Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia eutropha in its reduced state by HYSCORE and ENDOR spectroscopy

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.     

Identification of the nitrogen donor hydrogen bonded with the semiquinone at the Q(H) site of the cytochrome bo3 from Escherichia coli

The selective (15)N isotope labeling was used for the identification of the nitrogen involved in a hydrogen bond formation with the semiquinone in the high-affinity Q(H) site in the cytochrome bo(3) ubiquinol oxidase. This nitrogen produces dominating contribution to X-Band (14)N ESEEM spectra. The 2D ESEEM (HYSCORE) experiments with the Q(H) site SQ in the series of selectively (15)N labeled bo(3) oxidase proteins have directly identified the N(epsilon) of R71 as an H-bond donor. In addition, selective (15)N labeling has allowed us for the first time to determine weak hyperfine couplings with the side-chain nitrogens from all residues around the SQ. Those are reflecting a distribution of the unpaired spin density over the protein in the SQ state of the quinone processing site.     

The role of the Mg2+ cation in ATPsynthase studied by electron paramagnetic resonance using VO2+ and Mn2+ paramagnetic probes

The electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectra of Mg2+-depleted chloroplast F1-ATPase substituted with stoichiometric VO2+ are reported. The ESEEM and HYSCORE spectra of the complex are dominated by the hyperfine and quadrupole interactions between the VO2+ paramagnet and two different nitrogen ligands with isotropic hyperfine couplings /A1/ = 4.11 MHz and /A2/ = 6.46 MHz and nuclear quadrupole couplings e2qQ1 approximately 3.89-4.49 MHz and e2qQ2 approximately 1.91-2.20 MHz, respectively. Aminoacid functional groups compatible with these magnetic couplings include a histidine imidazole, the epsilon-NH2 of a lysine residue, and the guanidinium group of an arginine. Consistent with this interpretation, very characteristic correlations are detected in the HYSCORE spectra between the 14N deltaM1 = 2 transitions in the negative quadrant, and also between some of the deltaM1 = 1 transitions in the positive quadrant. The interaction of the substrate and product ADP and ATP nucleotides with the enzyme has been studied in protein complexes where Mg2+ is substituted for Mn2+. Stoichiometric complexes of Mn x ADP and Mn x ATP with the whole enzyme show distinct and specific hyperfine couplings with the 31P atoms of the bonding phosphates in the HYSCORE (ADP, A(31Pbeta) = 5.20 MHz: ATP, A(31Pbeta) = 4.60 MHz and A(31Pgamma) = 5.90 MHz) demonstrating the role of the enzyme active site in positioning the di- or triphosphate chain of the nucleotide for efficient catalysis. When the complexes are formed with the isolated alpha or beta subunits of the enzyme, the HYSCORE spectra are substantially modified, suggesting that in these cases the nucleotide binding site is only partially structured.     

Pulsed EPR and DFT characterization of radicals produced by photo-oxidation of zeaxanthin and violaxanthin on silica-alumina

Pulsed electron nuclear double resonance (ENDOR) and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) studies in combination with density functional theory (DFT) calculations revealed that photo-oxidation of natural zeaxanthin (ex Lycium halimifolium) and violaxanthin (ex Viola tricolor) on silica-alumina produces the carotenoid radical cations (Car*+) and also the neutral carotenoid radicals (#Car*) as a result of proton loss (indicated by #) from the C4(4') methylene position or one of the methyl groups at position C5(5'), C9(9'), or C13(13'), except for violaxanthin where the epoxide at positions C5(5')-C6(6') raises the energy barrier for proton loss, and the neutral radicals #Car*(4) and #Car*(5) are not observed. DFT calculations predict the largest isotropic beta-methyl proton hyperfine couplings to be 8 to 10 MHz for Car*+, in agreement with previously reported hyperfine couplings for carotenoid pi-conjugated radicals with unpaired spin density delocalized over the whole molecule. Anisotropic alpha-proton hyperfine coupling tensors determined from the HYSCORE analysis were assigned on the basis of DFT calculations with the B3LYP exchange-correlation functional and found to arise not only from the carotenoid radical cation but also from carotenoid neutral radicals, in agreement with the analysis of the pulsed ENDOR data. The formation of the neutral radical of zeaxanthin should provide another effective nonphotochemical quencher of the excited state of chlorophyll for photoprotection in the presence of excess light.     

