure of the simpler BRC provided a model for the functional core of PS I and PS II, in particular the processes of charge separation and progressive radical pair migration. It did not, however, provide any information on the water oxidizing complex (WOC) which is also called the oxygen-evolving complex (OEC), as this component is not found in the BRC that instead uses another electron source.
A crystal structure of a functional oxygen-evolving PS II core complex took almost two decades to be obtained. This was first achieved by the groups of Horst T. Witt and Wolfram Saenger in Berlin who reported the first crystallization of PS II from the cyanobacterium Synechococcus (S.) elongatus (later renamed Thermosynechococcus (T.) elongatus) and obtained a structure with 3.8 Å resolution in 2001 (Zouni et al. 2001). Although this first crystallographic structure provided valuable insight into the arrangement of the protein subunits and the cofactors of PS II it did not allow the positions of the atoms in the OEC, a tetranuclear manganese cluster, to be determined. This is because of radiation damage induced by the intense X-ray beam used for X-ray diffraction (XRD) data collection in modern synchrotrons. X-ray absorption spectroscopy (XAS) experiments with varying beam intensity by Yano and coworkers have shown photoreduction of the oxidized Mn ions, disintegration of the complex and blurring of the electron density associated with the cluster (Yano et al. 2005). Before the advent of a PS II crystal structure with sufficient resolution attempts have been made to obtain models of the tetranuclear Mn cluster from spectroscopic experiments, in particular from XAS spectroscopy (Yachandra et al. 1996; Yano et al. 2006). In 2004 the group of James Barber (Imperial College London) presented an improved structure of PS II from S. elongatus at 3.5 Å resolution (Ferreira et al. 2004). Based on their refined data, including anomalous diffraction to identify the Ca, and also on previous EXAFS data (Robblee et al. 2001) the authors proposed a heterometallic cubane-type structure of the OEC, which contained a Mn3Ca unit along with a more distant dangler Mn. Such a “dangler model” had been proposed earlier by David Britt’s group (UC Davis) based on constraints from EPR and ENDOR experiments and the distances obtained from EXAFS (Peloquin and Britt 2001; Peloquin et al. 2000). Finally, in 2011 the groups of Jian-Ren Shen (Okayama, Japan) and Nobuo Kamija (Osaka, Japan), who had reported a low resolution structure of PS II in 2003 (Kamiya and Shen 2003) published a significantly improved structure from Thermosynechococcus (T.) vulcanus at 1.9 Å resolution, in which the atomic arrangement of the ions in the OEC was finally resolved (see below) (Umena et al. 2011). Subsequent theoretical studies showed that the actual oxidation state of the OEC in this structure was a mixture of overreduced states, not present in the catalytic cycle (Galstyan et al. 2012; Luber et al. 2011). The Shen group also succeeded in obtaining a structure with reduced radiation damage using femtosecond (fs) pulsed X-ray crystallography with a free electron laser (XFEL) (Suga et al. 2015). More recently, time resolved XFEL measurements (serial femtosecond crystallography—SFX) near ambient temperature has emerged as an important tool for the investigation of the various states of the catalytic cycle of the OEC (see below) (Kern et al. 2012, 2013, 2014, 2018; Kupitz et al. 2014; Suga et al. 2015, 2017).
