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.

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli.

The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz' = 11.8 MHz, whereas for H2, Tz' = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.

Manganese Oxidation State Assignment for Manganese Catalase.

The oxidation state assignment of the manganese ions present in the superoxidized manganese (III/IV) catalase active site is determined by comparing experimental and broken symmetry density functional theory calculated (14)N, (17)O, and (1)H hyperfine couplings. Experimental results have been interpreted to indicate that the substrate water is coordinated to the Mn(III) ion. However, by calculating hyperfine couplings for both scenarios we show that water is coordinated to the Mn(IV) ion and that the assigned oxidation states of the two manganese ions present in the site are the opposite of that previously proposed based on experimental measurements alone.

Regulation of the primary quinone binding conformation by the H subunit in reaction centers from Rhodobacter sphaeroides.

Unlike photosystem II (PSII) in higher plants, bacterial photosynthetic reaction centers (bRCs) from Proteobacteria have an additional peripheral membrane subunit "H". The H subunit is necessary for photosynthetic growth, but can be removed chemically in vitro. The remaining LM dimer retains its activity to perform light-induced charge separation. Here we investigate the influence of the H subunit on interactions between the primary semiquinone and the protein matrix, using a combination of site-specific isotope labeling, pulsed electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. The data reveal substantially weaker binding interactions between the primary semiquinone and the LM dimer than observed for the intact bRC; the amount of electron spin transferred to the nitrogen hydrogen bond donors is significantly reduced, the methoxy groups are more free to rotate, and the spectra indicate a heterogeneous mixture of bound semiquinone states. These results are consistent with a loosening of the primary quinone binding pocket in the absence of the H subunit.