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A W-band (94 GHz) electron paramagnetic resonance (EPR) study of synthetic fluorapatite with 57 ± 4 ppm Gd has been made on single crystals at ~287 K. The spectra disclosed the presence of a previously unreported type of Gd3+ center denoted by “b” herein (S = 7/2), in addition to the Gd3+ center “a” assigned to the Ca2 site using the results of a previous X-band (9.5 GHz) EPR study (Chen et al. 2002). In particular, the single-crystal W-band EPR spectra from three orthogonal-rotation planes allowed determination of an appropriate spin-Hamiltonian for center “b,” including the spin terms of type BS (matrix g ) and S2 (matrix D ) and the parameters associated with the high-spin terms of type S4 and S6 as well as BS3 and BS5. Agreement between the observed and simulated single-crystal spectra confirmed the validity of the spin-Hamiltonian analysis. The principal values of the matrices g and D [e.g., D/geβe = 1069.2(1) G and E/geβe = 52.4(3) G] suggest a considerably distorted rhombic local environment for the Gd3+ ions in center “b.” The principal directions of D suggest that “b” corresponds to Gd3+ at the Ca1 site. This site assignment is supported by a pseudo-symmetry analysis of the term S4, i.e., approximate matching of the directions of the calculated pseudo-symmetry axes to the bond directions and face normals of the coordination polyhedron of the ideal Ca1 site. The data suggest that the incorporation of Gd3+ into the Ca1 site is achieved by a coupled substitution (2Gd3+ + □ ↔ 3Ca2+) involving a Ca2+ vacancy □ and that the vacancy is located at a next-nearest-neighbor Ca2 site, resulting in a Gd3+-- □ \---| Gd3+ arrangement, with the cations well separated.

The Mark II W-band (94 GHz) EPR spectrometer with a low-noise millimeter-wave amplifier is described. The microwave bridge is of a high-sensitivity homodyne design. Signal-to-noise ratios were measured for a number of detectors with and without the low-noise amplifier. The signal-to-noise ratio was determined not only by the type of detector but also how well it was matched. Without a microwave preamplifier, a hot-electron bolometer provides the best signal-to-noise ratio. Addition of a low-noise microwave preamplifier to the CW homodyne bridge gives a 10 dB improvement in the noise figure of the receiver at a modulation frequency of 100 kHz. A greater improvement in the signal-to-noise ratio is seen at low modulation frequencies (1–10 kHz), making the low-noise amplifier useful for systems with large linewidths. This allows larger modulation amplitudes to be used without causing significant cavity heating or microphonics. The W-band spectrometer is capable of rapid sweeps from 0 to 7 T, as well as narrower (0.1 T) high-resolution sweeps. It is suitable for a wide variety of samples including liquids and samples cooled to sub-liquid-helium temperatures.

We report the synthesis, crystal structure, physical properties and hydrolysis kinetics of [(O)(tmpa)VIV(μ-O)VV(tmpa)(O)]3+ (tmpa = tris(2-pyridylmethyl)amine), the first cation in its class for which both solid state and solution-phase characteristics are fully consistent with expectations for a Robin−Day type III mixed-valence complex. [V2(tmpa)2(O)3](ClO4)3·CH3CN crystallizes in the triclinic, P1 (No. 2) space group with a = 10.719(2) A, b = 11.609(1) A, c = 19.516(2) A, α = 87.876(9)°, β = 79.295(9)°, γ = 68.743(9)°, and V = 2222.6(6) A3; Z = 2. The two vanadium centers are crystallographically equivalent in a linear, oxo-bridged cation with V−Ob and VO bond lengths of 1.800(1) and 1.597(4) A, respectively. A 15-hyperfine line EPR spectrum (g = 1.9704, A = 46.4 × 10-4 cm-1) was observed for [V2(tmpa)2(O)3]3+, consistent with coupling of a single electron to two equivalent 51V atoms. The mixed-valence complex exhibits an intervalence transfer (IT) band at 1.05 × 104 cm-1 (e = 2.1 × 103 M-1 cm-1, CH3CN...

There are many advantages to carrying out electron paramagnetic resonance experiments at very high frequencies, either as part of a multifrequency strategy for solving problems or for special characteristics of high frequencies. These special characteristics include the potential for high point sensitivity, enhanced resolution, separation of similar species, altered sensitivity to motion, suppression of motional effects, and many others. This paper describes a three-millimeter-wavelength (W-band, 94 GHz) EPR spectrometer built for a multi-user facility and illustrates with some examples, most of them being disordered systems. One significant example is the oxidized primary reaction center, P 865 + , isolated from the red photosynthetic bacteriaRhodobacter Sphaeroides R-26. The W-band technique applied to both centers isolated from bacteria grown from either deuterated or ordinary growth media allows extraction of the fullg anisotropy in these centers and sets the stage for multifrequency EPR spectroscopy to yield a full analysis of the various contributions to linewidths in these systems.

