No abstract

Green plants and algae oxidize water to molecular oxygen in photosystem II (PS II) within a calcium/tetramanganese site known as the water-oxidizing complex (WOC). Oxygen is generated by the WOC in a four-electron process involving a series of intermediate states (S states, labeled S 0 …S 4 ) of increasingly higher mean oxidation level. [1] Over the past decade, X-ray crystallographic (XRD) structures of PS II at progressively improved resolution [2] have revealed much detail of the WOC. At present, only PS II from thermophilic cyanobacteria has been crystallized for XRD study and the enzyme is presumed to be in the dark stable S 1 state. The first PS II structure (at 3.5 resolution) to resolve side chain positions was presented by Barber and co-workers. [2c] Consistent with subsequent studies at higher resolution, it revealed the compact Mn 3 Ca "cube" structure of the WOC connected more distantly to a single Mn, referred to as the "dangler". More recent improved structures at 3.0 and 2.9 , [2d,e] substantially clarified the metal-and proteinsupplied ligand positions within the WOC, but were still of insufficient resolution to reveal the positions of bridging oxo groups and water molecules (including the substrate water molecules). Finally, Umena et al., [2f] using a new crystallization method, produced an atomic resolution structure at 1.9 , the most resolved to date. Despite this remarkable achievement, revealing, for the first time, the positions of bridging O atoms within the Mn 4 Ca core of the WOC, aspects of the new structure have been met with scepticism. [3,4] Central concerns over this structure involve 1) the identity and unexpected placement of the O(5) moiety (Figure 1), which appears to be either a weakly bound oxo, hydroxo, or water ligand at distances of 2.4-2.7 from four of the metal atoms in the WOC, and 2) the disparity in some key metalmetal distances when compared with earlier, high-precision extended X-ray absorption fine structure (EXAFS) results [5,6] and the previous lower-resolution XRD structures (see Table 1). Although the Mn EXAFS data do not unambiguously assign the individual near (less than 3 ) metal-metal distances within the cluster, they clearly indicate that two Mn-Mn vectors of a magnitude of approximately 2.7 exist within the functional WOC in the S 1 and S 2 states. These are totally consistent with the Mn1-Mn2 and Mn2-Mn3 distances of 2.65 and 2.70 in the 2.9 resolution XRD structure, but are significantly shorter than the corresponding Mn-Mn distances of 2.80 and 2.90 seen in the 1.9 resolution XRD structure.The discrepancies between the XRD and EXAFS data have led some to suggest [3,4,7] that the crystal structures generally, and the 1.9 resolution structure in particular, have undergone photoreduction of the Mn atoms during data collection, increasing the Mn II content and distorting the cluster from the functional S 1 state to as low as S À3 . This is despite the fact that in the later studies great pains were taken to minimize such X-ray exposure. The claims of ph...

Density functional theory (DFT) calculations, at the Becke-Perdew/TZP level of theory, were used to investigate a set of CaMn(4)-containing clusters that model the active site of the water-oxidizing complex (WOC) of photosystem II (PSII). Metal-atom positions for three representative isomeric clusters of the formula [CaMn(4)C(9)N(2)O(16)H(10)](+)4 H(2)O are in good agreement with the disparate Mn(4) geometries of the three most recent X-ray crystal structures. Remarkably, interconversion between these three isomeric clusters is found to be facile, resulting from subtle changes in the coordination environment around the CaMn(4) centre. This result provides a clear rationalisation of the marked differences in reported crystal structures. Recent concerns have been raised regarding the opportunity for X-ray-damage-induced distortion of the metal-containing active centre during crystallographic analysis. Our calculations suggest that an even greater problem may be presented by the apparent fluxionality of the CaMn(4) skeleton within the active centre. Structural rearrangement may well precede crystallographic analysis, for example by the preferential "freezing-out" of one of several near-isoenergetic structures during the workup for crystallisation. This prospect, which our calculations cannot exclude, highlights the difficulties that will continue to be faced by experimentalists seeking unambiguous structural information on the WOC's active site.

Density functional theory (DFT) calculations are reported for a set of model compounds intended to represent the structure of the Photosystem II (PSII) water oxidising complex (WOC) as determined by the recent 1.9 Å resolution single crystal X-ray diffraction (XRD) study of Umena et al. In contrast with several other theoretical studies addressing this structure, we find that it is not necessary to invoke photoreduction of the crystalline sample below the S(1)'resting state' in order to rationalise the observed WOC geometry. Our results are consistent with crystallised PSII in the S(1) state, with S(1) corresponding to either (Mn(III))(4) or (Mn(III))(2)(Mn(IV))(2) as required by the two competing paradigms for the WOC oxidation state pattern. Of these two paradigms, the 'low-oxidation-state' paradigm provides a better match for the crystal structure, with the comparatively long Mn(2)-Mn(3) distance in particular proving difficult to reconcile with the 'high-oxidation-state' model. Best agreement with the set of metal-metal distances is obtained with a S(1) model featuring μ-O, μ-OH bridging between Mn(3) and Mn(4) and deprotonation of one water ligand on Mn(4). Theoretical modelling of the 1.9 Å structure is an important step in assessing the validity of this recent crystal structure, with implications for our understanding of the mechanism of water oxidation by PSII.

The most recent XRD studies of Photosystem II (PS II) reveal that the His337 residue is sufficiently close to the Mn(4)Ca core of the Water Oxidising Complex (WOC) to engage in H-bonding interactions with the μ(3)-oxo bridge connecting Mn(1), Mn(2) and Mn(3). Such interactions may account for the lengthening of the Mn-Mn distances observed in the most recent and highest resolution (1.9 Å) crystal structure of PS II compared to earlier, lower-resolution (2.9 Å or greater) XRD structures and EXAFS studies on functional PS II. Density functional theory is used to examine the influence on Mn-Mn distances of H-bonding interactions, mediated by the proximate His337 residue, which may lead to either partial or complete protonation of the μ(3)-oxo bridge on models of the WOC. Calculations were performed on a set of minimal-complexity models (in which WOC-ligating amino acid residues are represented as formate and imidazole ligands), and also on extended models in which a 13-peptide sequence (from His332 to Ala344) is treated explicitly. These calculations demonstrate that while the 2.9 Å structure is best described by models in which the μ(3)-oxo bridge is neither protonated nor involved in significant H-bonding, the 1.9 Å XRD structure is better reproduced by models in which the μ(3)-oxo bridge undergoes H-bonding interactions with the His337 residue leading to expansion of the 'close' Mn-Mn distances well known from EXAFS studies at ∼ 2.7 Å. Furthermore, full μ(3)-oxo-bridge protonation remains a distinct possibility during the process of water oxidation, as evidenced by the lengthening of the Mn-Mn vectors observed in EXAFS studies of the higher oxidation states of PS II. In this context, the Mn-Mn distances calculated in the protonated μ(3)-oxo bridge structures, particularly for the peptide extended models, are in close agreement with the EXAFS data.