Intramolecular Hydrogen Bonding Restricts Gd-Aqua-Ligand Dynamics.

Aqua ligands can undergo rapid internal rotation about the M-O bond. For magnetic resonance contrast agents, this rotation results in diminished relaxivity. Herein, we show that an intramolecular hydrogen bond to the aqua ligand can reduce this internal rotation and increase relaxivity. Molecular modeling was used to design a series of four Gd complexes capable of forming an intramolecular H-bond to the coordinated water ligand, and these complexes had anomalously high relaxivities compared to similar complexes lacking a H-bond acceptor. Molecular dynamics simulations supported the formation of a stable intramolecular H-bond, while alternative hypotheses that could explain the higher relaxivity were systematically ruled out. Intramolecular H-bonding represents a useful strategy to limit internal water rotational motion and increase relaxivity of Gd complexes.

Overcoming artificial broadening in Gd(3+)-Gd(3+) distance distributions arising from dipolar pseudo-secular terms in DEER experiments.

By providing accurate distance measurements between spin labels site-specifically attached to bio-macromolecules, double electron-electron resonance (DEER) spectroscopy provides a unique tool to probe the structural and conformational changes in these molecules. Gd(3+)-tags present an important family of spin-labels for such purposes, as they feature high chemical stability and high sensitivity in high-field DEER measurements. The high sensitivity of the Gd(3+) ion is associated with its high spin (S = 7/2) and small zero field splitting (ZFS), resulting in a narrow spectral width of its central transition at high fields. However, under the conditions of short distances and exceptionally small ZFS, the weak coupling approximation, which is essential for straightforward DEER data analysis, becomes invalid and the pseudo-secular terms of the dipolar Hamiltonian can no longer be ignored. This work further explores the effects of pseudo-secular terms on Gd(3+)-Gd(3+) DEER measurements using a specifically designed ruler molecule; a rigid bis-Gd(3+)-DOTA model compound with an expected Gd(3+)-Gd(3+) distance of 2.35 nm and a very narrow central transition at the W-band (95 GHz). We show that the DEER dipolar modulations are damped under the standard W-band DEER measurement conditions with a frequency separation, Δν, of 100 MHz between the pump and observe pulses. Consequently, the DEER spectrum deviates considerably from the expected Pake pattern. We show that the Pake pattern and the associated dipolar modulations can be restored with the aid of a dual mode cavity by increasing Δν from 100 MHz to 1.09 GHz, allowing for a straightforward measurement of a Gd(3+)-Gd(3+) distance of 2.35 nm. The increase in Δν increases the contribution of the |-5/2〉→|-3/2〉 and |-7/2〉→|-5/2〉 transitions to the signal at the expense of the |-3/2 〉→|-1/2〉 transition, thus minimizing the effect of dipolar pseudo-secular terms and restoring the validity of the weak coupling approximation. We apply this approach to the A93C/N140C mutant of T4 lysozyme labeled with two different Gd(3+) tags that have narrow central transitions and show that even for a distance of 4 nm there is still a significant (about two-fold) broadening that is removed by increasing Δν to 636 MHz and 898 MHz.

Pulsed electron paramagnetic resonance spectroscopy of (33)S-labeled molybdenum cofactor in catalytically active bioengineered sulfite oxidase.

Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d(1)) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant (32)S has no nuclear spin (I = 0). Here we describe direct incorporation of (33)S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from (33)S-labeled MPT in this catalytically active SO variant are dominated by the "interdoublet" transition arising from the strong nuclear quadrupole interaction, as also occurs for the (33)S-labeled exchangeable equatorial sulfite ligand [ Klein, E. L., et al. Inorg. Chem. 2012 , 51 , 1408 - 1418 ]. The estimated experimental hfi and nqi parameters for (33)S (aiso = 3 MHz and e(2)Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two (33)S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).

Applications of pulsed EPR spectroscopy to structural studies of sulfite oxidizing enzymes().

