Pulsed Electron Nuclear Double Resonance Research Articles

Unravelling electronic and structural requisites of triplet–triplet energy transfer by advanced electron paramagnetic resonance and density functional theory

Advanced electron paramagnetic resonance (EPR) techniques, in combination with Density Functional theory (DFT), have been applied to the comparative study of carotenoid triplet states in two major photosynthetic antenna complexes, the Peridinin-chlorophyll a–protein of dinoflagellates and the light-harvesting complex II of higher plants.Carotenoid triplet states are populated by triplet–triplet energy transfer (TTET) from chlorophyll molecules to photoprotect the system from singlet oxygen formation under light-stress conditions. The TTET process is strongly dependent on the relative arrangement and on the electronic properties of the triplet states involved.The proposed spectroscopic approach exploits the concept of spin conservation during TTET, which leads to recognisable spin polarisation effects in the time-resolved and field-swept echo-detected EPR spectra. The electron spin polarisation produced at the carotenoid acceptor site depends on the initial polarisation of the chlorophyll donor and on the relative geometrical arrangement of the donor-acceptor zero-field splitting axes. We have demonstrated that a proper analysis of the spectra in the framework of spin angular momentum conservation allows to derive the pathways of TTET and to gain insight into the structural requirements of this mechanism for those antenna complexes, whose X-ray structure is available. We have further proved that this method, developed for natural antenna complexes of known X-ray structure, can be extended to systems lacking structural information in order to derive the relative arrangement of the partners in the energy transfer process.The structural requirements for efficient TTET, obtained from time-resolved and pulse EPR, have been complemented by a detailed description of the electronic structure of the carotenoid triplet state, provided by pulse Electron–Nuclear DOuble Resonance (ENDOR) experiments. Triplet-state hyperfine couplings of the α- and β-protons of the carotenoid conjugated chain have been assigned with the aid of quantum chemical calculation. DFT predictions of the electronic structure of the carotenoid triplet state, in terms of spin density distribution, frontier orbital description and orbital excitation represent suitable building blocks toward a deeper understanding of electronic requirements for efficient TTET.

Structure and Nature of Manganese(II) Imidazole Complexes in Frozen Aqueous Solutions

A common feature of a large majority of the manganese metalloenzymes, as well as many synthetic biomimetic complexes, is the bonding between the manganese ion and imidazoles. This interaction was studied by examining the nature and structure of manganese(II) imidazole complexes in frozen aqueous solutions using 285 GHz high magnet-field continuous-wave electron paramagnetic resonance (cw-HFEPR) and 95 GHz pulsed electron-nuclear double resonance (ENDOR) and pulsed electron-double resonance detected nuclear magnetic resonance (PELDOR-NMR). The (55)Mn hyperfine coupling and isotropic g values of Mn(II) in frozen imidazole solutions continuously decreased with increasing imidazole concentration. ENDOR and PELDOR-NMR measurements demonstrated that the structural basis for this behavior arose from the imidazole concentration-dependent distribution of three six-coordinate and two four-coordinate species: [Mn(H2O)6](2+), [Mn(imidazole)(H2O)5](2+), [Mn(imidazole)2(H2O)4](2+), [Mn(imidazole)3(H2O)](2+), and [Mn(imidazole)4](2+). The hyperfine and g values of manganese proteins were also fully consistent with this imidazole effect. Density functional theory methods were used to calculate the structures, spin and charge densities, and hyperfine couplings of a number of different manganese imidazole complexes. The use of density functional theory with large exact-exchange admixture calculations gave isotropic (55)Mn hyperfine couplings that were semiquantitative and of predictive value. The results show that the covalency of the Mn-N bonds play an important role in determining not only magnetic spin parameters but also the structure of the metal binding site. The relationship between the isotropic (55)Mn hyperfine value and the number of imidazole ligands provides a quick and easy test for determining whether a protein binds an Mn(II) ion using histidine residues and, if so, how many are involved. Application of this method shows that as much as 40% of the Mn(II) ions in Deinococcus radiodurans are ligated to two histidines (Tabares, L. C.; Un, S. J. Biol. Chem 2013, in press).