17O ESEEM evidence for exchange of the axial oxo ligand in the molybdenum center of the high pH form of sulfite oxidase

A 17O ESEEM investigation of the high pH form of chicken sulfite oxidase using hyperfine sublevel correlation (HYSCORE) spectroscopy at 29.25 GHz has revealed a new type of exchangeable 17O ligand that is characterized by a significantly smaller hyperfine interaction ( approximately 5 MHz) than that previously detected by CW EPR. This new type of exchangeable oxygen ligand is assigned to the axial oxo group of the Mo(V) center.     

Protein-cofactor interactions and EPR parameters for the Q(H) quinone binding site of quinol oxidase. A density functional study

Recent multifrequency EPR studies of the "high-affinity" quinone binding site of quinol oxidase (Q(H) site) have suggested a very asymmetric hydrogen-bonding environment for the semiquinone radical anion state. Single-sided hydrogen bonding to the O1 carbonyl position was one of the proposals, which contrasts with some previous experimental indications. Here density functional calculations of the EPR parameters (g-tensors, 13C, 1H, and 17O hyperfine tensors) for a wide variety of supermolecular model complexes have been used to provide insight into the detailed relations among structure, environment, and EPR parameters of ubisemiquinone radical anions. A single-sided binding model is not able to account for the experimentally observed low g(x) component of the g-tensor or for the observed magnitude of the asymmetry of the 13C carbonyl HFC tensors. Based on the detailed comparison between computation and experiment, a model with two hydrogen bonds to O1 and one hydrogen bond to O4 is suggested for the Q(H) site, but a model with one more hydrogen bond on each side cannot be excluded. Several general conclusions on the interrelations between EPR parameters and hydrogen bond patterns of ubisemiquinones in proteins are provided.     

Pulsed EPR characterization of encapsulated atomic hydrogen in octasilsesquioxane cages

Hydrogen atoms encapsulated in molecular cages are potential candidates for quantum computing applications. They provide the simplest two-spin system where the 1s electron spin, S = 1/2, is hyperfine-coupled to the proton nuclear spin, I = 1/2, with a large isotropic hyperfine coupling (A = 1420.40575 MHz for a free atom). While hydrogen atoms can be trapped in many matrices at cryogenic temperatures, it has been found that they are exceptionally stable in octasilsesquioxane cages even at room temperature [Sasamori et al., Science, 1994, 256, 1691]. Here we present a detailed spin-lattice and spin-spin relaxation study of atomic hydrogen encapsulated in Si(8)O(12)(OSiMe(2)H)(8) using X-band pulsed EPR spectroscopy. The spin-lattice relaxation times T(1) range between 1.2 s at 20 K and 41.8 μs at room temperature. The temperature dependence of the relaxation rate shows that for T < 60 K the spin-lattice relaxation is best described by a Raman process with a Debye temperature of θ(D) = 135 K, whereas for T > 100 K a thermally activated process with activation energy E(a) = 753 K (523 cm(-1)) prevails. The phase memory time T(M) = 13.9 μs remains practically constant between 200 and 300 K and is determined by nuclear spin diffusion. At lower temperatures T(M) decreases by an order of magnitude and exhibits two minima at T = 140 K and T = 60 K. The temperature dependence of T(M) between 20 and 200 K is attributed to dynamic processes that average inequivalent hyperfine couplings, e.g. rotation of the methyl groups of the cage organic substituents. The hyperfine couplings of the encapsulated proton and the cage (29)Si nuclei are obtained through numerical simulations of field-swept FID-detected EPR spectra and HYSCORE experiments, respectively. The results are discussed in terms of existing phenomenological models based on the spherical harmonic oscillator and compared to those of endohedral fullerenes.     