Basic function of photosystem II: kinetics and energetics of water oxidation
Light excitation of the reaction center of PS II, a multi-pigment assembly of four chlorophyll-a and two pheophytin-a molecules, leads to primary charge separation on a picosecond timescale. This is a single electron transfer process resulting in a charge-separated radical pair (RP) state comprising the radical cation P680·+ and the radical anion Pheo·− (Cardona et al. 2012). During primary charge separation, the positive charge is initially localized on the accessory chlorophyll ChlD1 and it is stabilized by subsequent transfer on PD1 (see Fig. 2A) (Groot et al. 2005; Holzwarth et al. 2006; Kammel et al. 2003). The primary acceptor Pheo is the pheophytin pigment bound to the D1 protein (Holzwarth et al. 2006). The oxidation potential of P680·+ is estimated to + 1.2 to + 1.3 V, the highest known in biology (Rappaport et al. 2002). The primary RP P680·+Pheo·− is stabilized by subsequent electron/hole transfer steps. This is important as the radical cation can, in principle, oxidize neighboring chlorophylls, and the protein itself. To suppress both side and back reactions, nature has placed secondary donor/acceptors in close proximity to the primary RP. On the donor side of the protein (lumen), it is the redox-active tyrosine residue D1-Tyr161 (known as TyrZ or Yz) next to P680·+ that is able to quickly donate an electron to this species on a nanosecond (20–250 ns) time scale, thereby reducing the radical cation to its initial state P680. The tyrosine radical cation Y z ·+ is stabilized by losing a proton to the neighboring D1-His190 in a reversible fashion, forming a neutral tyrosine radical (Y z · ) (Berthomieu et al. 1998; Chu et al. 1995; Saito et al. 2011). Y z · in turn oxidizes the adjacent Mn cluster in 40–1600 µs dependent on the Si transition (Babcock et al. 1976; Brettel et al. 1984; Dekker et al. 1984a; Haumann et al. 2005a; Karge et al. 1997; Klauss et al. 2012a; Noguchi et al. 2012; Rappaport et al. 1994; Razeghifard and Pace 1997). On the acceptor side of the protein (cytoplasm), the acceptor Pheo·− passes its electron to the bound plastoquinone molecules (to QA and subsequently to QB). Reducing equivalents derived from this process are temporarily stored as reduced plastoquinol (QBH2), a mobile electron carrier, generated after two light absorption/charge separation and protonation events.
Other light-induced processes that occur in PS II are equally dangerous, for which nature has not found a perfect solution to circumvent them. This is the creation of triplet states, e.g., of the chlorophylls in the core antenna via intersystem crossing or in the RC via (triplet) radical pair recombination. While carotenoid molecules are present in PS II to suppress (quench) triplet chlorophyll formation, these states can still react with triplet oxygen (3O2) formed in the water oxidation process yielding singlet oxygen (1O2) (Macpherson et al. 1993). This very aggressive species can then react with the pigments and/or the protein, resulting in damage of the D1 protein subunit of the PSII supercomplex. The production of such reactive oxygen species (ROS) (Vass 2012) limits the lifetime of PS II to only 30 min under normal light conditions. Owing to its remarkable efficiency (the turnover time of the OEC is ≈ 2 ms, that of the whole PS II ≈ 10 ms)(Vinyard and Brudvig 2017), it performs more than 105 reaction cycles before it must be replaced. Fortunately, all organisms performing oxygenic photosynthesis have developed an efficient repair mechanism for the PS II supercomplex by the discrete replacement of the D1 protein (Nixon et al. 2010).
PS II thus acts as a water:plastoquinone oxidoreductase (Wydrzynski and Satoh 2005). The products are molecular (triplet) oxygen (3O2), protons and reduced plastohydroquinone. The light-induced process in PS II is very efficient, with a quantum yield of over 90% and an energy efficiency of about 20%. This value is, however, strongly attenuated when all the subsequent processes are considered, such that the total biomass generated often contains only less than 1% of the original light energy input (Blankenship et al. 2011; Michel 2008).
In this sequence, YZ promotes both electron and proton transfer in the catalytic cycle displaying a dual function (Bovi et al. 2013; Klauss et al. 2012b; Perez-Navarro et al. 2016). Between Si and Si+1 states (for i = 0–3) the short-lived SiYZ˙ intermediates exist. Studying these intermediates is critical for understanding the role of YZ˙ in water oxidation and especially in proton removal from the Mn4OxCa cluster (Boussac et al. 2008; Havelius et al. 2010; Nugent et al. 2002; Peloquin et al. 1998; Petrouleas et al. 2005; Retegan et al. 2014; Styring et al. 2012).
The electronic structure of the Mn4OxCa cluster
A detailed understanding of the catalytic process of water oxidation in PS II requires knowledge of the electronic structure, i.e., the distribution of the electrons in the cluster, in all consecutive reaction steps. The oxidation and spin states of the Mn ions, representing the total number and configuration of electrons in the Mn valence orbitals, give a basic description thereof. These together with the magnetic interactions between the spin-bearing Mn ions, depending to a large part on the metal ligands, provide a comprehensive picture of the respective electronic state, which governs the chemical and catalytic properties of each S state. Thus, the spin states provide information about how the structure of the cofactor evolves during the S-state cycle, for a recent review see (Krewald et al. 2016).
