Sergey Milikisiyants - Academia.edu (original) (raw)

Papers by Sergey Milikisiyants

Biophysical Journal, 2015

The Type I photosynthetic reaction center, Photosystem I, is an exquisitely tuned protein complex... more The Type I photosynthetic reaction center, Photosystem I, is an exquisitely tuned protein complex comprised of multiple polypeptide subunits and protein-bound electron-transfer (ET) cofactors. Upon illumination, the special chlorophylls, P 700 , are photoexcited which results in the rapid formation of the chargeseparated state, P 700

Journal of Magnetic Resonance

High-field EPR provides significant advantages for studying structure and dynamics of molecular s... more High-field EPR provides significant advantages for studying structure and dynamics of molecular systems possessing an unpaired electronic spin. However, routine use of high-field EPR in biophysical research, especially for aqueous biological samples, is still facing substantial technical difficulties stemming from high dielectric millimeter wave (mmW) losses associated with non-resonant absorption by water and other polar molecules. The strong absorbance of mmW's by water also limits the penetration depth to just fractions of mm or even less, thus making fabrication of suitable sample containers rather challenging. Here we describe a radically new line of high Q-factor mmW resonators that are based on forming lattice defects in one-dimensional photonic band-gap (PBG) structures composed of low-loss ceramic discs of λ/4 in thickness and having alternating dielectric constants. A sample (either liquid or solid) is placed within the E = 0 node of the standing mm wave confined within the defect. A resonator prototype has been built and tested at 94.3 GHz. The resonator performance is enhanced by employing ceramic nanoporous membranes as flat sample holders of controllable thickness and tunable effective dielectric constant. The experimental Q-factor of an empty resonator was ≈ 420. The Q-factor decreased slightly to ≈ 370 when loaded with a water-containing nanoporous disc of 50 μm in thickness. The resonator has been tested with a number of liquid biological samples and demonstrated about tenfold gain in concentration sensitivity vs. a high-Q cylindrical TE012-type cavity. Detailed HFSS Ansys simulations have shown that the resonator structure could be further optimized by properly choosing the thickness of the aqueous sample and employing metallized surfaces. The PBG resonator design is readily scalable to higher mmW frequencies and is capable of accommodating significantly larger sample volumes than previously achieved with either Fabry-Perot or cylindrical resonators.

Biophysical Journal, 2016

FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of pr... more FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of protein structure. Both methods are based on measuring dipolar interactions-electrical dipoles in case of FRET and magnetic dipoles in case of DEER-between specifically labeled protein sites. The experimental data are then analyzed to derive the distance between the interacting dipoles and relate this distance to the structure of biomacromolecule(s). Molecular volume of EPR labels is generally smaller vs. that of the fluorescent probes and

Journal of the American Chemical Society

The Journal of Physical Chemistry B, 2015

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transfer... more Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ∼17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

The Journal of Physical Chemistry B, 2015

The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energ... more 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 (Mn 4 Ca-oxo) cluster at the heart of the oxygen-evolving complex (OEC) of PSII. Previous studies have determined the magnetic interactions between the paramagnetic Mn 4 Ca-oxo cluster and its environment in the S 2 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 Mn 4 Ca-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. Based on the values of the hyperfine interaction parameters of the dimanganese model, manganese catalase, and those of the S 2 state of the OEC of PSII, for the first time, we discuss the impact of a proteinaceous environment on the coordination geometry of multi-nuclear manganese clusters.