Sulfite oxidizing enzymes (SOEs), including sulfite oxidase (SO) and bacterial sulfite dehydrogenase (SDH), catalyze the oxidation of sulfite (SO(3) (2-)) to sulfate (SO(4) (2-)). The active sites of SO and SDH are nearly identical, each having a 5-coordinate, pseudo-square-pyramidal Mo with an axial oxo ligand and three equatorial sulfur donor atoms. One sulfur is from a conserved Cys residue and two are from a pyranopterindithiolene (molybdopterin, MPT) cofactor. The identity of the remaining equatorial ligand, which is solvent-exposed, varies during the catalytic cycle. Numerous in vitro studies, particularly those involving electron paramagnetic resonance (EPR) spectroscopy of the Mo(V) states of SOEs, have shown that the identity and orientation of this exchangeable equatorial ligand depends on the buffer pH, the presence and concentration of certain anions in the buffer, as well as specific point mutations in the protein. Until very recently, however, EPR has not been a practical technique for directly probing specific structures in which the solvent-exposed, exchangeable ligand is an O, OH(-), H(2)O, SO(3) (2-), or SO(4) (2-) group, because the primary O and S isotopes ((16)O and (32)S) are magnetically silent (I = 0). This review focuses on the recent advances in the use of isotopic labeling, variable-frequency high resolution pulsed EPR spectroscopy, synthetic model compounds, and DFT calculations to elucidate the roles of various anions, point mutations, and steric factors in the formation, stabilization, and transformation of SOE active site structures.

Identity of the exchangeable sulfur-containing ligand at the Mo(V) center of R160Q human sulfite oxidase.

In our previous study of the fatal R160Q mutant of human sulfite oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc.2008, 130, 8471-8480), a new Mo(V) species, denoted "species 1", was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and (33)S and (17)O labeling. Here we report new results for species 1 of R160Q, based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in K(a)- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial sulfite as the only exchangeable ligand. The two types of (17)O signals that are observed arise from the coordinated and remote oxygen atoms of the sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the observed and calculated EPR parameters.

Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella.

Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDH(R55M)) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053-2063, 2009). For SDH(R55M) only the high-pH form is observed, and samples prepared in H(2)(17)O-enriched buffer show essentially the same (17)O hyperfine interaction and nuclear quadrupole interaction parameters as SDH(WT) enzyme. However, the pH dependence of the EPR spectra of SDH(R55Q), in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDH(WT). For SDH(R55Q) the blocked form with bound sulfate is generated at low pH, as verified by (33)S couplings observed upon reduction with (33)S-labeled sulfite. This observation of bound sulfate for SDH(R55Q) supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156-2163, 2009). At pH > or = 8 the high-pH form dominates for SDH(R55Q).

Structural studies of the molybdenum center of the pathogenic R160Q mutant of human sulfite oxidase by pulsed EPR spectroscopy and 17O and 33S labeling.

Electron paramagnetic resonance (EPR) investigation of the Mo(V) center of the pathogenic R160Q mutant of human sulfite oxidase (hSO) confirms the presence of three distinct species whose relative abundances depend upon pH. Species 1 is exclusively present at pH < or = 6, and remains in significant amounts even at pH 8. Variable-frequency electron spin echo envelope modulation (ESEEM) studies of this species prepared with (33)S-labeled sulfite clearly show the presence of coordinated sulfate, as has previously been found for the "blocked" form of Arabidopsis thaliana at low pH (Astashkin, A. V.; Johnson-Winters, K.; Klein, E. L.; Byrne, R. S.; Hille, R.; Raitsimring, A. M.; Enemark, J. H. J. Am. Chem. Soc. 2007, 129, 14800). The ESEEM spectra of Species 1 prepared in (17)O-enriched water show both strongly and weakly magnetically coupled (17)O atoms that can be assigned to an equatorial sulfate ligand and the axial oxo ligand, respectively. The nuclear quadrupole interaction (nqi) of the axial oxo ligand is substantially stronger than those found for other oxo-Mo(V) centers studied previously. Additionally, pulsed electron-nuclear double resonance (ENDOR) measurements reveal a nearby weakly coupled exchangeable proton. The structure for Species 1 proposed from the pulsed EPR results using isotopic labeling is a six-coordinate Mo(V) center with an equatorial sulfate ligand that is hydrogen bonded to an exchangeable proton. Six-coordination is supported by the (17)O nqi parameters for the axial oxo group of the model compound, (dttd)Mo(17)O((17)Otms), where H2dttd = 2,3:8,9-dibenzo-1,4,7,10-tetrathiadecane; tms = trimethylsilyl. Reduction of R160Q to Mo(V) with Ti(III) gives primarily Species 2, another low pH form, whereas reduction with sulfite at higher pH values gives a mixture of Species 1 and 2, as well as the "primary" high pH form of wild-type SO. The occurrence of significant amounts of the "sulfate-blocked" form of R160Q (Species 1) at physiological pH suggests that this species may be a contributing factor to the lethality of this mutation.