Superhyperfine Interactions of the Nitrogen Donors in 4H SiC Studied by Pulsed ENDOR and TRIPLE ENDOR Spectroscopy

The nitrogen donors residing at quasi-cubic lattice site (Nk) in 4H SiC were investigated by field sweep electron spin echo (FS ESE), pulsed electron nuclear double resonance (ENDOR) and pulsed General TRIPLE ENDOR spectroscopy. The 29Si and 13C superhyperfine lines observed in the FS ESE and ENDOR spectra of Nk in n-type 6H SiC were assigned by pulsed General TRIPLE resonance spectroscopy to the specific carbon (C) and silicon (Si) atoms located in the nearest environment of Nk in 4H SiC. The superhyperfine interaction constants and their relative signs for Nk with 29Si and 13C nuclei located in the nearest-neighbor shells are found from the General TRIPLE ENDOR spectra to be positive for C atoms and negative for Si atoms.

Arsenic speciation in synthetic gypsum (CaSO4·2H2O): A synchrotron XAS, single-crystal EPR, and pulsed ENDOR study

Gypsum (CaSO4·2H2O) is a major by-product of mining and milling processes of borate, phosphate and uranium deposits worldwide and, therefore, potentially plays an important role in the stability and bioavailability of heavy metalloids, including As, in tailings and surrounding areas. Gypsum containing 1900 and 185ppm As, synthesized with Na2HAsO4·7H2O and NaAsO2 in the starting materials, respectively, have been investigated by synchrotron X-ray absorption spectroscopy (XAS), single-crystal electron paramagnetic resonance spectroscopy (EPR), and pulsed electron nuclear double resonance spectroscopy (ENDOR). Quantitative analyses of As K edge XANES and EXAFS spectra show that arsenic occurs in both +3 and +5 oxidation states and the As3+/As5+ value varies from 0.35 to 0.79. Single-crystal EPR spectra of gamma-ray-irradiated gypsum reveal two types of arsenic-associated oxyradicals: [AsO3]2− and an [AsO2]2−. The [AsO3]2− center is characterized by principal 75As hyperfine coupling constants of A1=1952.0(2) MHz, A2=1492.6(2) MHz and A3=1488.7(2) MHz, with the unique A axis along the S–O1 bond direction, and contains complex 1H superhyperfine structures that have been determined by pulsed ENDOR. These results suggest that the [AsO3]2− center formed from electron trapping on the central As5+ ion of a substitutional (AsO4)3− group after removal of an O1 atom. The [AsO2]2− center is characterized by its unique A(75As) axis approximately perpendicular to the O1–S–O2 plane and the A2 axis along the S–O2 bond direction, consistent with electron trapping on the central As3+ ion of a substitutional (AsO3)3− group after removal of an O2 atom. These results confirm lattice-bound As5+ and As3+ in gypsum and point to potential application of this mineral for immobilization and removal of arsenic pollution.

Measuring central-spin interaction with a spin bath by pulsed ENDOR: Towards suppression of spin diffusion decoherence

We present pulsed electron-nuclear double resonance (ENDOR) experiments which enable us to characterize the coupling between bismuth donor spin-qubits in Si and the surrounding spin-bath of (29)Si impurities which provides the dominant decoherence mechanism (nuclear spin diffusion) at low temperatures (< 16 K). Decoupling from the spin-bath is predicted and cluster correlation expansion simulations show near-complete suppression of spin diffusion, at optimal working points. The suppression takes the form of sharply peaked divergences of the spin diffusion coherence time, in contrast with previously identified broader regions of insensitivity to classical fluctuations. ENDOR data shows anisotropic contributions are comparatively weak, so the form of the divergences is independent of crystal orientation.

Electronic structure of the nitrogen donors in 6H SiC as studied by pulsed ENDOR and TRIPLE ENDOR spectroscopy