EPR and ENDOR study of radiation-induced radical formation in purines: hypoxanthine hydrochloride monohydrate crystals X-irradiated at 10 K

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) study of hypoxanthine.HCl.H(2)O crystals irradiated at low temperatures (10 K) identified three radical species. In these crystals, the parent molecules exist in a cationic form with a proton at N7. R1 was the product of net hydrogen addition to N3 and exhibited alpha-proton hyperfine couplings to HC2, HN1, HC8, and HN3. The coupling to HC2 has an isotropic component smaller than usual, evidently an indication that the bonds to C2 are nonplanar. R2 was the product of net hydrogen loss from N7, equivalent to the one-electron oxidation product of neutral hypoxanthine, and exhibited alpha-proton hyperfine couplings to HC2 and HC8. Both couplings are characteristic of planar bonding arrangements at the centers of spin. R3 was provisionally identified as the product of net hydrogen addition to O6 and exhibited hyperfine alpha-proton couplings to HC8 and NH1. To identify the set of radicals, the experiments employed four crystal types: normal, deuterated only at NH positions, deuterated at HC8 and NH positions, and deuterated at HC8 only. The low-temperature data also showed clear evidence for H/D isotope effects in formation and/or stabilization of all radicals. To aid and support the identifications, the experimental results were compared to DFT calculations performed on a variety of radical structures plausible for the parent molecule and molecular packing within the crystal.     

Hydrogen bonds not only provide a structural scaffold to assemble donor and acceptor moieties of zinc porphyrin-quinone dyads but also control the photoinduced electron transfer to afford the long-lived charge-separated states

A series of zinc porphyrin-quinone linked dyads [ZnP-CONH-Q, ZnP-NHCO-Q, and ZnP-n-Q (n = 3, 6, 10)] were designed and synthesized to investigate the effects of hydrogen bonds which can not only provide a structural scaffold to assemble donor and acceptor moieties but also control the photoinduced electron-transfer process. In the case of ZnP-CONH-Q and ZnP-NHCO-Q, the hydrogen bond between the N-H proton and the carbonyl oxygen of Q results in the change in the reduction potential of Q. The strong hydrogen bond between the N-H proton and the carbonyl oxygen of Q*- in ZnP-CONH-Q*-,ZnP-NHCO-Q*-, and ZnP-n-Q*- (n = 3, 6, 10) generated by the chemical reduction has been confirmed by the ESR spectra, which exhibit hyperfine coupling constants in agreement those predicted by the density functional calculations. In the case of ZnP-n-Q (n = 3, 6, 10), on the other hand, the hydrogen bond between two amide groups provides a structural scaffold to assemble the donor (ZnP) and the acceptor (Q) moiety together with the hydrogen bond between the N-H proton and the carbonyl oxygen of Q, leading to attainment of the charge-separated state with a long lifetime up to a microsecond.     

Coordination of solvent molecules to VO(acac)<Subscript>2</Subscript> complexes in solution studied by hyperfine sublevel correlation spectroscopy and pulsed electron nuclear double resonance

Interactions and binding sites of the solvent molecules chloroform and ethanol to bis(acetylacetonate)oxovanadium(IV) (VO(acac)₂) complexes in (frozen) solutions have been investigated by pulsed electron nuclear double resonance, sum peak electron spin echo envelope modulation and hyperfine sublevel correlation spectroscopy. The experimental proton hyperfine coupling data of coordinating solvent molecules have been interpreted using quantum chemical calculations (density functional theory). Experimental and computed hyperfine couplings indicate that ethanol coordinates to vanadium in the equatorial plane of VO(acac)₂ and chloroform interacts via hydrogen bonding to oxygens of the acac ligands.     

Electronic structure of binuclear mixed valence copper azacryptates derived from integrated advanced EPR and DFT calculations

Binuclear, mixed valence copper complexes with a [Cu(+1)(.5), Cu(+1)(.5)] redox state and S = (1)/(2) can be stabilized with rigid azacryptand ligands. In this system the unpaired electron is delocalized equally over the two copper ions, and it is one of the very few synthetic models for the electron mediating Cu(A) site of nitrous oxide reductase and cytochrome c oxidase. The spatial and electronic structures of the copper complex in frozen solution were obtained from the magnetic interactions, namely the g-tensor and the (63,65)Cu, (14)N, (2)H, and (1)H hyperfine couplings, in combination with density functional theory (DFT) calculations. The magnetic interactions were determined from continuous wave (CW) electron paramagnetic resonance (EPR), pulsed electron nuclear double resonance (ENDOR), two-dimensional TRIPLE, and hyperfine sublevel correlation spectroscopy (HYSCORE) carried out at W-band or/and X-band frequencies. The DFT calculated g and Cu hyperfine values were in good agreement with the experimental values showing that the structure in solution is indeed close to that of the optimized structure. Then, the DFT calculated hyperfine parameters were used as guidelines and starting points in the simulations of the various experimental ENDOR spectra. A satisfactory agreement with the experimental results was obtained for the (14)N hyperfine and quadrupole interactions. For (1)H the DFT calculations gave good predictions for the hyperfine tensor orientations and signs, and they were also successful in reproducing trends in the magnitude of the various proton hyperfine couplings. These, in turn, were very useful for ENDOR signals assignments and served as constraints on the simulation parameters.     