Structures and reaction pathways of the molybdenum centres of sulfite-oxidizing enzymes by pulsed EPR spectroscopy.

SOEs (sulfite-oxidizing enzymes) are physiologically vital and occur in all forms of life. During the catalytic cycle, the five-co-ordinate square pyramidal oxo-molybdenum active site passes through the Mo(V) state, and intimate details of the structure can be obtained from variable frequency pulsed EPR spectroscopy through the hyperfine and nuclear quadrupole interactions of nearby magnetic nuclei. By employing variable spectrometer operational frequencies, it is possible to optimize the measurement conditions for difficult quadrupolar nuclei of interest (e.g. (17)O, (33)S, (35)Cl and (37)Cl) and to simplify the interpretation of the spectra. Isotopically labelled model Mo(V) compounds provide further insight into the electronic and geometric structures and chemical reactions of the enzymes. Recently, blocked forms of SOEs having co-ordinated sulfate, the reaction product, were detected using (33)S (I=3/2) labelling. This blocking of product release is a possible contributor to fatal human sulfite oxidase deficiency in young children.

Studies of the Mo(V) Center of the Y343F Mutant of Human Sulfite Oxidase by Variable Frequency Pulsed EPR Spectroscopy.

The Mo(V) forms of the Tyr343Phe (Y343F) mutant of human sulfite oxidase (SO) have been investigated by continuous wave (CW) and variable frequency pulsed EPR spectroscopies as a function of pH. The CW EPR spectrum recorded at low pH (∼6.9) has g-values similar to those known for the low-pH form of the native vertebrate SO (original lpH form); however, unlike the spectrum of original lpH SO, it does not show any hyperfine splittings from a nearby exchangeable proton. The detailed electron spin echo (ESE) envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) experiments also did not reveal any nearby protons that could belong to an exchangeable ligand at the molybdenum center. These results suggest that under low-pH conditions the active site of Y343F SO is in the "blocked" form, with the Mo(V) center coordinated by sulfate. With increasing pH the EPR signal from the "blocked" form decreases, while a signal similar to that of the original lpH form appears and becomes the dominant signal at pH>9. In addition, both the CW EPR and ESE-detected field sweep spectra reveal a considerable contribution from a signal similar to that usually detected for the high-pH form of native vertebrate SO (original hpH form). The nearby exchangeable protons in both of the component forms observed at high pH were studied by the ESEEM spectroscopy. These results indicate that the Y343F mutation increases the apparent pK(a) of the transition from the lpH to hpH forms by ∼2 pH units.

Direct demonstration of the presence of coordinated sulfate in the reaction pathway of Arabidopsis thaliana sulfite oxidase using 33S labeling and ESEEM spectroscopy.

Sulfite oxidase from Arabidopsis thaliana has been reduced at pH = 6 with sulfite labeled with 33S (nuclear spin I = 3/2), followed by reoxidation by ferricyanide to generate the Mo(V) state of the active center. To obtain information about the hyperfine interaction (hfi) of 33S with Mo(V), continuous-wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) experiments have been performed. The interpretation of the EPR and ESEEM spectra was facilitated by a theoretical analysis of the nuclear transition frequencies expected for the situation of the nuclear quadrupole interaction being much stronger than the Zeeman and hyperfine interactions. The isotropic hfi constant of 33S determined in these experiments was about 3 MHz, which demonstrates the presence of coordinated sulfate in the sulfite-reduced low-pH form of the plant enzyme.

Double electron electron resonance as a method for characterization of micelles.