The Journal of Physical Chemistry B, 2011

The solar water-splitting protein complex, photosystem II, catalyzes one of the most energeticall... more The solar water-splitting protein complex, photosystem II, 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 manganeseÀcalciumÀoxo cluster that is present in the oxygen-evolving complex of photosystem II. The tetranuclear manganeseÀcalciumÀoxo cluster is comprised of mixedvalence Mn(III) and Mn(IV) ions in the ground state. The oxoÀmanganese dimer, [H 2 O(terpy)Mn III (μ-O) 2 Mn IV (terpy)OH 2 ](NO 3 ) 3 (terpy = 2,2 0 :6 0 ,2 00terpyridine) (1), is an excellent biomimetic model that has been extensively used to gain insight on the molecular structure and mechanism of water oxidation in photosystem II. In this work, weak magnetic interactions between the protons of the two terminal water ligands and the paramagnetic dimanganese "di-μ-oxo" core of 1 are quantitatively characterized using two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy. For the water molecule that is directly coordinated at the Mn(III) ion, the two protons are found to be magnetically equivalent and exhibit near axial hyperfine anisotropy. In contrast, for the first time, we demonstrate that the two protons of the water molecule that is directly coordinated at the Mn(IV) ion are inequivalent. We obtain the isotropic and anisotropic components of the hyperfine interaction for each proton. A comparison of the HYSCORE spectra measured in the presence and absence of acetate ions provides unambiguous evidence that only one molecule of acetate binds to 1 by replacing a terminal water molecule that is coordinated at the Mn(III) ion.

The Journal of Physical Chemistry B, 2013

Quinones are widely used electron transport cofactors in photosynthetic reaction centers. Previou... more Quinones are widely used electron transport cofactors in photosynthetic reaction centers. Previous studies have suggested that the structure of the quinone cofactors and the protein interactions or "smart" matrix effects from the surrounding environment govern the redox potential and hence the function of quinones in photosynthesis. In the present study, a series of 1,4-benzoquinone models are examined via differential pulse voltammetry to provide relative redox potentials. In parallel, CW and pulsed EPR methods are used to directly determine the electronic properties of each benzoquinone in aprotic and protic environments. The shifts in the redox potential of the quinones are found to be dependent on the nature of the substituent group and the number of substituent groups on the quinone molecule. Further, we establish a direct correlation between the nature of the substituent group and the change in electronic properties of the benzosemiquinone by analysis of the isotropic and anisotropic components of the electron-nuclear hyperfine interactions observed by CW and pulsed EPR studies, respectively. Examination of an extensive library of model quinones in both aprotic and protic solvents indicates that hydrogen-bonding interactions consistently accentuate the effects of the substituent groups of the benzoquinones. This study provides direct support for the tuning and control of quinone cofactors in biological solar energy transduction through interactions with the surrounding protein matrix.

Energy & Environmental Science, 2012

The water-splitting protein, photosystem II, catalyzes the light-driven oxidation of water to dio... more The water-splitting protein, photosystem II, catalyzes the light-driven oxidation of water to dioxygen. The solar water oxidation reaction takes place at the catalytic center, referred to as the oxygen-evolving complex, of photosystem II. During the catalytic cycle, the oxygen-evolving complex cycles through five distinct intermediate states, S 0 -S 4 . In this study, we trap the oxygen-evolving complex in the S 2 intermediate state by low temperature illumination of photosystem II isolated from three different species, Thermosynechococcus vulcanus, the PsbB variant of Synechocystis PCC 6803 and spinach. We apply two-dimensional hyperfine sublevel correlation spectroscopy to detect weak magnetic interactions between the paramagnetic tetra-nuclear manganese cluster of the S 2 state of the OEC and the surrounding protons. We identify five groups of protons that are interacting with the tetra-nuclear manganese cluster. From the values of hyperfine interactions and using the recently reported 1.9