Pulsed ENDOR study of Cu(I)-NO adsorption complexes in Cu-L zeolite

The local environments of Cu(I)-NO adsorption complexes formed in zeolites Cu-L and Cu-ZSM-5 were studied by electron spin resonance (ESR), pulsed electron nuclear double resonance (ENDOR), and hyperfine sublevel correlation spectroscopy (HYSCORE). Cu(I)-NO complexes have attracted special interest because they are important intermediates in the catalytic decomposition of nitric oxide over copper exchanged zeolites. Recently, detailed structures of the complexes in Cu-ZSM-5 zeolites, O2-Al-O2-Cu(I)-NO, have been proposed on the basis of quantum chemical calculations (Pietrzyk, et al. J. Phys. Chem. B 2003, 107, 6105. Dedecek, et al. Phys. Chem. Chem. Phys. 2002, 4, 5406). 27Al pulsed ENDOR and HYSCORE experiments allowed the hyperfine coupling parameters of an aluminum nuclei found in the vicinity of the Cu(I)-NO complex formed in zeolite Cu-L to be estimated. The data indicate that the aluminum atom is located in the third coordination sphere of the adsorbed NO molecule in agreement with the suggested geometry of the adsorption sites. Broad distributions of aluminum nuclear quadrupole and hyperfine coupling parameters and short electron spin relaxation times of the Cu(I)-NO species prevented the determination of the 27Al hyperfine couplings for zeolite Cu-ZSM-5.     

The electronic structure of the H-cluster in the [FeFe]-hydrogenase from Desulfovibrio desulfuricans: a Q-band 57Fe-ENDOR and HYSCORE study

The active site of the (57)Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examined using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (H(ox)) and the CO inhibited form (H(ox)-CO) all six (57)Fe hyperfine couplings were detected. The analysis shows that the apparent spin density extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six (57)Fe nuclei in the H-cluster of the H(ox)-CO state. Four large 57Fe hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S](H) (cubane) subcluster. Two weak (57)Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe](H) subcluster. For the H(ox) state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An (57)Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all six (57)Fe nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe](H) assumes a [Fe(I)Fe(II)] redox configuration where the paramagnetic Fe(I) atom is attached to the [4Fe-4S](H) subcluster. The (57)Fe hyperfine interactions of the formally diamagnetic [4Fe-4S](H) are due to an exchange interaction between the two subclusters as has been discussed earlier by Popescu and Münck [Popescu, C.V.; Münck, E., J. Am. Chem. Soc. 1999, 121, 7877-7884]. This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the observed modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.     

Protein-cofactor interactions in bacterial reaction centers from Rhodobacter sphaeroides R-26: effect of hydrogen bonding on the electronic and geometric structure of the primary quinone. A density functional theory study