Double electron electron resonance (DEER) is an experimental technique used to determine distance between electron spins. In this work, we show that it can be used to study the properties of micelles in solution, specifically their volume and the aggregation number. The feasibility of the method is tested on micelles of Pluronic block copolymers, PEO(x)-PPO(y)-PEO(x), built from chains of poly(ethylene oxide) (PEO), comprising the more hydrophilic corona, and a poly(propylene oxide) (PPO) block constituting the hydrophobic core. In this work, the dimensions of the hydrophobic core of micelles of Pluronic L64 (x = 13, y = 30), P123 (x = 20, y = 70), and F127 (x = 106, y = 70) and their aggregation number were studied. This was done using the spin-probe 4-hydroxy-tempo-benzoate (4HTB), which is hydrophobic and is localized in the hydrophobic core of the micelles and does not dissolve in aqueous solution. The measurements were carried out on frozen solutions, freeze quenched after equilibration at 50 degrees C. It was found that the hydrophobic core radii occupied by 4HTB in 7.5 wt % F127 and 6 wt % L64 are 4.0 +/- 0.05 and 3.8 +/- 0.1 nm, respectively, and the corresponding aggregation numbers are 57 +/- 2 and 206 +/- 14. The micelles of 6 wt % P123 were found to have a rod shape, and the addition of 4HTB at concentrations higher than 0.7 mM resulted in a phase transitioned to spherical micelles. Finally, this study also showed that the micelle structure is preserved upon rapid freezing.

Oxomolybdenum(V)/Iron(III) Porphyrinate Complexes: Effect of Axial Ligand Plane Orientation on Complex Stability, Reduction Potential, and NMR and EPR Spectra.

The compounds {5,10,15-tri-p-tolyl-20-[[2,3-[((hydrotris(3,5-dimethylpyrazolyl)borato)oxomolybdenio)dioxy]phenyl]porphyrinato}bis(2-methylimidazole)iron(III) chloride, Fe(2,3-Mo-TTP)(2MeImH)(2)Cl (1), and {5,10,15-tri-p-tolyl-20-[3,4-[((hydrotris(3,5-dimethylpyrazolyl)borato)oxomolybdenio)dioxy]phenyl]porphyrinato}bis(2-methylimidazole)iron(III) chloride, Fe(3,4-Mo-TTP)(2MeImH)(2)Cl) (2), have been prepared in order to assess the effect of axial ligand plane orientation upon the stability, reduction potential, and NMR and EPR spectra of these novel (porphryinato)iron(III)-Mo(V) systems that possess two S = (1)/(2) metal centers. The proton NMR spectra of 1 and 2 are characteristic of perpendicular orientation of the planes of the axial 2MeImH ligands. These results contrast with those previously reported (Basu, P.; Shokirev, N. V.; Enemark, J. H.; Walker, F. A. J. Am. Chem. Soc. 1995, 117, 9042-9055) for the analogous compounds with NMeIm as the axial base (3, 4) whose (1)H NMR spectra are characteristic of one or both axial ligands in parallel planes. The equilibrium constants (beta(2)) for binding the bulky 2MeImH ligands of 1 and 2 are more than an order of magnitude smaller than those for NMeIm binding to 3 and 4. Three distinct pseudo-Nernstian one-electron couples are observed for 1 and 2 in DMF that can be assigned to the Fe(III/II), Mo(V/IV), and Fe(II/I) reductions, with the Fe(III/II) couple being most positive. The Fe(III/II) and Mo(V/IV) potentials are similar to those for 3 and 4 and only slightly perturbed from those of the individual isolated components. The EPR spectrum of 1 shows features due to Mo(V) and low-spin Fe(III) that are perturbed by weak exchange coupling (2.6 GHz, 0.078 cm(-)(1)) between the two metal centers which are separated by approximately 7.9 Å. The "large g(max)" feature characteristic of the 2MeImH adducts of Fe(III) tetraphenylporphyrinates is shifted toward the Mo(V) signal to 2.85; the anisotropy of the Mo(V) signal is lost, and no molybdenum hyperfine can be detected. The EPR spectrum of 2, which has a metal-metal separation of approximately 9.4 Å, shows an unperturbed "large g(max)" value of 3.41 for the Fe(III) center. The Mo(V) part of the spectrum is slightly perturbed from that of the precursor catecholate complex but is essentially identical to that of 4, which exhibits a rhombic Fe(III) signal.

A New Chelating Cyclophane and Its Complexation with Ni(2+), Cu(2+), and Zn(2+): Spectroscopic Properties and Allosterism via Ring Contraction.

