Rupal Gupta

Reversible Switching of Magnetism in Thiolate-Protected Au25 Superatoms

We report reversible switching of paramagnetism in a well-defined gold nanoparticle system consisting of atomically monodisperse nanoparticles containing 25 gold atoms protected by 18 thiolates [abbreviated as Au(25)(SR)(18)]. The magnetism in these nanoparticles can be switched on or off by precisely controlling the charge state of the nanoparticle, that is, the magnetic state of the Au(25)(SR)(18) nanoparticles is charge-neutral while the nonmagnetic state is an anionic form of the particle. Electron paramagnetic resonance (EPR) spectroscopy measurements establish that the magnetic state of the Au(25)(SR)(18) nanoparticles possess one unpaired spin per particle. EPR studies also imply an unusual electronic structure of the Au(25)(SR)(18) nanoparticle. Density functional theory calculations coupled with the experiments successfully explain the origin of the observed magnetism in a Au(25)(SR)(18) nanoparticle as arising from one unpaired spin having distinct P-like character and delocalized among the icosahedral Au(13) core of the particle in the highest occupied molecular orbital. The results suggest that the Au(25)(SR)(18) nanoparticles are best considered as ligand-protected superatoms.

Formation, Structure, and EPR Detection of a High Spin FeIV—Oxo Species Derived from Either an FeIII—Oxo or FeIII—OH Complex

High spin oxoiron(IV) complexes have been proposed to be a key intermediate in numerous nonheme metalloenzymes. The successful detection of similar complexes has been reported for only two synthetic systems. A new synthetic high spin oxoiron(IV) complex is now reported that can be prepared from a well-characterized oxoiron(III) species. This new oxoiron(IV) complex can also be prepared from a hydroxoiron(III) species via a proton-coupled electron transfer process--a first in synthetic chemistry. The oxoiron(IV) complex has been characterized with a variety of spectroscopic methods: FTIR studies showed a feature associated with the Fe-O bond at nu(Fe(16)O) = 798 cm(-1) that shifted to 765 cm(-1) in the (18)O complex; Mossbauer experiments show a signal with an delta = 0.02 mm/s and |DeltaE(Q)| = 0.43 mm/s, electronic parameters consistent with an Fe(IV) center, and optical spectra had visible bands at lambda(max) = 440 (epsilon(M) = 3100), 550 (epsilon(M) = 1900), and 808 (epsilon(M) = 280) nm. In addition, the oxoiron(IV) complex gave the first observable EPR features in the parallel-mode EPR spectrum with g-values at 8.19 and 4.06. A simulation for an S = 2 species with D = 4.0(5) cm(-1), E/D = 0.03, sigma(E/D) = 0.014, and g(z) = 2.04 generates a fit that accurately predicted the intensity, line shape, and position of the observed signals. These results showed that EPR spectroscopy can be a useful method for determining the properties of high spin oxoiron(IV) complexes. The oxoiron(IV) complex was crystallized at -35 degrees C, and its structure was determined by X-ray diffraction methods. The complex has a trigonal bipyramidal coordination geometry with the Fe-O unit positioned within a hydrogen bonding cavity. The Fe(IV)-O unit bond length is 1.680(1) A, which is the longest distance yet reported for a monomeric oxoiron(IV) complex.

High-spin Mn–oxo complexes and their relevance to the oxygen-evolving complex within photosystem II

Significance Metal complexes with terminal oxido ligands are important in a wide variety of transformations, including a high valent manganese-oxido unit that is involved in the O–O bond-forming step in photosynthetic water oxidation. Theoretical proposals suggest that a MnIV–oxyl radical species is present, yet such species have not been observed experimentally. Using a combination of experimental measurements and theoretical calculations, we show here that the bonding within the Mn–oxido unit is best described as highly covalent, with 0.45 spins on the oxido ligand. These findings offer a counter explanation for the putative high valent manganese species in photosynthesis as an energetically accessible, high-spin MnV–oxido unit instead of a MnIV–oxyl radical species. The structural and electronic properties of a series of manganese complexes with terminal oxido ligands are described. The complexes span three different oxidation states at the manganese center (III–V), have similar molecular structures, and contain intramolecular hydrogen-bonding networks surrounding the Mn–oxo unit. Structural studies using X-ray absorption methods indicated that each complex is mononuclear and that oxidation occurs at the manganese centers, which is also supported by electron paramagnetic resonance (EPR) studies. This gives a high-spin MnV–oxo complex and not a MnIV–oxy radical as the most oxidized species. In addition, the EPR findings demonstrated that the Fermi contact term could experimentally substantiate the oxidation states at the manganese centers and the covalency in the metal–ligand bonding. Oxygen-17–labeled samples were used to determine spin density within the Mn–oxo unit, with the greatest delocalization occurring within the MnV–oxo species (0.45 spins on the oxido ligand). The experimental results coupled with density functional theory studies show a large amount of covalency within the Mn–oxo bonds. Finally, these results are examined within the context of possible mechanisms associated with photosynthetic water oxidation; specifically, the possible identity of the proposed high valent Mn–oxo species that is postulated to form during turnover is discussed.

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of l-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔEQ = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of l-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated l-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.

