Magnetic and Electronic Structural Properties of the S3 State of Nature's Water Oxidizing Complex: A Combined Study in ELDOR-Detected Nuclear Magnetic Resonance Spectral Simulation and Broken-Symmetry Density Functional Theory.

ELDOR-detected nuclear magnetic resonance (EDNMR) spectral simulations combined with broken-symmetry density functional theory (BS-DFT) calculations are used to obtain and to assign the 55Mn hyperfine coupling constants (hfcs) for modified forms of the water oxidizing complex in the penultimate S3 state of the water oxidation cycle. The study shows that an open cubane form of the core Mn4CaO6 cluster explains the magnetic properties of the dominant S = 3 species in all cases studied experimentally with no need to invoke a closed cubane intermediate possessing a distorted pentacoordinate Mn4 ion as recently suggested. EDNMR simulations found that both the experimental bandwidth and multinuclear transitions may alter relative EDNMR peak intensities, potentially leading to incorrect assignment of hfcs. The implications of these findings for the water oxidation mechanism are discussed.

S3 State Models of Nature's Water Oxidizing Complex: Analysis of Bonding and Magnetic Exchange Pathways, Assessment of Experimental Electron Paramagnetic Resonance Data, and Implications for the Water Oxidation Mechanism.

Broken symmetry density functional theory (BS-DFT) calculations on large models of Nature's water oxidizing complex (WOC) are used to investigate the electronic structure and associated magnetic interactions of this key intermediate state. The electronic origins of the ferromagnetic and antiferromagnetic couplings between neighboring Mn ions are investigated and illustrated by using corresponding orbital transformations. Protonation of the O4 and/or O6 atoms leads to large variation in the distribution of spin around the complex with associated changes in its magnetic resonance properties. Models for Sr2+ exchange and methanol addition indicate minor perturbations reflected in slightly altered spin projection coefficients for the Mn1 and Mn2 ions. These are shown to account for the observed changes observed experimentally via electron paramagnetic resonance methods and suggest a reinterpretation of the experimental findings. By comparison with experimental determinations, we show that the spin projections and resulting calculated 55Mn hyperfine couplings support the open cubane form of an oxo (O5)-hydroxo (O6) cluster in all cases with no need to invoke a closed cubane intermediate. The implications of these findings for the water oxidation mechanism are discussed.

Bonding and Magnetic Exchange Pathways in Nature's Water-Oxidizing Complex.

The nature of the bonding and magnetic exchange pathways of the water-oxidizing complex of photosystem 2 is explored using broken symmetry density functional theory. The electronic structure and superexchange pathways are illustrated and analyzed using corresponding orbitals and intrinsic bond orbitals. These demonstrate a dominating influence on the bonding and magnetic interactions by both the geometrical structure of the Mn4CaO5 core complex and the ionic interactions of the oxo bridges with the neighboring Ca2+ ion. The demonstrated ionic nature of the Ca2+ bonds is proposed to contribute to the stabilization of the oxygen atoms participating in O-O bond formation.

Localized Bond Orbital Analysis of the Bonds of O2.

A localized bond orbital analysis of the bonding in dioxygen and related species provides a unique fundamental insight into its bonding characteristics. It reveals the coalescence of the molecular orbital and valence bond/Lewis approaches and clearly demonstrates that the often stated inability of valence bond theory to describe the bonding of O2 is a myth. The analysis indicates that the σ-bond strength of 3O2 is not weak as previously believed and accounts for much of its enhanced stability compared with hydroperoxyl. We attribute the stability and persistence of 3O2 to a combination of this attribute and favorable maximization of exchange coupling between the valence electrons.

Molecular Identification of a High-Spin Deprotonated Intermediate during the S2 to S3 Transition of Nature's Water-Oxidizing Complex.