AbstractThe nitrogen (N) donors in 6H SiC were investigated by field sweep electron spin echo (FS ESE), pulsed electron nuclear double resonance (ENDOR) and pulsed General TRIPLE ENDOR spectroscopy. The 29Si and 13C superhyperfine (shf) lines observed in the FS ESE and ENDOR spectra of N in n‐type 6H SiC were assigned by pulsed General TRIPLE resonance spectroscopy to the specific carbon (C) and silicon (Si) atoms located in the environment of N donors residing at two quasi‐cubic lattice sites (Nk1, Nk2) in 6H SiC. As a result, the largest value of the shf interaction was found with Si atoms for the N donors at the hexagonal (Nh) and quasi‐cubic site Nk1, while for Nk2 the largest value of the shf interaction was found with C atoms. It gives us the argument to consider that N substitute different lattice sites in 6H SiC lattice. The relative signs of the shf interaction (shfi) constants for Nk1 and Nk2 with 29Si and 13C nuclei located in the nearest‐neighbor, next nearest‐neighbor, and outer shells are found from the TRIPLE ENDOR spectra to be positive for C atoms and negative for Si atoms. From the comparison of the experimentally obtained shfi constants with the theory, the electronic spin‐density distribution over the 29Si and 13C nuclei located in the nearest neighbor shells of N donors has been obtained taking into account the Kohn–Luttinger interference effect. The position of the conduction band minimum along the ML‐line was determined to be at k0z/kmax = 0.2 ± 0.05.

EPR and 57Fe ENDOR investigation of 2Fe ferredoxins from Aquifex aeolicus

We have employed EPR and a set of recently developed electron nuclear double resonance (ENDOR) spectroscopies to characterize a suite of [2Fe-2S] ferredoxin clusters from Aquifex aeolicus (Aae Fd1, Fd4, and Fd5). Antiferromagnetic coupling between the Fe(II), S = 2, and Fe(III), S = 5/2, sites of the [2Fe-2S](+) cluster in these proteins creates an S = 1/2 ground state. A complete discussion of the spin-Hamiltonian contributions to g includes new symmetry arguments along with references to related FeS model compounds and their symmetry and EPR properties. Complete (57)Fe hyperfine coupling (hfc) tensors for each iron, with respective orientations relative to g, have been determined by the use of "stochastic" continuous wave and/or "random hopped" pulsed ENDOR, with the relative utility of the two approaches being emphasized. The reported hyperfine tensors include absolute signs determined by a modified pulsed ENDOR saturation and recovery (PESTRE) technique, RD-PESTRE-a post-processing protocol of the "raw data" that comprises an ENDOR spectrum. The (57)Fe hyperfine tensor components found by ENDOR are nicely consistent with those previously found by Mössbauer spectroscopy, while accurate tensor orientations are unique to the ENDOR approach. These measurements demonstrate the capabilities of the newly developed methods. The high-precision hfc tensors serve as a benchmark for this class of FeS proteins, while the variation in the (57)Fe hfc tensors as a function of symmetry in these small FeS clusters provides a reference for higher-nuclearity FeS clusters, such as those found in nitrogenase.

EPR/ENDOR, Mössbauer, and quantum-chemical investigations of diiron complexes mimicking the active oxidized state of [FeFe]hydrogenase.

Understanding the catalytic process of the heterolytic splitting and formation of molecular hydrogen is one of the key topics for the development of a future hydrogen economy. With an interest in elucidating the enzymatic mechanism of the [Fe(2)(S(2)C(2)H(4)NH)(CN)(2)(CO)(2)(μ-CO)] active center uniquely found in [FeFe]hydrogenases, we present a detailed spectroscopic and theoretical analysis of its inorganic model [Fe(2)(S(2)X)(CO)(3)(dppv)(PMe(3))](+) [dppv = cis-1,2-bis(diphenylphosphino)ethylene] in two forms with S(2)X = ethanedithiolate (1edt) and azadithiolate (1adt). These complexes represent models for the oxidized mixed-valent Fe(I)Fe(II) state analogous to the active oxidized "H(ox)" state of the native H-cluster. For both complexes, the (31)P hyperfine interactions were determined by pulse electron paramagnetic resonance and electron nuclear double resonance (ENDOR) methods. For 1edt, the (57)Fe parameters were measured by electron spin-echo envelope modulation and Mössbauer spectroscopy, while for 1adt, (14)N and selected (1)H couplings could be obtained by ENDOR and hyperfine sublevel correlation spectroscopy. The spin density was found to be predominantly localized on the Fe(dppv) site. This spin distribution is different from that of the H-cluster, where both the spin and charge densities are delocalized over the two Fe centers. This difference is attributed to the influence of the "native" cubane subcluster that is lacking in the inorganic models. The degree and character of the unpaired spin delocalization was found to vary from 1edt, with an abiological dithiolate, to 1adt, which features the authentic cofactor. For 1adt, we find two (14)N signals, which are indicative for two possible isomers of the azadithiolate, demonstrating its high flexibility. All interaction parameters were also evaluated through density functional theory calculations at various levels.