An experimental method to investigate the electronic structure of transition metal complexes is electron paramagnetic resonance (EPR) spectroscopy (Goldfarb and Stoll 2018; Schweiger and Jeschke 2001; Weil and Bolton 2007). It exploits a fundamental property of matter, which is that unpaired electrons have an intrinsic angular momentum (spin), which can be excited by microwave radiation in a magnetic field. The unpaired electron spin also interacts with other electron and nuclear spins as well as with local electric field gradients, making it a sensitive reporter of its chemical environment. It is thus analogous to other magnetic spectroscopies such as nuclear magnetic resonance (NMR). Since the Mn ions are open-shell species, i.e., exhibit orbitals with single electron occupancy, whereas most of the electrons of the protein and solvent surrounding are paired, the EPR signals of the Mn4OxCa cluster can be detected selectively. It has also been shown that all S states in the Kok cycle can be trapped (except for the elusive S4 state) and that all exhibit paramagnetism (Haddy 2007).
In the S2 state, in addition to the low-spin Seff = ½ form showing the characteristic multiline signal around g ≈ 2, the cluster can also be found in a high-spin Seff = 5/2 state under certain conditions. This is evident from an EPR signal around g = 4.1 (see Fig. 5), which has been observed earlier by several research groups (Boussac et al. 1996; Casey and Sauer 1984; Haddy et al. 2004; Zimmermann and Rutherford 1984). Pantazis et al. could show computationally that the two electronic structures are a direct consequence of two different spatial conformations of the manganese cluster, namely a “closed cubane” (Seff = 5/2) and an “open cubane” (Seff = ½) form, which have almost the same energy (Bovi et al. 2013; Isobe et al. 2012; Pantazis et al. 2012). In these two structures one of the oxygen bridges, O5, is occupying different positions. In turn this also led to a change of the MnIII position (Jahn–Teller ion) and thus of the open coordination site of the cluster resulting in a change of the precise electronic properties of the cofactor. EXAFS data also support this model (Chatterjee et al. 2019). This shows that the effective spin Seff is a crucial parameter for describing a particular state of the cluster and assigning it a spatial structure. It is postulated that O5 has not a fixed position but toggles between two structures in the dynamic S2 state, which is important for the water binding and the catalytic mechanism (see below).
Modifications of the Mn4OxCa cluster, such as exchange of Ca2+ with Sr2+ (Boussac and Rutherford 1988; Boussac et al. 2015; Chu et al. 2000; Cox et al. 2011; Koua et al. 2013; Yachandra and Yano 2011), its complete removal (Boussac et al. 1989; Lohmiller et al. 2012; Styring et al. 2003; Vrettos et al. 2001b) and the binding of small molecules (Beck et al. 1986; Perez-Navarro et al. 2013; Oyala et al. 2015; Oyala et al. 2014; Retegan and Pantazis 2016; Su et al. 2011), provided further insight into the conformation and number of ligands of individual Mn ions (coordination geometry) and substrate access in the S2 state. Upon Ca depletion water oxidation functionality is lost, but the S2 state (S2′) is still formed (Boussac et al. 1989, 1990). Interestingly, the EPR and 55Mn ENDOR data of S2′ show that the calcium has no substantial effect on the magnetic parameters of the S2 state; this ion is thus not crucial for maintaining the electronic structure of the tetranuclear Mn cluster (Lohmiller et al. 2012). Instead, it might serve as stage for the delivery of water molecules to the reaction site (Nakamura et al. 2016; Service et al. 2014; Ugur et al. 2016), affect the function of YZ, (Miqyass et al. 2008; Retegan et al. 2014) and introduce some structural flexibility allowing the cofactor to toggle between the different motifs of the open and closed cubane structures (see Fig. 5). The interactions of small molecules which mimic the substrate water (methanol, ammonia) also support this potential role for Ca2+. These molecules associate with the Ca2+ and Asp61 water channels that lead to the Mn4OxCa cofactor (Marchiori et al. 2018; Navarro et al. 2013; Oyala et al. 2014; Retegan and Pantazis 2016; Schraut and Kaupp 2014) (see Fig. 2D). In addition, both of these molecules reduce turnover efficiency; these results implicate that at least one (or possibly both) of these channels are involved in substrate delivery.