Biophysical Journal, 2010

The solar water-splitting protein complex, photosystem II, catalyzes the light-driven oxidation o... more The solar water-splitting protein complex, photosystem II, catalyzes the light-driven oxidation of water to dioxygen in Nature. The four-electron oxidation reaction of water occurs at the tetranuclear manganese-calcium-oxo catalytic cluster that is present in the oxygen-evolving complex of photosystem II. The mechanism of light-driven water oxidation has been a subject of intense interest, and the oxygenevolving complex of photosystem II has been studied extensively by structural and biochemical methods. While the recent X-ray crystal structures and single-crystal EXAFS investigations provide a model for the geometry of the tetranuclear manganese-calcium-oxo catalytic cluster, there is limited knowledge of the protein environment that surrounds the catalytic cluster. In this study, we demonstrate the application of two-dimensional hyperfine sublevel correlation spectroscopy to determine the magnetic couplings of the catalytic cluster with the 14 N atoms of surrounding amino acid residues in the S 2 state of the oxygen-evolving complex of photosystem II. We utilize two-dimensional difference spectroscopy to facilitate unambiguous assignments of the spectral features and identify at least three separate 14 N atoms that are interacting with the catalytic cluster in the S 2 state. The results presented here, for the first time, identify previously unknown ligands to the catalytic cluster of photosystem II and provide avenues for the assignment of residues by site-directed mutagenesis and the refinement of computational and mechanistic models of photosystem II.

Biochemistry, 2013

The solar water-splitting protein complex, photosystem II, catalyzes one of the most energeticall... more 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, Y D and Y Z , one on each subunit of the heterodimeric core. The Y Z residue is kinetically competent and is proposed to be directly involved in the proton-coupled electron transfer reactions of water oxidation. In contrast, the Y D proton-coupled electron transfer redox poises the catalytic tetranuclear manganese cluster and may electrostatically tune the adjacent monomeric redox-active chlorophyll and β-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 doubleresonance (ENDOR) spectroscopy to study the photochemical proton-coupled electron transfer (PCET) intermediates of Y D . We detect the "unrelaxed" and "relaxed" photoinduced PCET intermediates of Y D 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; 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 Y D residue and (ii) there are negligible changes in the conformation of the tyrosyl ring in the unrelaxed and relaxed PCET intermediates of Y D .

Biochemistry, 2011

The phylloquinones of photosystem I (PS I), A(1A) and A(1B), exist in near-equivalent protein env... more The phylloquinones of photosystem I (PS I), A(1A) and A(1B), exist in near-equivalent protein environments but possess distinct thermodynamic and kinetic properties. Although the determinants responsible for the different properties of the phylloquinones are not completely understood, the strength and geometry of hydrogen bond interactions are significant factors in tuning and control of function. This study focuses on characterizing the hydrogen-bonding interactions of the phylloquinone acceptor, A(1A), by (1)H and (14)N HYSCORE spectroscopy. Photoaccumulation of PS I complexes at pH 8.0 results in the trapping of the phyllosemiquinone anion, A(1A)(-), on the A-branch of cofactors. The experiments described here indicate that A(1A)(-) forms a single H-bond. Using a simple point dipole approximation, we estimate its length to be 1.6 ± 0.1 Å. The value of the (1)H isotropic hyperfine coupling constant suggests that the H-bond has significant out-of-plane character. The (14)N HYSCORE spectroscopy experiments support the assignment of a H-bond wherein, the (14)N quadrupolar coupling constant is consistent with a backbone amide nitrogen as the hydrogen bond donor.

Biochemistry, 2011

Quinones are naturally occurring isoprenoids that are widely exploited by photosynthetic reaction... more 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.

Biophysical Journal, 2015

Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in d... more Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in diameter enable two-dimensional, solid-state nuclear magnetic resonance studies of lipid-induced conformational changes of uniformly (15)N-labeled Pf1 coat protein in native-like bilayers. The Pf1 helix tilt angles in bilayers composed of two different lipids are not entirely governed by the membrane thickness but could be rationalized by hydrophobic interactions of lysines at the bilayer interface. The anodic aluminum oxide alignment method is applicable to a broader repertoire of lipids versus bicelle bilayer mimetics currently employed in solid-state nuclear magnetic resonance of oriented samples, thus allowing for elucidation of the role played by lipids in shaping membrane proteins.