The Basic Properties of the Electronic Structure of the Oxygen-evolving Complex of Photosystem II Are Not Perturbed by Ca2+ Removal

Ca(2+) is an integral component of the Mn(4)O(5)Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S(2)' state of the Ca(2+)-depleted cluster from spinach is examined by X- and Q-band EPR and (55)Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca(2+) removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S(2)' state in spinach PS II is very similar to the native S(2) state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca(2+) a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y(Z) and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel (55)Mn(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca(2+)-depleted PS II. Mn(2+) titration, monitored by (55)Mn ENDOR, revealed a specific Mn(2+) binding site with a submicromolar K(D). Ca(2+) titration of Mn(2+)-loaded, Ca(2+)-depleted PS II demonstrated that the site is reversibly made accessible to Mn(2+) by Ca(2+) depletion and reconstitution. Mn(2+) is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn(4)O(5)Ca cluster during photoassembly and/or D1 repair.

Pulsed ENDOR Determination of the Arginine Location in the Ferrous–NO Form of Neuronal NOS

Mammalian nitric oxide synthases (NOSs) are enzymes responsible for oxidation of L-arginine (L-Arg) to nitric oxide (NO). Mechanisms of reactions at the catalytic heme site are not well understood, and it is of current interest to study structures of the heme species that activates O(2) and transforms the substrate. The NOS ferrous-NO complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this work, pulsed electron-nuclear double resonance (ENDOR) was used to probe the position of the l-Arg substrate at the NO(•)-coordinated ferrous heme center(s) in the oxygenase domain of rat neuronal NOS (nNOS). The analysis of (2)H and (15)N ENDOR spectra of samples containing d(7)- or guanidino-(15)N(2) labeled L-Arg has resulted in distance estimates for the nearby guanidino nitrogen and the nearby proton (deuteron) at C(δ). The L-Arg position was found to be noticeably different from that in the X-ray crystal structure of nNOS ferrous-NO complex [Li et al. J. Biol. Inorg. Chem.2006, 11, 753-768], with the nearby guanidino nitrogen being ~0.5 Å closer to, and the nearby H(δ) about 1 Å further from, the NO ligand than in the X-ray structure. The difference might be related to the structural constraints imposed on the protein by the crystal. Importantly, in spite of its closer position, the guanidino nitrogen does not form a hydrogen bond with the NO ligand, as evidenced by the absence of significant isotropic hfi constant for N(g1). This is consistent with the previous reports that it is not the L-Arg substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (NO and O(2)) bound to the heme.

Characterization of the electronic structure of [formula omitted] defect in quartz by pulsed EPR spectroscopy

The electronic structure of the E2′ defect in quartz is investigated for the first time by the pulsed electron paramagnetic resonance spectroscopy. The temperature dependence of the longitudinal and transverse relaxations and the possible mechanisms lying behind are carefully characterized. With the pulsed electron–nuclear double resonance spectroscopy, a magnetically coupled proton is confirmed to exist in the E2′ center. The observed long T2 over 260 μs at 60 K, as well as the well-separated energy levels arising from the proton-coupled electron–nuclear structure, makes E2′ center a good candidate for quantum memory in quantum computing.

NH3 and O2interaction with tetrahedral Ti3+ions isomorphously substituted in the framework of TiAlPO-5. A combined pulse EPR, pulse ENDOR, UV-Vis and FT-IR study

Continuous Wave (CW), pulse Electron Paramagnetic Resonance (EPR) and pulse Electron Nuclear Double Resonance (ENDOR) spectroscopies, in conjunction with UV-Vis and Infrared (IR) spectroscopies, are used to investigate the chemical reactivity of tetrahedrally coordinated Ti(3+) ions isomorphously substituted in the framework of AlPO-5 towards NH(3) and O(2). The coordination of ammonia to Ti(3+) centres is followed in detail by complementary vibrational and electron magnetic resonance techniques. In particular HYSCORE spectra allow identifying the coordination of two ammonia molecules to Ti(3+) centres resolving the full hyperfine and quadrupole (14)N coupling tensors. The reactivity of the reduced TiAlPO sample towards molecular oxygen is detailed by means of CW-EPR and pulse ENDOR spectroscopy. (17)O(2) is employed, allowing to establish the formation of a "side-on" η(2) O(2)(-)-Ti(4+) electrostatic complex. Pulse ENDOR spectra provide detailed information on the local environment of the formed superoxide radical anion which acts as a paramagnetic probe, providing evidence for Ti-O-Ti oligomeric species.