A condensation reaction between ethylenediaminetetraacetic dianhydride and p-xylenediamine gave a new chelating cyclophane, 3,10,21,28-tetraoxo-5,8,23,26-tetrakis(carboxymethyl)-2,5,8,11,20,23,26,29-octaaza[12.12]paracyclophane, abbreviated as (32edtaxan)H(4), which has three types of electron-donor groups, i.e., amine, carboxylate, and amide groups. The formation of the cyclophane has been confirmed by a single-crystal X-ray analysis of its Zn(2+) complex, [Zn(2)(32edtaxan)].7.5H(2)O, which crystallized in the monoclinic space group P2(1)/c with a = 19.818(1) Å, b = 13.169(1) Å, c = 18.134(1) Å, beta = 104.491(6) degrees, and Z = 4. Each cyclophane molecule coordinates two Zn(2+) ions and results in the formation of a binuclear chelate molecule. The coordination geometry around each metal ion is distorted octahedral, the donor atoms being two carboxylate oxygen atoms, two amine nitrogen atoms, and two amide oxygen atoms. The new cyclophane exhibited a well-defined fluorescence band at 290 nm with 210 nm excitation. The emission intensity was markedly increased in the Zn(2+) complex, in which the coordination of Zn(2+) ions increases the rigidity of the cyclophane leading to a high fluorescence quantum yield. When the cyclophane was coordinated to Cu(2+) ions, the molar absorptivity of a pi-pi transition band observed at 260 nm was increased by a factor of about 10. Such a large spectral change was not observed for the Zn(2+) and Ni(2+) complexes. In the Cu(2+) complex, the two phenyl rings of the cyclophane are expected to be brought closer, as a result of the coordination of deprotonated amide nitrogens to the central metal ion. This allosterism via ring contraction is responsible for the novel behavior of the absorption spectrum. The emission band of the cyclophane was weakened by coordination of copper and nickel as a result of fluorescence quenching caused by a photo-induced electron transfer.

Multifrequency ESEEM Spectroscopy of Sulfite Oxidase in Phosphate Buffer: Direct Evidence for Coordinated Phosphate.

The molybdenum(V) coordination environment of sulfite oxidase has been investigated by multifrequency ESEEM spectroscopy in approximately 70 mM phosphate buffer at pH = 6.5 in both H(2)O and D(2)O. The FT-ESEEM spectra in H(2)O typically consist of three lines. One of these lines is always close to twice the Larmor frequency of the P atom (2nu(P)) and is assigned to one or more coordinated phosphates, providing the first direct unambiguous detection of such coordination. Extensive simulations of this phosphate signal at the various operational frequencies indicated that the coordinated phosphate group(s) probably does (do) not adopt a fixed orientation, and as a result, a description of the Mo.P hyperfine interaction required the introduction of a distribution of such orientations, with Mo.P distance(s) of 3.2-3.3 Å. The other two lines in the FT-ESEEM spectra in H(2)O, located at nu(H) and 2nu(H), were assigned to matrix protons. In D(2)O buffer two additional lines, assigned to matrix deuterons, were also seen.

Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for "difficult" nuclei.

The catalytic mechanisms of sulfite oxidizing enzymes (SOEs) have been investigated by multi-frequency pulsed EPR measurements of "difficult" magnetic nuclei (35.37Cl, 33S, 17O) associated with the Mo(v) center. Extensive DFT calculations have been used to relate the experimental magnetic resonance parameters of these nuclei to specific active site structures. This combined spectroscopic and computational approach has provided new insights concerning the structure/function relationships of the active sites of SOEs, including: (i) the exchange of oxo ligands; (ii) the nature of the blocked forms; and (iii) the role of Cl- in low pH forms.

Distribution of guest molecules in Pluronic micelles studied by double electron electron spin resonance and small angle X-ray scattering.

Pulse double electron-electron spin resonance (DEER) measurements were applied to characterize the distribution and average number of guest-molecules (in the form of spin-probes) in Pluronic P123 micelles. Two types of spin-probes were used, one of which is a spin-labeled P123 (P123-NO), which is similar to the micelles constituent molecules, and the other is spin-labeled Brij56 (Brij56-NO) which is significantly different. Qualitative information regarding the relative location of the spin-labels within the micelles was obtained from the isotropic hyperfine coupling and the correlation times, determined from continuous wave EPR measurements. In addition, complementary small angle X-ray scattering (SAXS) measurements on the P123 micellar solutions, with and without the spin-probes, were carried out for an independent determination of the size of the core and corona of the micelles and to ensure that the spin-probes do not alter the size or shape of the micelles. Two approaches were used for the analysis of the DEER data. The first is model free, which is based on the determination of the leveling off value of the DEER kinetics. This provided good estimates of the number of radicals per micelle (low limit) which, together with the known concentration of the P123 molecules, gave the aggregation number of the P123 micelles. In addition, it provided an average distance between radicals which is within the range expected from the micelles' size determined by SAXS. The second approach was to analyze the full kinetic form which is model dependent. This analysis showed that both spin-labels are not homogeneously distributed in either a sphere or a spherical shell, and that large distances are preferred. This analysis yielded a slightly larger occupation volume within the micelle for P123-NO than for Brij56-NO, consistent with their chemical character.