Magic Angle Spinning NMR Spectroscopy: A Versatile Technique for Structural and Dynamic Analysis of Solid-Phase Systems

Magic Angle Spinning (MAS) NMR spectroscopy is a powerful method for analysis of a broad range of systems, including inorganic materials, pharmaceuticals, and biomacromolecules. The recent developments in MAS NMR instrumentation and methodologies opened new vistas to atomic-level characterization of a plethora of chemical environments previously inaccessible to analysis, with unprecedented sensitivity and resolution.

The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation.

We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV-visible absorbance, electron paramagnetic resonance spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the case for the canonical Pseudomonas type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP fusion and tag-free So CcP and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis, these data suggest that So CcP can accommodate electron donor binding in several possible orientations and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP, we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form.

Dynamic Nuclear Polarization enhanced MAS NMR spectroscopy for structural analysis of HIV-1 protein assemblies

Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

Phase-modulated LA-REDOR: a robust, accurate and efficient solid-state NMR technique for distance measurements between a spin-1/2 and a quadrupole spin.

Distances between a spin-1/2 and a spin>1/2 can be efficiently measured by a variety of magic-angle spinning solid state NMR methods such as Rotational Echo Adiabatic Passage Double Resonance (REAPDOR), Low-Alpha/Low-Amplitude REDOR (LA-REDOR) and Rotational-Echo Saturation-Pulse Double-Resonance (R/S-RESPDOR). In this manuscript we show that the incorporation of a phase modulation into a long quadrupolar recoupling pulse, lasting 10 rotor periods that are sandwiched between rotor-synchronized pairs of dipolar recoupling π pulses, extends significantly the range of the values of the quadrupole moments that can be accessed by the experiment. We show by a combination of simulations and experiments that the new method, phase-modulated LA-REDOR, is very weakly dependent on the actual value of the radio-frequency field, and is highly robust with respect to off-resonance irradiation. The experimental results can be fitted by numerical simulations or using a universal formula corresponding to an equal-transition-probability model. Phase-modulated LA-REDOR (13)C{(11)B} and (15)N{(51)V} dipolar recoupling experiments confirm the accuracy and applicability of this new method.

Preparation and properties of an MnIV–hydroxide complex: proton and electron transfer at a mononuclear manganese site and its relationship to the oxygen evolving complex within photosystem II

Photosynthetic water oxidation is catalyzed by a Mn4O5Ca cluster with an unprecedented arrangement of metal ions in which a single manganese center is bonded to a distorted Mn3O4Ca cubane-like structure. Several mechanistic proposals describe the unique manganese center as a site for water binding and subsequent formation of a high valent Mn-oxo center that reacts with a M-OH unit (M = Mn or CaII) to form the O-O bond. The conversion of low valent Mn-OHn (n = 1,2) to a Mn-oxo species requires that a single manganese site be able to accommodate several oxidation states as the water ligand is deprotonated. To study these processes, the preparation and characterization of a new monomeric MnIV-OH complex is described. The MnIV-OH complex completes a series of well characterized Mn-OH and Mn-oxo complexes containing the same primary and secondary coordination spheres; this work thus demonstrates that a single ligand can support mononuclear Mn complexes spanning four different oxidation states (II through V) with oxo and hydroxo ligands that are derived from water. Moreover, we have completed a thermodynamic analysis based on this series of manganese complexes to predict the formation of high valent Mn-oxo species; we demonstrated that the conversion of a MnIV-OH species to a MnV-oxo complex would likely occur via a stepwise proton transfer-electron transfer mechanism. The large dissociation energy for the MnIVO-H bond (~95 kcal/mol) diminished the likelihood that other pathways are operative within a biological context. Furthermore, these studies showed that reactions between Mn-OH and Mn-oxo complexes lead to non-productive, one-electron processes suggesting that initial O-O bond formation with the OEC does not involve an Mn-OH unit.

EPR and Mössbauer Spectroscopy Show Inequivalent Hemes in Tryptophan Dioxygenase

Tryptophan 2,3-dioxygenase (TDO) is an essential enzyme in the pathway of NAD biosynthesis and important for all living organisms. TDO catalyzes oxidative cleavage of the indole ring of L-tryptophan (L-Trp), converting it to N-formylkynurenine (NFK). The crystal structure of TDO shows a dimer of dimer quaternary structure of the homotetrameric protein. The four catalytic sites of the protein, one per subunit, contain a heme that catalyzes the activation and insertion of dioxygen into L-Trp. Because of the alpha(4) structure and because only one type of heme center has been identified in previous spectroscopic studies, the four hemes sites have been presumed to be equivalent. The present work demonstrates that the heme sites of TDO are not equivalent. Quantitative interpretation of EPR and Mössbauer spectroscopic data indicates the presence of two dominant inequivalent heme species in reduced and oxidized states of the enzyme, which is consistent with a dimer of dimer protein quaternary structure that now extends to the electronic properties of the hemes. The electronic properties of the hemes in the reduced state of TDO change significantly upon L-Trp addition, which is attributed to a change in the protonation state of the proximal histidine to the hemes. The binding of O(2) surrogates NO or CO shows two inequivalent heme sites. The heme-NO complexes are 5- and 6-coordinate without L-Trp, and both 6-coordinate with L-Trp. NO can be selectively photodissociated from only one of the heme-NO sites and only in the presence of L-Trp. Cryoreduction of TDO produces a novel diamagnetic heme species, tentatively assigned as a reduced heme-OH complex. This work presents a new description of the heme interactions with the protein, and with the proximal His, which must be considered during the general interpretation of physical data as it relates to kinetics, mechanism, and function of TDO.