Manganese Cation Reports on the Number of Coordinated Phosphates from the Nucleotide at the Active Site in Myosin ATPase

ATP is an important cofactor for myosin ATPase. ATP binding initiates myosin conformation change (recovery stroke), priming it for force generation. In the presence of a divalent cation (usually Mg2+), myosin hydrolyzes ATP to ADP and phosphate, and their subsequent release presumably triggers the power stroke in actomyosin. What is the role of ATP hydrolysis in myosin? What is the relation between the kinetics of ATP hydrolysis and those of myosin conformation changes? To address these problems, we propose to use pulsed electron paramagnetic resonance (EPR) techniques and study the coordination environment of the divalent cation complexed with a nucleotide at the myosin active site. To enable the EPR investigations, Mn2+ can be used instead of Mg2+ without interruption of the myosin ATPase activity. In this preliminary work, we studied the myosin.Mn complexes with non-hydrolyzable nucleotide analogs, AMPPNP (triphosphate) and ADP.AlF4 (diphosphate), as well as Mn.nucleotide complexes without myosin. Mn2+ binding to myosin was monitored by pulsed electron-electron double resonance (ELDOR), which detected the magnetic dipole interaction between Mn2+ and a nitroxide spin probe attached to the A639C myosin mutant. The number of coordinated phosphates in myosin.Mn.AMPPNP complex determined by 31P pulsed electron-nuclear double resonance (ENDOR) was twice that detected for the myosin.Mn.ADP.AlF4 complex. These results demonstrate the potential of pulsed EPR for distinguishing between the myosin.ATP and myosin.ADP biochemical states and study the role of ATP hydrolysis in myosin structural kinetics.

Probing Conformational Variations at the ATPase Site of the RNA Helicase DbpA by High-Field Electron–Nuclear Double Resonance Spectroscopy

The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein's ATPase site in solution. Specifically, we substituted the essential Mg(2+) cofactor in the ATPase active site for paramagnetic Mn(2+) and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn(2+) interactions with the nucleotide phosphates through the (31)P hyperfine couplings and the coordination by protein residues through (13)C hyperfine coupling from (13)C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the (13)C and the (31)P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn(2+) ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn(2+) cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein's ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn(2+) ion. Finally, a competitive Mn(2+) binding site was found for single-stranded RNA construct.

Pulsed ENDOR determination of relative orientation of g-frame and molecular frame of imidazole-coordinated heme center of iNOS.

Mammalian nitric oxide synthase (NOS) is a flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide. Information about the relative alignment of the heme and FMN domains of NOS is important for understanding the electron transfer between the heme and FMN centers, but no crystal structure data for NOS holoenzyme are available. In our previous work [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059-12067], the distance between the imidazole-coordinated low-spin Fe(III) heme and FMN semiquinone in a human inducible NOS (iNOS) oxygenase/FMN construct has been determined by pulsed electron paramagnetic resonance (EPR). The orientation of the Fe-FMN radius vector, R(Fe-FMN), with respect to the heme g-frame was also determined. In the present study, pulsed electron-nuclear double resonance (ENDOR) investigation of the deuterons at carbons C2 and C5 in the deuterated coordinated imidazole was used to determine the relative orientation of the heme g-frame and molecular frame, from which R(Fe-FMN) can be referenced to the heme molecular frame. Numerical simulations of the ENDOR spectra showed that the g-factor axis corresponding to the low-field EPR turning point is perpendicular to the heme plane, whereas the axis corresponding to the high-field turning point is in the heme plane and makes an angle of about 80° with the coordinated imidazole plane. The FMN-heme domain docking model obtained in the previous work was found to be in qualitative agreement with the combined experimental results of the two pulsed EPR works.