The magnetic resonance experiments described above together with theoretical calculations allowed a reliable characterization of the S0, S2 and S3 states with respect to oxidation and spin states of individual ions and their spin coupling in the tetranuclear Mn cluster summarized by Krewald et al. (2015, 2016). Together with information about binding of the two substrate water molecules described below spatial models of the S states could be obtained that form the basis for developing a catalytic mechanism for the OEC (Fig. 8).
Identification of the substrate water molecules in water oxidation
Knowledge of the binding and dynamics of the substrate water molecules is crucial for formulating the reaction mechanism of photosynthetic water oxidation. Candidates for substrate waters, besides the four H2O/OH− molecules (Fig. 2C) directly attached to the cluster (Ames et al. 2011; Suga et al. 2015; Umena et al. 2011), are oxygen bridges within the cluster as well as possible water molecules binding at a later point in the catalytic cycle. Apart from X-ray diffraction spectroscopic techniques are used to investigate water binding, in particular EPR (Cox et al. 2013b; Fiege et al. 1996; Kawamori et al. 1989; McConnell et al. 2012; Nagashima and Mino 2013; Rapatskiy et al. 2012) and vibrational (infrared) spectroscopy (Debus 2014, 2015; Kim et al. 2018; Kim and Debus 2017; Nakamura et al. 2016; Noguchi 2008; Sakamoto et al. 2017; Suzuki et al. 2008). These are used in conjunction with time-resolved mass spectrometry (MIMS) which detects the uptake of H 2 18 O labeled water into the product O2; for a comprehensive review on the identification of possible water substrates by MIMS see (Cox and Messinger 2013). MIMS experiments showed that the two substrate water molecules exchange at different rates in all of the S states (Hillier and Wydrzynski 2000, 2004, 2008). The more slowly exchanging water (Ws) has an exchange rate of the order of seconds (Messinger et al. 1995) while the faster exchanging water (Wf) has an exchange rate of milliseconds (Hillier et al. 1998) (see Fig. 3B). The observation of two rates implies that the two substrates bind at two chemically distinct sites. They also demonstrate that the O–O bond is not formed until the (S4) state is reached. Interestingly, it was recently observed that during the lifetime of the S3YZ˙ state, that lies between S3 and the elusive S4 state while the YZ˙ radical exists, the water exchange is slowing down, which may suggest that the two oxygen species are “arrested” in a bonding formation (Nilsson et al. 2014).
Similar timescales for the water exchange were found both for Ws in the MIMS, see ref. (Hillier and Wydrzynski 2008), and for O5 in the EPR experiments, i.e., complete exchange in < 10 s (Rapatskiy et al. 2012).
Orientation dependent EDNMR studies showed that the measured hyperfine coupling can only be assigned to O5 or O4) (Rapatskiy et al. 2012).
This is in agreement with earlier low frequency FTIR studies of the OEC that showed that a µ2-oxo or µ3-oxo bridge is exchangeable (Chu et al. 2000). It is remarkable that in the OEC a µ-oxo bridge is readily exchangeable. For structurally similar model compounds an exchange has only been observed on much longer timescales (Rapatskiy et al. 2015; Tagore et al. 2006, 2007). These results further demonstrate the dynamic nature of this O5 ligand in the OEC (see above, Fig. 5).