The effect of hydrogen bonding to the primary quinone (Q(A) and Q(*)(-)(A)) in bacterial reaction centers was studied using density functional theory (DFT) calculations. The charge neutral state Q(A) was investigated by optimizing the hydrogen atom positions of model systems extracted from 15 different X-ray structures. From this analysis, mean values of the H-bond lengths and directions were derived. It was found that the N(delta)-H of His M219 forms a shorter H-bond to Q(A) than the N-H of Ala M260. The H-bond of His M219 is linear and more twisted out of the quinone plane. The radical anion Q(*)(-)(A) in the protein environment was investigated by using a mixed quantum mechanics/molecular mechanics (QM/MM) approach. Two geometry optimizations with a different number of flexible atoms were performed. H-bond lengths were obtained and spectroscopic parameters calculated, i.e. the hyperfine and nuclear quadrupole couplings of magnetic nuclei coupled to the radical. Good agreement was found with the results provided by EPR/ENDOR spectroscopy. This implies that the calculated lengths and directions of the H-bonds to Q(*)(-)(A) are reliable values. From a comparison of the neutral and reduced state of Q(A) it was concluded that the H-bond distances are shortened by approximately 0.17 Angstroms (His M219) and approximately 0.13 Angstroms (Ala M260) upon single reduction of the quinone. It is shown that the point-dipole approximation can not be used for an estimation of H-bond lengths from measured hyperfine couplings in a system with out-of-plane H-bonding. In contrast, the evaluation of the nuclear quadrupole couplings of (2)H nuclei substituted in the hydrogen bonds yields H-bond lengths close to the values that were deduced from DFT geometry optimizations. The significance of hydrogen bonding to the quinone cofactors in biological systems is discussed.     

Two-dimensional (2D) pulsed electron paramagnetic resonance study of VO(2+)-triphosphate interactions: evidence for tridentate triphosphate coordination, and relevance to bone uptake and insulin enhancement by vanadium pharmaceuticals

Two- and four-pulse electron spin echo envelope modulation (ESEEM) and four-pulse two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopies have been used to determine the solution structure of a 3:1 triphosphate:vanadyl solution at pH 5.0. Limited quantitative data were extracted from the two pulse spectra; however, HYSCORE proved to be more useful in the detection and interpretation of the (31)P and (1)H couplings. Three sets of cross-peaks were observed for each nucleus. For the (31)P couplings, three sets of cross-peaks were observed in the HYSCORE spectrum, and contour line shape analysis yielded coupling constants of approximately 15, 9, and 1 MHz. HYSCORE cross-peaks in the proton region were partially overlapping; however, interpretation of the proton coupling was simplified through the use of one-dimensional four-pulse ESEEM and subsequent analysis of the sum combination peaks. Comparison of the derived isotropic and anisotropic coupling constants with results from earlier ESEEM and electron nuclear double resonance (ENDOR) studies was consistent with the presence of at least one, and most likely two, water molecules coordinated in the equatorial plane of the vanadyl cation. The vanadyl-triphosphate system was shown to be an accurate model of the in vivo vanadyl-phosphate coupling constants determined in an earlier study (Dikanov, S. A.; Liboiron, B. D.; Thompson, K. H.; Vera, E.; Yuen, V. G.; McNeill, J. H.; Orvig, C. J. Am. Chem. Soc. 1999, 121, 11004.) Comparison of these values to those found in previous spectroscopic studies of vanadyl-triphosphate interactions, along with a detailed structural interpretation, are presented. This work represents the first detection of tridentate polyphosphate coordination to the vanadyl ion, and the first observation of an axial phosphate interaction not previously reported in earlier ENDOR and pulsed electron paramagnetic resonance studies.     

CH stretching vibration of N-methylformamide as a sensitive probe of its complexation: infrared matrix isolation and computational study

The complexes between trans-N-methylformamide (t-NMF) and Ar, N(2), CO, H(2)O have been studied by infrared matrix isolation spectroscopy and/or ab initio calculations. The infrared spectra of NMF/Ne, NMF/Ar and NMF/N(2)(CO,H(2)O)/Ar matrices have been measured and the effect of the complexation on the perturbation of t-NMF frequencies was analyzed. The geometries of the complexes formed between t-NMF and Ar, N(2), CO and H(2)O were optimized in two steps at the MP2/6-311++G(2d,2p) level of theory. The four structures, found for every system at this level, were reoptimized on the CP-corrected potential energy surface; both normal and CP corrected harmonic frequencies and intensities were calculated. For every optimized structure the interaction energy was partitioned according to the SAPT scheme and the topological distribution of the charge density (AIM theory) was performed. The analysis of the experimental and theoretical results indicates that the t-NMF-N(2) and CO complexes present in the matrices are stabilized by very weak N-H···N and N-H···C hydrogen bonds in which the N-H group of t-NMF serves as a proton donor. In turn, the t-NMF-H(2)O complex present in the matrix is stabilized by O-H···O(C) hydrogen bonding in which the carbonyl group of t-NMF acts as a proton acceptor. Both, the theoretical and experimental results indicate that involvement of the NH group of t-NMF in formation of very weak hydrogen bonds with the N(2) or CO molecules leads to a clearly noticeable red shift of the CH stretching wavenumber whereas engagement of the CO group as a proton acceptor triggers a blue shift of this wavenumber.     

