Patrick J. O’Malley

Density functional calculated spin densities and hyperfine couplings for hydrogen bonded 1,4-naphthosemiquinone and phyllosemiquinone anion radicals: a model for the A1 free radical formed in Photosystem I

The B3LYP hybrid density functional method is used to calculate spin densities and hyperfine couplings for the 1,4-naphthosemiquinone anion radical and a model of the phyllosemiquinone anion radical. The effect of hydrogen bonding on the spin density distribution is shown to lead to a redistribution of pi spin density from the semiquinone carbonyl oxygens to the carbonyl carbon atoms. The effect of in plane and out of plane hydrogen bonding is examined. Out of plane hydrogen bonding is shown to give rise to a significant delocalisation of spin density on to the hydrogen bond donor heavy atom. Excellent agreement is observed between calculated and experimental hyperfine couplings. Comparison of calculated hyperfine couplings with experimental determinations for the A1 phyllosemiquinone anion radical present in Photosystem I (PS I) of higher plant photosynthesis indicates that the in vivo radical may have a hydrogen bond to the O4 atom only as opposed to hydrogen bonds to each oxygen atom in alcohol solvents. The hydrogen bonding situation appears to be the reverse of that observed for QA in the bacterial type II reaction centres where the strong hydrogen bond occurs to the quinone O1 oxygen atom. For different types of reaction centre the presence or absence of the non-heme Fe(II) atom may well determine which type of hydrogen bonding situation prevails at the primary quinone site which in turn may influence the direction of subsequent electron transfer.

The reaction profile for hydrogen atom transfer from phenol to peroxyl free radicals

The reaction profile for hydrogen atom transfer from phenol to the methylperoxyl free radical is calculated using B3LYP hybrid density functional calculations. The electronic structure of the transition state reveals partial delocalisation of unpaired spin density onto the phenol and electron density removal from the aromatic ring of the phenol. The results provide a clear rationale for the rate enhancing effects of spin delocalising and/or electron releasing substituents on the phenol ring.

Hyperfine and Nuclear Quadrupole Tensors of Nitrogen Donors in the Q A Site of Bacterial Reaction Centers: Correlation of the Histidine N δ Tensors with Hydrogen Bond Strength

X- and Q-band pulsed EPR spectroscopy was applied to study the interaction of the QA site semiquinone (SQA) with nitrogens from the local protein environment in natural abundance 14N and in 15N uniformly labeled photosynthetic reaction centers of Rhodobacter sphaeroides. The hyperfine and nuclear quadrupole tensors for His-M219 Nδ and Ala-M260 peptide nitrogen (Np) were estimated through simultaneous simulation of the Q-band 15N Davies ENDOR, X- and Q-band 14,15N HYSCORE, and X-band 14N three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a(14N) = 2.3 MHz, T = 0.3 MHz for His-M219 Nδ and a(14N) = 2.6 MHz, T = 0.3 MHz for Ala-M260 Np. Despite that His-M219 Nδ is established as the stronger of the two H-bond donors, Ala-M260 Np is found to have the larger value of a(14N). The nuclear quadrupole coupling constants were estimated as e2Qq/4h = 0.38 MHz, η = 0.97 and e2Qq/4h = 0.74 MHz, η = 0.59 for His-M219 Nδ and Ala-M260 Np, respectively. An analysis of the available data on nuclear quadrupole tensors for imidazole nitrogens found in semiquinone-binding proteins and copper complexes reveals these systems share similar electron occupancies of the protonated nitrogen orbitals. By applying the Townes–Dailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter η to be a sensitive probe of the histidine Nδ–semiquinone hydrogen bond strength. This is supported by a strong correlation observed between η and the isotropic coupling constant a(14N) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a(14N) and η will provide an important structural characterization tool in future studies of semiquinone-binding proteins.

Density functional calculations modelling tyrosine oxidation in oxygenic photosynthetic electron transfer

Hybrid density functional calculations are used to model tyrosine oxidation during electron transfer reactions of photosystem II. The predicted frequency values for the 7a and deltaCOH modes of the reduced form and the 7a mode of the oxidised radical form are in excellent agreement with experimental data obtained for Mn and Ca depleted systems by Hienerwadel et al. [Biochemistry 36 (1997) 15447] and Berthomieu et al. [Biochemistry 37 (1998) 10547]. The calculations confirm that the two tyrosines Y(D) and Y(Z) are protonated in the reduced form. On oxidation the larger 7a frequency value observed experimentally for Y(Z*) can be best explained by a greater localisation of the protonic charge released on formation of this tyrosyl free radical.

The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.

The geometry and spin density distribution of the tyrosyl radical: a molecular orbital study

Abstract The oxidation of the amino acid tyrosine to the tyrosyl radical is now known to be important in many electron transfer reactions in biology. Electron Paramagnetic Resonance (EPR) and Electron Nuclear Double Resonance (ENDOR) have previously been used to obtain proton hyperfine couplings for the radical in vivo. This study uses AM 1 molecular orbital calculations to provide a detailed insight into the geometry and electronic makeup of this important radical. Molecular orbital studies are first used to obtain an optimised geometry for the tyrosyl radical. This is shown to differ significantly from the unoxidised form. The extent of the singly occupied molecular orbital is then examined and a theoretical estimate of the unpaired electron spin distribution is obtained. This is then used to calculate the anisoptopic hyperfine coupling components for comparison with experimental determinations.

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.

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 Natures 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.

Electronic Structure Studies of the Spin Density Distribution of the QA Plastosemiquinone Free Radical of Photosystem II

Density functional calculations are used to calculate the spin density distribution for the plastosemiquinone anion radical in the QA binding site of Photosystem II. A number of models are examined which explore the effect of iron depletion on the QA site semiquinone spin density distribution and resultant hyperfine couplings. For a model system with a divalent metal ion in the non-heme site the calculated spin density in the QA site model suggests that differential hydrogen-bonding strength to the O1 and O4 oxygen atoms of the radical results in an asymmetric spin density distribution in the semiquinone anion free radical form. The hydrogen bond to the proximal O1 atom is significantly stronger. This is similar to the situation shown to exist previously in the bacterial reaction centre of Rba sphaeroides. Various models of depleted non-heme site metal show the profound effect that the presence of a divalent ion in this site has on the spin density distribution of the QA site semiquinone. The variation in calculated spin density distribution of the QA site plastosemiquinone as a function of the occupancy of the non-heme site needs to be taken into account in the interpretation of experimental paramagnetic resonance data. For Type II reaction centres a major role for Fe2+ in the non-heme site may be the raising of the redox potential of the QA/QA − couple to ensure that electron transfer from the (bacterio)pheophytin anion free radical occurs at a sufficient rate to compete with wasteful back-reactions.