Biophysical Journal, 2015

The Type I photosynthetic reaction center, Photosystem I, is an exquisitely tuned protein complex... more The Type I photosynthetic reaction center, Photosystem I, is an exquisitely tuned protein complex comprised of multiple polypeptide subunits and protein-bound electron-transfer (ET) cofactors. Upon illumination, the special chlorophylls, P 700 , are photoexcited which results in the rapid formation of the chargeseparated state, P 700

Journal of Magnetic Resonance

High-field EPR provides significant advantages for studying structure and dynamics of molecular s... more High-field EPR provides significant advantages for studying structure and dynamics of molecular systems possessing an unpaired electronic spin. However, routine use of high-field EPR in biophysical research, especially for aqueous biological samples, is still facing substantial technical difficulties stemming from high dielectric millimeter wave (mmW) losses associated with non-resonant absorption by water and other polar molecules. The strong absorbance of mmW's by water also limits the penetration depth to just fractions of mm or even less, thus making fabrication of suitable sample containers rather challenging. Here we describe a radically new line of high Q-factor mmW resonators that are based on forming lattice defects in one-dimensional photonic band-gap (PBG) structures composed of low-loss ceramic discs of λ/4 in thickness and having alternating dielectric constants. A sample (either liquid or solid) is placed within the E = 0 node of the standing mm wave confined within the defect. A resonator prototype has been built and tested at 94.3 GHz. The resonator performance is enhanced by employing ceramic nanoporous membranes as flat sample holders of controllable thickness and tunable effective dielectric constant. The experimental Q-factor of an empty resonator was ≈ 420. The Q-factor decreased slightly to ≈ 370 when loaded with a water-containing nanoporous disc of 50 μm in thickness. The resonator has been tested with a number of liquid biological samples and demonstrated about tenfold gain in concentration sensitivity vs. a high-Q cylindrical TE012-type cavity. Detailed HFSS Ansys simulations have shown that the resonator structure could be further optimized by properly choosing the thickness of the aqueous sample and employing metallized surfaces. The PBG resonator design is readily scalable to higher mmW frequencies and is capable of accommodating significantly larger sample volumes than previously achieved with either Fabry-Perot or cylindrical resonators.

Biophysical Journal, 2016

FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of pr... more FRET and DEER are two spectroscopic methods that are widely applied for biophysical studies of protein structure. Both methods are based on measuring dipolar interactions-electrical dipoles in case of FRET and magnetic dipoles in case of DEER-between specifically labeled protein sites. The experimental data are then analyzed to derive the distance between the interacting dipoles and relate this distance to the structure of biomacromolecule(s). Molecular volume of EPR labels is generally smaller vs. that of the fluorescent probes and

Journal of the American Chemical Society

The Journal of Physical Chemistry B, 2015

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transfer... more Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ∼17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

The Journal of Physical Chemistry B, 2015

The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energ... more 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 (Mn 4 Ca-oxo) cluster at the heart of the oxygen-evolving complex (OEC) of PSII. Previous studies have determined the magnetic interactions between the paramagnetic Mn 4 Ca-oxo cluster and its environment in the S 2 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 Mn 4 Ca-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. Based on the values of the hyperfine interaction parameters of the dimanganese model, manganese catalase, and those of the S 2 state of the OEC of PSII, for the first time, we discuss the impact of a proteinaceous environment on the coordination geometry of multi-nuclear manganese clusters.