Electrical Detection of Coherent Nuclear Spin Oscillations in Phosphorus-Doped Silicon using Pulsed ENDOR

We demonstrate the electrical detection of pulsed X-band electron nuclear double resonance (ENDOR) in phosphorus-doped silicon at 5 K. A pulse sequence analogous to Davies ENDOR in conventional electron spin resonance is used to measure the nuclear spin transition frequencies of the (31)P nuclear spins, where the (31)P electron spins are detected electrically via spin-dependent transitions through Si/SiO(2) interface states, thus not relying on a polarization of the electron spin system. In addition, the electrical detection of coherent nuclear spin oscillations is shown, demonstrating the feasibility to electrically read out the spin states of possible nuclear spin qubits.

Hydration Structure of the Ti(III) Cation as Revealed by Pulse EPR and DFT Studies: New Insights into a Textbook Case

The (17)O and (1)H hyperfine interactions of water ligands in the Ti(III) aquo complex in a frozen solution were determined using Hyperfine Sublevel Correlation (HYSCORE) and Pulse Electron Nuclear Double Resonance (ENDOR) spectroscopies at 9.5 GHz. The isotropic hyperfine interaction (hfi) constant of the water ligand (17)O was found to be about 7.5 MHz. (1)H Single Matched Resonance Transfer (SMART) HYSCORE spectra allowed resolution of the hfi interactions of the two inequivalent water ligand protons and the relative orientations of their hfi tensors. The magnetic and geometrical parameters extracted from the experiments were compared with the results of DFT computations for different geometrical arrangements of the water ligands around the cation. The theoretical observable properties (g tensor (1)H and (17)O hfi tensors and their orientations) of the [Ti(H(2)O)(6)](3+) complex are in quantitative agreement with the experiments for two slightly different geometrical arrangements associated with D(3d) and C(i) symmetries.

Dynamic Hydrogen-Bonding Network in the Distal Pocket of the Nitrosyl Complex of Pseudomonas aeruginosa cd1 Nitrite Reductase

cd(1) nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d(1)-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d(1)-heme to avoid enzyme inhibition by the formation of ferrous-nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d(1)-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d(1)-heme NO complex from Pseudomonas aeruginosa cd(1) NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the "distal" side of the heme (Tyr(10), His(327), and His(369)), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron-nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d(1)-heme complex of cd(1) NIR forms H-bonds with Tyr(10) and His(369), whereas the second conserved histidine, His(327), appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr(10) is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His(369) is closer to the NO, whereas mutation of both distal histidines displaces Tyr(10), removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.

ChemInform Abstract: EPR and ENDOR Studies of Shallow Donors in SiC

AbstractReview: 85 refs.

Giant titanium electron wave function in gallium oxide: A potential electron-nuclear spin system for quantum information processing

The hyperfine interactions of the unpaired electron with eight surrounding $^{69}\text{G}\text{a}$ and $^{71}\text{G}\text{a}$ nuclei in Ti-doped $\ensuremath{\beta}{\text{-Ga}}_{2}{\text{O}}_{3}$ were analyzed by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopies. They are dominated by strong isotropic hyperfine couplings due to a direct Fermi contact interaction with Ga nuclei in octahedral sites of rutile-type chains oriented along $\mathbf{b}$ axis, revealing a large anisotropic spatial extension of the electron wave function. Titanium in $\ensuremath{\beta}{\text{-Ga}}_{2}{\text{O}}_{3}$ is thus best described as a diffuse $({\text{Ti}}^{4+}\text{\ensuremath{-}}{e}^{\ensuremath{-}})$ pair rather than as a localized ${\text{Ti}}^{3+}$. Both electron and $^{69}\text{G}\text{a}$ nuclear spin Rabi oscillations could be observed by pulsed EPR and pulsed ENDOR, respectively. The electron spin decoherence time is about $1\text{ }\ensuremath{\mu}\text{s}$ (at 4 K) and an upper bound of $520\text{ }\ensuremath{\mu}\text{s}$ (at 8 K) is estimated for the nuclear decoherence time. Thus, $\ensuremath{\beta}{\text{-Ga}}_{2}{\text{O}}_{3}:\text{Ti}$ appears to be a potential spin-bus system for quantum information processing with a large nuclear spin quantum register.