As described above, recent spectroscopic results, including high field EPR indicate that a new water molecule (O6) is inserted into the manganese cluster in the S3 state as an OH− at the open coordination site of Mn1 (Cox et al. 2014). Its close proximity to O5 suggests it is the second substrate Wf (Fig. 6B). How it is exactly inserted remains an open question. Recent SFX data (Kern et al. 2018; Suga et al. 2017) indicate that the Glu189, which bridges the Ca2+ ion and Mn1 in the S1 state detaches from the Ca2+ ion upon formation of the S3 state. This removes steric hindrance to direct water binding via the Ca2+ channel, i.e., it is originally one of the Ca2+ waters. Another possibility is that this OH− (O6) is introduced in an indirect fashion by toggling between the open and closed cubane structures and is thus derived from W2, one of the terminal ligands of Mn4 (compare structure S3′ in Fig. 8). Within this sequence the water that binds during the S2 to S3 transition is not one of the substrates of the current cycle, but a substrate of the next cycle (Cox and Messinger 2013). It also implies that O5 (the existing μ-oxo bridge) and O6 (the new water-derived oxygen ligand) may switch identities. There is significant theoretical support for this sequence, which explains how redox tuning of the cofactor can precede water binding (Retegan et al. 2016). This hypothesis also better agrees with MIMS data which shows both substrates are bound to the cofactor (most likely to Mn ions) in all S-states and that the rate of exchange does not dramatically change upon moving from S2 to S3 (Cox and Messinger 2013; Hendry and Wydrzynski 2002, 2004).
The water oxidation cycle in PS II
As a consequence of the above results, it is assumed in the following discussion of the catalytic cycle of the OEC that the O5 bridge derives from one of the substrate waters (Ws). A possible cycle is shown in Fig. 8 (Cox et al. 2015; Krewald et al. 2016). The first water Ws binds as an OH− (O5) in the S0 state, being deprotonated during the subsequent oxidation step to S1. In the S3 state the second substrate water enters the reaction as OH− (O6) to Mn4 (or Mn1) as suggested by the results obtained from EPR and XFEL experiments on the S3 state (Cox et al. 2014; Kern et al. 2018; Suga et al. 2017). A further oxidation and deprotonation step leads to S4. Experimental evidence shows that formation of S4 is triggered by proton release (Haumann et al. 2005a; Klauss et al. 2012a). Current theoretical models suggest that a proton is transferred from W1 (H2O) to Asp61, as in the preceding S2 to S3 transition (Siegbahn 2012). Mutants of PSII lacking Asp61 show a pH dependency of the lag phase during the S3–S0 transition which has been attributed to a deprotonation of the Mn4CaO5 (Bao and Burnap 2015; Dilbeck et al. 2012) in support of this model.
In the current literature, there are several proposed mechanisms that suggest the early onset of O–O bond formation in the S3 state (Corry and O’Malley 2018). This is mainly based on the SFX crystal structure of Suga et al. which modeled a short distance between the O5 and O6 bridges in the S3 state (Suga et al. 2017). We note that the more recent higher resolution SFX structure of the S3 state (Kern et al. 2018), does not exhibit this short O5–O6 distance, and is instead more consistent with the high field EPR structure described above. Furthermore, earlier biophysical measurements categorically rule out O–O bond formation in the S3 state. The MIMS data described above shows that both substrates still rapidly exchange with bulk water in the S3 state (Messinger et al. 1995). It is only upon oxidation and proton release to form the S3Y z · state directly preceding S4 that substrate exchange is ‘arrested’ indicating the onset of O–O bond formation, i.e., formation of a peroxo type intermediate (Nilsson et al. 2014).
How the O–O bond is precisely formed in the S4 state is still unknown, owing to a lack of experimental data. There are two popular chemical mechanisms for this transition that differ with regard to what component of the cofactor is oxidized to form S4: (i) a ligand (one of the substrate waters); or (ii) a metal (one of the Mn ions). In the case of a ligand oxidation event, forming a MnIV-oxyl moiety, O–O bond formation is proposed to proceed by a radical coupling mechanism described below. In the case of a metal oxidation event, forming a MnV-oxo type moiety, O–O bond formation is instead proposed to proceed by nucleophilic attack of the electrophilic oxygen (O5) bound to the MnV by a nearby water, e.g., W3 bound to the Ca2+. Unfortunately the nature of the last oxidation (ligand vs. metal centered) does not resolve this question. XAS data which monitors an oxidation state change of the Mn ions during the S3 to S0 transition appears to rule out a MnV intermediate (Haumann et al. 2005a), favoring instead a ligand (oxygen) oxidation. We note that while the nucleophilic attack mechanism initially envisaged the involvement of a MnV intermediate, it was later suggested to have substantial MnIV oxyl character in the S4 state based on theoretical studies (Vinyard and Brudvig 2017).
