Determining the Electronic Structure of Paramagnetic Intermediates in membrane proteins: A high-resolution 2D 1H hyperfine sublevel correlation study of the redox-active tyrosines of photosystem II.

The photosynthetic reaction center, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese­calcium-oxo (Mn4Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The water oxidation reaction is facilitated by proton-coupled electron transfer (PCET) at the redox-active tyrosine residue, YZ, in the OEC which is one of the two symmetric tyrosine residues, YZ and YD, in PSII. Although YZ and YD are chemically identical, their redox properties and reaction kinetics are very different. In the present study, we apply high-resolution two-dimensional (2D) 1H hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic structure of YZ and YD to understand better the functional tuning of PCET at each tyrosine. Most importantly, the 2D HYSCORE measurements that are described here are applicable for the study of paramagnetic cofactors in a wide variety of membrane-bound proteins.

Two-dimensional HYSCORE spectroscopy of superoxidized manganese catalase: a model for the oxygen-evolving complex of photosystem II.

The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction takes place at the tetra-nuclear manganese calcium-oxo (Mn4Ca-oxo) cluster at the heart of the oxygen-evolving complex (OEC) of PSII. Previous studies have determined the magnetic interactions between the paramagnetic Mn4Ca-oxo cluster and its environment in the S2 state of the OEC. The assignments for the electron-nuclear magnetic interactions that were observed in these studies were facilitated by the use of synthetic dimanganese di-µ-oxo complexes. However, there is an immense need to understand the effects of the protein environment on the coordination geometry of the Mn4Ca-oxo cluster in the OEC of PSII. In the present study, we use a proteinaceous model system to examine the protein ligands that are coordinated to the dimanganese catalytic center of manganese catalase from Lactobacillus plantarum. We utilize two-dimensional hyperfine sublevel correlation (2D HYSCORE) spectroscopy to detect the weak magnetic interactions of the paramagnetic dinuclear manganese catalytic center of superoxidized manganese catalase with the nitrogen and proton atoms of the surrounding protein environment. We obtain a complete set of hyperfine interaction parameters for the protons of a water molecule that is directly coordinated to the dinuclear manganese center. We also obtain a complete set of hyperfine and quadrupolar interaction parameters for two histidine ligands as well as a coordinated azide ligand, in azide-treated superoxidized manganese catalase. On the basis of the values of the hyperfine interaction parameters of the dimanganese model, manganese catalase, and those of the S2 state of the OEC of PSII, for the first time, we discuss the impact of a proteinaceous environment on the coordination geometry of multinuclear manganese clusters.

The structure and activation of substrate water molecules in Sr(2+)-substituted photosystem II.

The mechanism of solar water oxidation by photosystem II (PSII) is of fundamental interest and it is the object of extensive studies both in the past and present. The solar water oxidation reaction of PSII occurs in the oxygen-evolving complex (OEC). The OEC consists of a tetranuclear manganese calcium-oxo (Mn4Ca-oxo) cluster that is surrounded by amino acid residues and inorganic cofactors. The role of the Ca(2+) ion in the water oxidation reaction is one of the most interesting questions that is yet to be answered. In this study, we probe the structural and functional differences induced by metal ion substitution in the Mn4Ca-oxo cluster by substituting the Ca(2+) ion in the OEC by a Sr(2+) ion. We apply two-dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy to detect weak magnetic interactions between the paramagnetic Mn4Sr-oxo cluster and the surrounding protons in the S2 state of the OEC of Sr(2+)-substituted PSII. We identify three groups of protons that are magnetically interacting with the Mn4Sr-oxo cluster. Using the recently reported 1.9 Å resolution X-ray structure of the OEC in the S1 state [Umena et al.] and the high-resolution 2D HYSCORE spectroscopy studies of the S2 state of the OEC of Ca(2+)-containing PSII [Milikisiyants et al., Energy Environ. Sci., 2012, 5, 7747], we discuss the assignments of the three groups of protons that are magnetically coupled to the Mn4Sr-oxo cluster. Since hyperfine interactions are highly sensitive to small perturbations in the electronic and geometric structure of paramagnetic centers, a comparison of the 2D HYSCORE spectra of Sr(2+)-substituted and Ca(2+)-containing PSII allows us to draw important conclusions with respect to the structure of the substrate water molecules in the OEC and the role of the Ca(2+) ion in the water oxidation reaction. In addition, for the first time, we determine the experimental value of the spin projection factor for the Mn(III) ion of the Mn4Ca-oxo cluster as ρ1 = ±1.7 from the assignment of the hyperfine interaction of the paramagnetic cluster with the protons of the D1-His332 residue of PSII.

High-frequency electron nuclear double-resonance spectroscopy studies of the mechanism of proton-coupled electron transfer at the tyrosine-D residue of photosystem II.