The Journal of Physical Chemistry B, 2011

The solar water-splitting protein complex, photosystem II, catalyzes one of the most energeticall... more The solar water-splitting protein complex, photosystem II, 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 manganeseÀcalciumÀoxo cluster that is present in the oxygen-evolving complex of photosystem II. The tetranuclear manganeseÀcalciumÀoxo cluster is comprised of mixedvalence Mn(III) and Mn(IV) ions in the ground state. The oxoÀmanganese dimer, [H 2 O(terpy)Mn III (μ-O) 2 Mn IV (terpy)OH 2 ](NO 3 ) 3 (terpy = 2,2 0 :6 0 ,2 00terpyridine) (1), is an excellent biomimetic model that has been extensively used to gain insight on the molecular structure and mechanism of water oxidation in photosystem II. In this work, weak magnetic interactions between the protons of the two terminal water ligands and the paramagnetic dimanganese "di-μ-oxo" core of 1 are quantitatively characterized using two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy. For the water molecule that is directly coordinated at the Mn(III) ion, the two protons are found to be magnetically equivalent and exhibit near axial hyperfine anisotropy. In contrast, for the first time, we demonstrate that the two protons of the water molecule that is directly coordinated at the Mn(IV) ion are inequivalent. We obtain the isotropic and anisotropic components of the hyperfine interaction for each proton. A comparison of the HYSCORE spectra measured in the presence and absence of acetate ions provides unambiguous evidence that only one molecule of acetate binds to 1 by replacing a terminal water molecule that is coordinated at the Mn(III) ion.

The Journal of Physical Chemistry B, 2013

Quinones are widely used electron transport cofactors in photosynthetic reaction centers. Previou... more Quinones are widely used electron transport cofactors in photosynthetic reaction centers. Previous studies have suggested that the structure of the quinone cofactors and the protein interactions or "smart" matrix effects from the surrounding environment govern the redox potential and hence the function of quinones in photosynthesis. In the present study, a series of 1,4-benzoquinone models are examined via differential pulse voltammetry to provide relative redox potentials. In parallel, CW and pulsed EPR methods are used to directly determine the electronic properties of each benzoquinone in aprotic and protic environments. The shifts in the redox potential of the quinones are found to be dependent on the nature of the substituent group and the number of substituent groups on the quinone molecule. Further, we establish a direct correlation between the nature of the substituent group and the change in electronic properties of the benzosemiquinone by analysis of the isotropic and anisotropic components of the electron-nuclear hyperfine interactions observed by CW and pulsed EPR studies, respectively. Examination of an extensive library of model quinones in both aprotic and protic solvents indicates that hydrogen-bonding interactions consistently accentuate the effects of the substituent groups of the benzoquinones. This study provides direct support for the tuning and control of quinone cofactors in biological solar energy transduction through interactions with the surrounding protein matrix.

Energy & Environmental Science, 2012

The water-splitting protein, photosystem II, catalyzes the light-driven oxidation of water to dio... more The water-splitting protein, photosystem II, catalyzes the light-driven oxidation of water to dioxygen. The solar water oxidation reaction takes place at the catalytic center, referred to as the oxygen-evolving complex, of photosystem II. During the catalytic cycle, the oxygen-evolving complex cycles through five distinct intermediate states, S 0 -S 4 . In this study, we trap the oxygen-evolving complex in the S 2 intermediate state by low temperature illumination of photosystem II isolated from three different species, Thermosynechococcus vulcanus, the PsbB variant of Synechocystis PCC 6803 and spinach. We apply two-dimensional hyperfine sublevel correlation spectroscopy to detect weak magnetic interactions between the paramagnetic tetra-nuclear manganese cluster of the S 2 state of the OEC and the surrounding protons. We identify five groups of protons that are interacting with the tetra-nuclear manganese cluster. From the values of hyperfine interactions and using the recently reported 1.9