Only a nucleophilic attack mechanism has been observed for first row molecular transition metal water oxidation catalysts, for which there is mechanistic data, (Codola et al. 2015) with radical coupling to be thus far only described for second row transition metals, e.g., ruthenium (Cox and Lubitz 2013; Romain et al. 2009). Nucleophilic attack mechanisms have thus been proposed for the OEC by a number of groups (Ferreira et al. 2004; Pecoraro et al. 1998); see (Barber 2017; Vinyard and Brudvig 2017; Vinyard et al. 2015) for recent reviews. In this sequence, a nearby water (e.g., W3) attacks a manganese bound oxygen, most likely O5. Upon re-reduction of the four Mn ion and loss of the product O2, the newly inserted O6 bridge presumably fills the site vacated by O5 leading to rapid recovery of the S0 state.
In contrast the oxo-oxyl radical coupling mechanism involving two Mn bound oxygens, is most compatible with the assignment of the two substrates sites described in the previous section (O5 and O6). Such a mechanism was first proposed ten years ago by Per Siegbahn (Siegbahn 2009). Importantly high field S3 state EPR data has demonstrated that there is the requisite spin alignment of the two putative substrate oxygens and the Mn ions to which they are bound (Mn4-O5-O6-Mn1 = βαβα) on which this mechanism is based.
There are also other alternative mechanisms. A very recent example, proposed by the Yamaguchi group, involves an O–O˙ radical intermediate, formed following electron transfer to YZ (Shoji et al. 2018). Similarly, there are proposed mechanisms that do not fall into either of the two categories, such as a recent proposal from the Sun group. In this novel mechanism, the dangler Mn acts as the site of catalysis, as in a nucleophilic attack like mechanism, forming a MnVII-dioxo intermediate following charge rearrangement of the Mn cluster in the S4 state (Zhang and Sun 2018). As above for the nucleophilic attack mechanism, it is unclear if this mechanism is compatible with XAS data (Haumann et al. 2005a). For a more detailed comparison of alternative mechanisms the reader is directed to a recent in-depth review (Pantazis 2018).
Subsequently, a peroxo product (a bound O2 with a single σ bond) is formed bridging Mn3 and Mn1 (Fig. 10 [S4 peroxo]). Mn4 is now five coordinate with the product peroxide sitting along its Jahn–Teller (JT) axis (Li and Siegbahn 2015; Siegbahn 2009). Cleavage of the two remaining Mn–O bonds leads to formation of the final O2 product. Importantly the cleavage of each Mn–O bond results in an α spin electron being transferred to each of the two Mn ions that were attached to the peroxide and a β spin electron being transferred to O5 and O6. As before, this is an energetically favorable situation as the electron gained by each metal is of the same spin (α) as their existing d-electrons. The two new β spin electrons occupy two π* orbitals giving rise to the triplet O2 product. The displacement of O2, via binding of the first substrate water of the next S cycle (coupled to its deprotonation), leads to formation of the observed (metastable) S0 state (Fig. 10, S0). The O2 may detach in a stepwise fashion, during its concerted replacement with OH−, passing through a transient superoxo intermediate (Li and Siegbahn 2015).
New experimental data is needed to probe these key stages in O–O bond formation and release. SFX crystallography (Kern et al. 2014, 2018; Kupitz et al. 2014; Suga et al. 2015, 2017; Young et al. 2016) is poised to address structural changes during the S3 to S0 transition, and may suggest an alternative proton transfer sequence. Specifically, as described above, the Siegbahn mechanism starts with internal proton transfer from O6 to W1. We note, however, that recent SFX measurements indicate that Glu189 detaches from the Ca2+ ion during S-state progression. Glu189 is proximal to Mn1 and could act as an internal base, accepting a proton from O6, similar to old ideas put forward by Gerry Babcock (Hoganson and Babcock 1997). Within this revised scheme W1 does not need to undergo deprotonation—with proton transfer to Asp61 switched off due to a conformational change (gating) (Kern et al. 2018).