The solar water-splitting protein complex, photosystem II, catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive the catalytic oxidation of water. Photosystem II contains two symmetrically placed tyrosine residues, YD and YZ, one on each subunit of the heterodimeric core. The YZ residue is kinetically competent and is proposed to be directly involved in the proton-coupled electron transfer reactions of water oxidation. In contrast, the YD proton-coupled electron transfer redox poises the catalytic tetranuclear manganese cluster and may electrostatically tune the adjacent monomeric redox-active chlorophyll and ß-carotene in the secondary electron transfer pathway of photosystem II. In this study, we apply pulsed high-frequency electron paramagnetic resonance (EPR) and electron nuclear double-resonance (ENDOR) spectroscopy to study the photochemical proton-coupled electron transfer (PCET) intermediates of YD. We detect the "unrelaxed" and "relaxed" photoinduced PCET intermediates of YD using high-frequency EPR spectroscopy and observe an increase of the g anisotropy upon temperature-induced relaxation of the unrelaxed intermediate to the relaxed state as previously observed by Faller et al. [(2002) Biochemistry 41, 12914-12920; (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 8732-8735]. This observation suggests the presence of structural differences between the two intermediates. We probe the possible structural differences by performing high-frequency (2)H ENDOR spectroscopy experiments. On the basis of numerical simulations of the experimental (2)H ENDOR spectra, we confirm that (i) there is a significant change in the H-bond length of the tyrosyl radical in the unrelaxed (1.49 Å) and relaxed (1.75 Å) PCET intermediates. This observation suggests that the D2-His189 residue is deprotonated prior to electron transfer at the YD residue and (ii) there are negligible changes in the conformation of the tyrosyl ring in the unrelaxed and relaxed PCET intermediates of YD.

The structure and function of quinones in biological solar energy transduction: a cyclic voltammetry, EPR, and hyperfine sub-level correlation (HYSCORE) spectroscopy study of model naphthoquinones.

Quinones function as electron transport cofactors in photosynthesis and cellular respiration. The versatility and functional diversity of quinones is primarily due to the diverse midpoint potentials that are tuned by the substituent effects and interactions with surrounding amino acid residues in the binding site in the protein. In the present study, a library of substituted 1,4-naphthoquinones are analyzed by cyclic voltammetry in both protic and aprotic solvents to determine effects of substituent groups and hydrogen bonds on the midpoint potential. We use continuous-wave electron paramagnetic resonance (EPR) spectroscopy to determine the influence of substituent groups on the electronic properties of the 1,4-naphthoquinone models in an aprotic solvent. The results establish a correlation between the presence of substituent group(s) and the modification of electronic properties and a corresponding shift in the midpoint potential of the naphthoquinone models. Further, we use pulsed EPR spectroscopy to determine the effect of substituent groups on the strength and planarity of the hydrogen bonds of naphthoquinone models in a protic solvent. This study provides support for the tuning of the electronic properties of quinone cofactors by the influence of substituent groups and hydrogen bonding interactions.

Structure and function of quinones in biological solar energy transduction: a high-frequency D-band EPR spectroscopy study of model benzoquinones.

Quinones are utilized as charge-transfer cofactors in a wide variety of reactions that are crucial for photosynthesis and respiration. In photosynthetic protein complexes, both Type I and Type II, including oxygenic and anoxygenic reaction centers contain quinone cofactors that are known to participate in electron- and proton-transfer processes. Type II reaction centers, purple bacterial reaction centers, and photosystem II utilize benzoquinone molecules, ubiquinone, and plastoquinone, respectively, to facilitate proton-coupled electron transfer reactions. Here, we report a systematic study of the principal components of the g-tensor of an extensive library of model benzosemiquinone anion radicals in both protic (2-isopropanol) and aprotic (dimethyl sulfoxide) solvents using high-frequency EPR spectroscopy. A detailed comparison of the experimental g-values of the benzosemiquinone models at D-band EPR frequency allows for the discrimination of substituent effects and solvent hydrogen bonds on the principal components of the g-tensor. Further, we compare the primary plastosemiquinone, Q(A)(-), of photosystem II with the substituent and solvent hydrogen bond effects of benzosemiquinone models in vitro. This study significantly extends the experimental basis for elucidating the role of both molecular structure and interactions with environment on the functional tuning of quinone cofactors in biological solar energy transduction.

High-resolution two-dimensional 1H and 14N hyperfine sublevel correlation spectroscopy of the primary quinone of photosystem II.

Quinones are naturally occurring isoprenoids that are widely exploited by photosynthetic reaction centers. Protein interactions modify the properties of quinones such that similar quinone species can perform diverse functions in reaction centers. Both type I and type II (oxygenic and nonoxygenic, respectively) reaction centers contain quinone cofactors that serve very different functions as the redox potential of similar quinones can operate at up to 800 mV lower reduction potential when present in type I reaction centers. However, the factors that determine quinone function in energy transduction remain unclear. It is thought that the location of the quinone cofactor, the geometry of its binding site, and the "smart" matrix effects from the surrounding protein environment greatly influence the functional properties of quinones. Photosystem II offers a unique system for the investigation of the factors that influence quinone function in energy transduction. It contains identical plastoquinones in the primary and secondary quinone acceptor sites, Q(A) and Q(B), which exhibit very different functional properties. This study is focused on elucidating the tuning and control of the primary semiquinone state, Q(A)(-), of photosystem II. We utilize high-resolution two-dimensional hyperfine sublevel correlation spectroscopy to directly probe the strength and orientation of the hydrogen bonds of the Q(A)(-) state with the surrounding protein environment of photosystem II. We observe two asymmetric hydrogen bonding interactions of reduced Q(A)(-) in which the strength of each hydrogen bond is affected by the relative nonplanarity of the bond. This study confirms the importance of hydrogen bonds in the redox tuning of the primary semiquinone state of photosystem II.