Biophysical Journal, 2010

The solar water-splitting protein complex, photosystem II, catalyzes the light-driven oxidation o... more The solar water-splitting protein complex, photosystem II, catalyzes the light-driven oxidation of water to dioxygen in Nature. The four-electron oxidation reaction of water occurs at the tetranuclear manganese-calcium-oxo catalytic cluster that is present in the oxygen-evolving complex of photosystem II. The mechanism of light-driven water oxidation has been a subject of intense interest, and the oxygenevolving complex of photosystem II has been studied extensively by structural and biochemical methods. While the recent X-ray crystal structures and single-crystal EXAFS investigations provide a model for the geometry of the tetranuclear manganese-calcium-oxo catalytic cluster, there is limited knowledge of the protein environment that surrounds the catalytic cluster. In this study, we demonstrate the application of two-dimensional hyperfine sublevel correlation spectroscopy to determine the magnetic couplings of the catalytic cluster with the 14 N atoms of surrounding amino acid residues in the S 2 state of the oxygen-evolving complex of photosystem II. We utilize two-dimensional difference spectroscopy to facilitate unambiguous assignments of the spectral features and identify at least three separate 14 N atoms that are interacting with the catalytic cluster in the S 2 state. The results presented here, for the first time, identify previously unknown ligands to the catalytic cluster of photosystem II and provide avenues for the assignment of residues by site-directed mutagenesis and the refinement of computational and mechanistic models of photosystem II.

Biochemistry, 2013

The solar water-splitting protein complex, photosystem II, catalyzes one of the most energeticall... more 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, Y D and Y Z , one on each subunit of the heterodimeric core. The Y Z residue is kinetically competent and is proposed to be directly involved in the proton-coupled electron transfer reactions of water oxidation. In contrast, the Y D proton-coupled electron transfer redox poises the catalytic tetranuclear manganese cluster and may electrostatically tune the adjacent monomeric redox-active chlorophyll and β-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 doubleresonance (ENDOR) spectroscopy to study the photochemical proton-coupled electron transfer (PCET) intermediates of Y D . We detect the "unrelaxed" and "relaxed" photoinduced PCET intermediates of Y D 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; 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 Y D residue and (ii) there are negligible changes in the conformation of the tyrosyl ring in the unrelaxed and relaxed PCET intermediates of Y D .

Biochemistry, 2011

The phylloquinones of photosystem I (PS I), A(1A) and A(1B), exist in near-equivalent protein env... more The phylloquinones of photosystem I (PS I), A(1A) and A(1B), exist in near-equivalent protein environments but possess distinct thermodynamic and kinetic properties. Although the determinants responsible for the different properties of the phylloquinones are not completely understood, the strength and geometry of hydrogen bond interactions are significant factors in tuning and control of function. This study focuses on characterizing the hydrogen-bonding interactions of the phylloquinone acceptor, A(1A), by (1)H and (14)N HYSCORE spectroscopy. Photoaccumulation of PS I complexes at pH 8.0 results in the trapping of the phyllosemiquinone anion, A(1A)(-), on the A-branch of cofactors. The experiments described here indicate that A(1A)(-) forms a single H-bond. Using a simple point dipole approximation, we estimate its length to be 1.6 ± 0.1 Å. The value of the (1)H isotropic hyperfine coupling constant suggests that the H-bond has significant out-of-plane character. The (14)N HYSCORE spectroscopy experiments support the assignment of a H-bond wherein, the (14)N quadrupolar coupling constant is consistent with a backbone amide nitrogen as the hydrogen bond donor.

Biochemistry, 2011

Quinones are naturally occurring isoprenoids that are widely exploited by photosynthetic reaction... more 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.

Biophysical Journal, 2015

Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in d... more Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in diameter enable two-dimensional, solid-state nuclear magnetic resonance studies of lipid-induced conformational changes of uniformly (15)N-labeled Pf1 coat protein in native-like bilayers. The Pf1 helix tilt angles in bilayers composed of two different lipids are not entirely governed by the membrane thickness but could be rationalized by hydrophobic interactions of lysines at the bilayer interface. The anodic aluminum oxide alignment method is applicable to a broader repertoire of lipids versus bicelle bilayer mimetics currently employed in solid-state nuclear magnetic resonance of oriented samples, thus allowing for elucidation of the role played by lipids in shaping membrane proteins.