In biology, there is only one catalytic site that is able to split water—the OEC—and its structure and function is identical (Su et al. 2011) in cyanobacteria, algae and all higher plants; however, differences in the second coordination sphere between higher plants and cyanobacteria have been reported (Retegan and Pantazis 2017). Many other enzymes have found different ways to perform their task—but not the unique, highly optimized OEC in PS II. The work compiled above shows that this unique metal cluster requires the presence and concerted action of all four manganese ions in the Mn4OxCa complex. The oxidation states of the Mn ions are always III or IV—there exists no MnII in the whole cycle. The possible presence of a MnV in the last step (S4) has, however, not yet been finally clarified. The importance of the total electron spin state of the cluster has been highlighted—and is understandable considering the necessary restriction to form and release triplet dioxygen 3O2. The Ca2+ is probably important for efficient water delivery and the Cl− for maintaining the correct charge balance of the OEC. Water delivery is optimized through highly efficient water channels leading to the Mn cluster. In the favored model the two substrate waters bind to neighboring redox-active Mn ions, and deprotonation of these waters is coupled to the oxidation events of the cluster. Close proximity and proper alignment of the two active oxygens are assured to efficiently form the O–O bond and finally release the 3O2 molecule with high efficiency.
In spite of the great progress in the understanding of the OEC in PS II there are still several challenges to be tackled. One pertinent open question is the structure of the elusive S4 state. A possible stabilization and characterization of this state would finally complete our knowledge of the S states of the Kok cycle. A missing key experiment is further the detection of the 6th oxygen in the manganese cluster by EPR (using 17O labeling) to support the recent crystallographic XFEL (SFX) data on the S3 state (Kern et al. 2018; Suga et al. 2017). Furthermore the determination of the water binding kinetics should be followed for all state transitions by rapid freeze quench (RFQ) techniques combined with advanced EPR techniques. First experiments along these lines have successfully been performed in our laboratory using time resolved RFQ 17O EDNMR measurements (Rapatskiy et al., unpublished data). The exchange of certain amino acids in the vicinity of the OEC could shed light on the fine-tuning of its electronic structure in the S state cycle and would be important for explaining its high turnover frequency. Furthermore, a great challenge for chemists working in the field of water oxidation is the understanding and modeling of the sophisticated mechanism by which PS II repairs photo-damage of its protein, of the pigments and of the manganese cluster.
The material of the catalyst must be abundant, easily accessible, inexpensive (i.e., no precious metals), non-toxic, sufficiently stable under working conditions and should be scalable.
The required four oxidizing equivalents must be stored in the catalyst to couple the fast 1-electron photochemical reaction with the slow 4-electron chemical water oxidation process.
The binding of the two substrate water molecules should preferentially take place at two well-defined neighboring redox-active metal centers, accompanied by successive deprotonation and activation of the water molecules.
The redox steps of the catalytic metal centers should be of similar magnitude and in the range of 1 eV, and should finally lead to a concerted water oxidation, O2 formation and O2 release, to avoid reactive oxygen intermediates. This also requires a sequential release of the 4 protons.
A functional matrix mimicking the protein (in PS II) is needed for efficient transport of water to the reaction zone, release of dioxygen and for the correct proton management.
In the photochemical act, a light-induced species must be generated with sufficient oxidative power to oxidize water (+ 1.23 eV).
Effective coupling of the charge separation unit with the catalytic center (metal cluster) is necessary to diminish the overpotential (comparable to the function of the tyrosine YZ in PS II).
The catalytic unit should be stable; in case of deterioration/destruction a mechanism of self-repair or healing should be in place to avoid deactivation of the catalytic process.
At present, it is still a great challenge to create a catalyst that fulfills the above criteria and could thus compete with the Mn cluster in PS II. The future will show if at least part of the above points can be fulfilled. Attempts to synthesize water splitting chemical catalysts and devices are too numerous in the literature to be discussed here—but show the great importance of this research field for a future society using (only) renewable, sustainable energy based on sunlight driven processes (Andreiadis et al. 2011; Blakemore et al. 2015; Kanan and Nocera 2008; Kurz 2016; Najafpour et al. 2016; Tran et al. 2012; Zhang et al. 2015; Matheu et al. 2019).
Open access funding provided by Max Planck Society. Financial support of this work by the Max Planck Society and MANGAN (03EK3545) funded by the Bundesministeriums für Bildung und Forschung is gratefully acknowledged. N.C. acknowledges the support of the Australian Research Council (FT140100834).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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