The identity of a key intermediate in the S2 to S3 transition of nature's water-oxidizing complex (WOC) in Photosystem 2 is presented. Broken-symmetry density functional theory (BS-DFT) calculations and Heisenberg-Dirac-van Vleck (HDvV) spin ladder calculations show that an S2 state open cubane model of the WOC containing a µ-hydroxo O4 changes from an S = 5/2 form to an S = 7/2, form upon deprotonation of W1. Combined with X-band electron paramagnetic resonance (EPR) spectral analysis, this indicates that the g = 4.1 EPR signal corresponds to an S = 5/2 form of the WOC with W1 present as a water ligand to Mn4, while the g = 4.8/4.9 form observed at high pH values corresponds to an S = 7/2 form, with W1 as a hydroxo ligand. The latter is also likely to represent the form needed to progress to S3 in the functioning enzyme.

Electronic-Level View of O-O Bond Formation in Nature's Water Oxidizing Complex.

The crucial O-O bond forming step in the water oxidizing complex (WOC) of photosystem II is modeled using density functional theory calculations and compared with structural X-ray free electron laser (XFEL) determinations for the penultimate S3 state. Concerted electron flow between the Mn4O5 and Mn1O6 bonds of the complex and the nascent O-O bond is monitored using intrinsic bond orbital analysis along the reaction path. Concerted transfer to Mn1 and Mn4 of two electrons from the reactant oxos, O5 and O6, resulting in an unoccupied antibonding σ2p* orbital is the key to low barrier O-O bond formation. The potential energy surface for O-O bond formation shows a rather broad energy minimum for the oxo-oxo form ranging from 2.4-2.0 Å which may explain the relatively short O5-O6 bond distance reported in experimental structure studies. Alternatively the short O5-O6 bond distance may reflect a dynamic equilibrium model across the whole O-O potential energy surface.

Impurity analysis of 2-butynoic acid by ion chromatography-mass spectrometry.

Small organic acids are widely used within the pharmaceutical industry but can be difficult to analyse. Ion chromatography is a suitable technique for the analysis of these acids but method development can be hindered as mass spectrometry is not often used as a detector; this means that peak tracking and peak purity cannot be performed. The authors report method development for the analysis of 2-butynoic acid, where by using electrospray ionisation mass spectrometry, peak purity was investigated and the presence of co-eluting impurities determined. Optimisation of the additives in the make-up flow to the mass spectrometer was shown to have an impact on the response observed. A standard series of organic acids were analysed spiked in to 2-butynoic acid at levels representative of impurities, the presence of the 2-butynoic acid did not impact the linearity or limit of detection observed for the acids; R2 values greater than 0.98 were obtained for all acids with and without the presence of 2-butynoic acid with a limit of detection at 1 ppb for all but one of the acids.

Proton Isomers Rationalize the High- and Low-Spin Forms of the S2 State Intermediate in the Water-Oxidizing Reaction of Photosystem II.

A new paradigm for the high- and low-spin forms of the S2 state of nature's water-oxidizing complex in Photosystem II is found. Broken symmetry density functional theory calculations combined with Heisenberg-Dirac-van Vleck spin ladder calculations show that an open cubane form of the water-oxidizing complex changes from a low-spin, S = 1/2, to a high-spin, S = 5/2, form on protonation of the bridging O4 oxo. We show that such models are fully compatible with structural determinations of the S2 state by X-ray free-electron laser crystallography and extended X-ray absorption fine structure and provide a clear rationale for the effect of various treatments on the relative populations of each form observed experimentally in electron paramagnetic resonance studies.

Evidence of O-O Bond Formation in the Final Metastable S3 State of Nature's Water Oxidizing Complex Implying a Novel Mechanism of Water Oxidation.

A novel mechanism for the final stages of Nature's photosynthetic water oxidation to molecular oxygen is proposed. This is based on a comparison of experimental and broken symmetry density functional theory (BS-DFT) calculated geometries and magnetic resonance properties of water oxidizing complex models in the final metastable oxidation state, S3. We show that peroxo models of the S3 state are in vastly superior agreement with the current experimental structural determinations compared with oxo-hydroxo models. Comparison of experimental and BS-DFT calculated 55Mn hyperfine couplings for the electron paramagnetic resonance (EPR) visible form shows better agreement for the oxo-hydroxo model. An equilibrium between oxo-hydroxo and peroxo models is proposed for the S3 state and the major implications for the final steps in the water oxidation mechanism are analyzed and discussed.