HYSCORE and DEER with an upgraded 95GHz pulse EPR spectrometer.

The set-up of a new microwave bridge for a 95 GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7 W) that generates pi pulses of 25 ns and a microwave field, B(1)=0.71 mT. Such a high B(1) enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6 nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95 GHz, observing nuclear modulation frequencies of 14N and 17O as high as 40 MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides 14N quadrupolar splittings.

Investigation of the coordination structures of the molybdenum(v) sites of sulfite oxidizing enzymes by pulsed EPR spectroscopy.

Sulfite oxidizing enzymes (SOEs) are physiologically vital and occur in all forms of life. During the catalytic cycle the five-coordinate square-pyramidal oxo-molybdenum active site passes through the Mo(v) state, and intimate details of the structure can be obtained from pulsed EPR spectroscopy through the hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of nearby magnetic nuclei (e.g., (1)H, (2)H, (17)O, (31)P) of the ligands. By employing spectrometer operational frequencies ranging from approximately 4 to approximately 32 GHz, it is possible to make the nuclear Zeeman interaction significantly greater than the hfi and nqi, and thereby simplify the interpretations of the spectra. The SOEs exhibit three general types of Mo(v) structures which differ in the number of nearby exchangeable protons (one, two or zero). The observed structure depends upon the organism, pH, anions in the medium, and method of reduction. One type of structure has a single exchangeable Mo-OH proton approximately in the equatorial plane and a large isotropic hfi (e.g., low pH form of chicken SOE, low pH form of plant SOE reduced by Ti(iii)); the second type has two exchangeable protons with distributed orientations out of the equatorial plane and very small (or zero) isotropic hfi (e.g., high pH form of chicken SOE, high pH form of plant SOE reduced by sulfite); the third type has no nearby exchangeable protons and a coordinated oxyanion (e.g., phosphate inhibited chicken SOE, low pH form of plant SOE reduced by sulfite). An additional structural conclusion is that the orientation angle of any exchangeable equatorial ligand (OH, OH(2), PO(4)(3-)) is not uniquely fixed, but is distributed around its central value by up to +/-20 degrees (depending on pH, the type of the ligand and the type of enzyme). An unexpected finding was that the axial oxo group of SOEs exchanges with (17)O in solutions enriched in H(2)(17)O. The first determination of oxo (17)O nqi parameters for a well-characterized model compound, [Mo(17)O(SPh)(4)](-), clearly demonstrated that (17)O nqi parameters can distinguish between oxo and OH(2) ligands.

Determination of the hydration number of gadolinium(III) complexes by high-field pulsed 17O ENDOR spectroscopy.

Pulsed 17O Mims electron-nuclear double resonance (ENDOR) spectroscopy at the W band (95 GHz) and D band (130 GHz) is used for the direct determination of the water coordination number (q) of gadolinium-based magnetic resonance imaging (MRI) contrast agents. Spectra of metal complexes in frozen aqueous solutions at approximately physiological concentrations can be obtained either in the presence or absence of protein targets. This method is an improvement over the 1H ENDOR method described previously, which involved the difference ENDOR spectrum of exchangeable protons from spectra taken in H2O and D2O. In addition to exchangeable water protons, the 1H ENDOR method is also sensitive to other exchangeable protons, and it is shown here that this method can overestimate hydration numbers for complexes with exchangeable protons at GdH distances similar to that of the coordinated water, for example, from NH groups. The 17O method does not suffer from this limitation. 17O ENDOR spectroscopy is applied to Gd(III) complexes containing zero, one, or two inner-sphere water molecules. In addition, 13C and 1H ENDOR studies were performed to assess the extent of methanol coordination, since methanol is used to produce a glass in these experiments. Under the experimental conditions used for the hydration number determination (30 mol % methanol), fewer than 15 % of the coordination sites were found to be occupied by methanol.

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.