Ion-binding study by 17O solid-state NMR spectroscopy in the model peptide Gly-Gly-Gly at 19.6 T

Li(+) and Ca(2+) binding to the carbonyl oxygen sites of a model peptide system has been studied by (17)O solid-state NMR spectroscopy. (17)O chemical shift (CS) and quadrupole coupling (QC) tensors are determined in four Gly-(Gly-(17)O)-Gly polymorphs by a combination of stationary and fast magic-angle spinning (MAS) methods at high magnetic field, 19.6 T. In the crystal lattice, the carbonyl oxygen of the central glycyl residue in two gly-gly-gly polymorphs form intermolecular hydrogen bonds with amides, whereas the corresponding carbonyl oxygens of the other two polymorphs form interactions with Li(+) and Ca(2+) ions. This permits a comparison of perturbations on (17)O NMR properties by ion binding and intermolecular hydrogen bonding. High quality spectra are augmented by density functional theory (DFT) calculations on large molecular clusters to gain additional theoretical insights and to aid in the spectral simulations. Ion binding significantly decreases the two (17)O chemical shift tensor components in the peptide plane, delta(11) and delta(22), and, thus, a substantial change in the isotropic chemical shift. In addition, quadrupole coupling constants are decreased by up to 1 MHz. The effects of ion binding are found to be almost an order of magnitude greater than those induced by hydrogen bonding.     

Probing mode and site of substrate water binding to the oxygen-evolving complex in the S2 state of photosystem II by 17O-HYSCORE spectroscopy

In the oxygen-evolving complex (OEC) of photosystem II (PSII) molecular oxygen is formed from two substrate water molecules that are ligated to a mu-oxo bridged cluster containing four Mn ions and one Ca ion (Mn4OxCa cluster; Ox symbolizes the unknown number of mu-oxo bridges; x >or= 5). There is a long-standing enigma as to when, where, and how the two substrate water molecules bind to the Mn4OxCa cluster during the cyclic water-splitting reaction, which involves five distinct redox intermediates (Si-states; i = 0,...,4). To address this question we employed hyperfine sublevel correlation (HYSCORE) spectroscopy on H217O-enriched PSII samples poised in the paramagnetic S2 state. This approach allowed us to resolve the magnetic interaction between one solvent exchangeable 17O that is directly ligated to one or more Mn ions of the Mn4OxCa cluster in the S2 state of PSII. Direct coordination of 17O to Mn is supported by the strong (A approximately 10 MHz) hyperfine coupling. Because these are properties expected from a substrate water molecule, this spectroscopic signature holds the potential for gaining long-sought information about the binding mode and site of one of the two substrate water molecules in the S2 state of PSII.     

Influence of hybridization and cooperativity on the properties of Au-bonding interaction: comparison with hydrogen bonds

Quantum chemical calculations have been performed to study the hybridization effect in H(2)O-AuCH(2)CH(3), H(2)O-AuCHCH(2), and H(2)O-AuCCH dimers, and the cooperativity between the hydrogen bond and Au bonding in three trimers (T1, T2, and T3) composed of one AuCCH and two H(2)O molecules. With regard to the organic Au compounds, sp-hybridized AuCCH forms the strongest Au bonding, followed by sp(2) and then sp(3). The C-Au bond is elongated, and its elongation becomes larger with the increase of the s character in hybrid orbitals, whereas the corresponding stretch vibration displays a small blue shift. The positive cooperativity is present for the hydrogen bond and Au bonding in T1 and T2 trimers, whereas the negative cooperativity is found in T3 trimer. The results show that the hybridization effect and cooperative interaction in Au bonding are similar to those in hydrogen bonds. Additionally, an OH···Au hydrogen bond is suggested in T1 trimer.     

Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase

The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H. The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz. The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.