Comparison of Experimental and Broken Symmetry Density Functional Theory Calculated Electron Paramagnetic Resonance Parameters for the Manganese Catalase Active Site in the Superoxidized MnIII/MnIV State.

Broken symmetry density functional theory has been used to calculate g-tensor, 55Mn, 14N, and 17O hyperfine couplings for active site models of superoxidized MnIII/MnIV manganese catalase both in its native and azide-inhibited form. While a good agreement is found between the calculated and experimental g-tensor and 55Mn hyperfine couplings for all models, the active site geometry and Mn ion oxidation state can only be readily distinguished based on a comparison of the calculated and experimental 14N azide and 17O HFCs. This comparison shows that only models containing a Jahn-Teller distorted 5-coordinate (MnIII)2 site and a 6-coordinate (MnIV)1 site can satisfactorily reproduce the experimental 14N and 17O hyperfine couplings.

A Comparison of Experimental and Broken Symmetry Density Functional Theory (BS-DFT) Calculated Electron Paramagnetic Resonance (EPR) Parameters for Intermediates Involved in the S2 to S3 State Transition of Nature's Oxygen Evolving Complex.

A broken symmetry density functional theory (BS-DFT) magnetic analysis of the S2, S2YZ•, and S3 states of Nature's oxygen evolving complex is performed for both the native Ca and Sr substituted forms. Good agreement with experiment is observed between the tyrosyl calculated g-tensor and 1H hyperfine couplings for the native Ca form. Changes in the hydrogen bonding environment of the tyrosyl radical in S2YZ• caused by Sr substitution lead to notable changes in the calculated g-tensor of the tyrosyl radical. Comparison of calculated and experimental 55Mn hyperfine couplings for the S3 state presently favors an open cubane form of the complex with an additional OH ligand coordinating to MnD. In Ca models, this additional ligation can arise by closed-cubane form deprotonation of the Ca ligand W3 in the S2YZ• state accompanied by spontaneous movement to the vacant Mn coordination site or by addition of an external OH group. For the Sr form, no spontaneous movement of W3 to the vacant Mn coordination site is observed in contrast to the native Ca form, a difference which may lead to the reduced catalytic activity of the Sr substituted form. BS-DFT studies on peroxo models of S3 as indicated by a recent X-ray free electron laser (XFEL) crystallography study give rise to a structural model compatible with experimental data and an S = 3 ground state compatible with EPR studies.

Comparison between Experimental and Broken Symmetry Density Functional Theory (BS-DFT) Calculated Electron Paramagnetic Resonance (EPR) Parameters of the S2 State of the Oxygen-Evolving Complex of Photosystem II in Its Native (Calcium) and Strontium-Substituted Form.

A comparison between experimental and Broken Symmetry Density Functional theory (BS-DFT) calculated hyperfine couplings for the S2 state of the oxygen-evolving complex (OEC) has been performed. The effect of Ca substitution by Sr combined with the protonation state of two terminal hydroxo or aqua ligands, W1 and W2, on the calculated hyperfine couplings of 55Mn, 13C, 14N, 17O, and 1H nuclei has been investigated. Our findings show best agreement with experiment for OEC models which contain a hydroxide group at the W2 position and a water molecule at W1. For this model the agreement between calculated and experimental data for all hyperfine couplings is excellent. Models with a hydroxide group at W1 are particularly poor models. Sr substitution has a minor influence on calculated hyperfine couplings in agreement with experimental determinations. The sensitivity of the hyperfine couplings to relatively minor changes in the OEC structure demonstrates the power of this methodology in refining the details of its steric and electronic structure which is an essential step in formulating a complete mechanism for water oxidation by the OEC.