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple Cu(A) cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of Cu(A) sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the Cu(A) site of a biosynthetic Cu(A) model, Cu(A) azurin (Cu(A)Az) and found that both T1 Cu and T2 Cu intermediates form on the path to final Cu(A) reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native Cu(A) center of nitrous oxide reductase, the faster kinetics of copper incorporation into Cu(A)Az allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ~410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)-dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying Cu(A)Az variants where the Cys and His ligands are mutated, we have identified the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of Cu(A) centers.

Na + -NQR is a unique respiratory enzyme that couples the free energy of electron transfer reactions to electrogenic pumping of sodium across the cell membrane. This enzyme is found in many marine and pathogenic bacteria where it plays an analogous role to the H + -pumping complex I. It has generally been assumed that the sodium pump of Na + -NQR operates on the basis of thermodynamic coupling between reduction of a single redox cofactor and the binding of sodium at a nearby site. In this study, we have defined the coupling to sodium translocation of individual steps in the redox reaction of Na + -NQR. Sodium uptake takes place in the reaction step in which an electron moves from the 2Fe-2S center to FMN C , while the translocation of sodium across the membrane dielectric (and probably its release into the external medium) occurs when an electron moves from FMN B to riboflavin. This argues against a single-site coupling model because the redox steps that drive these two parts of the sodium pumping process do not have any redox cofactor in common. The significance of these results for the mechanism of coupling is discussed, and we proposed that Na + -NQR operates through a novel mechanism based on kinetic coupling, mediated by conformational changes.

Sco-like proteins contain copper bound by two cysteines and a histidine residue. Although their function is still incompletely understood, there is a clear involvement with the assembly of cytochrome oxidases that contain the Cu(A) center in subunit 2, possibly mediating the transfer of copper into the Cu(A) binuclear site. We are investigating the reaction chemistry of BSco, the homologue from Bacillus subtilis. Our studies have revealed that BSco behaves more like a redox protein than a metallochaperone. The essential H135 residue that coordinates copper plays a role in stabilizing the Cu(II) rather than the Cu(I) form. When H135 is mutated to alanine, the oxidation rate of both hydrogen peroxide and one-electron outer-sphere reductants increases by 3 orders of magnitude, suggestive of a redox switch mechanism between the His-on and His-off conformational states of the protein. Imidazole binds to the H135A protein, restoring the N superhyperfine coupling in the EPR, but is unable to rescue the redox properties of wild-type Sco. These findings reveal a unique role for H135 in Sco function. We propose a hypothesis that electron transfer from Sco to the maturing oxidase may be essential for proper maturation and/or protection from oxidative damage during the assembly process. The findings also suggest that interaction of Sco with its protein partner(s) may perturb the Cu(II)-H135 interaction and thus induce a sensitive redox activity to the protein.

Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer (ET) in photosynthesis and respiration to catalysis in industrial and fuel cell research. Nature accomplishes these functions by employing only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or ET properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centers1,2 or fuel cell catalysts3 for energy conversion. We have shown here that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin (Az) over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of Az is now predictable across the full range of cupredoxin potentials.

Here we present new evidence that riboflavin is present as one of four flavins in Na+-NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na+-NQR.

The one-electron oxidations of a series of diiron(I) dithiolato carbonyls were examined to evaluate the factors that affect the oxidation state assignments, structures, and reactivity of these low-molecular weight models for the H ox state of the [FeFe]-hydrogenases. The propanedithiolates Fe 2(S 2C 3H 6)(CO) 3(L)(dppv) (L = CO, PMe 3, P i-Pr 3) oxidize at potentials approximately 180 mV milder than the related ethanedithiolates ( Angew. Chem., Int. Ed. 2007, 46, 6152). The steric clash between the central methylene of the propanedithiolate and the phosphine favors the rotated structure, which forms upon oxidation. Electron Paramagnetic Resonance (EPR) spectra for the mixed-valence cations indicate that the unpaired electron is localized on the Fe(CO)(dppv) center in both [Fe 2(S 2C 3H 6)(CO) 4(dppv)]BF 4 and [Fe 2(S 2C 3H 6)(CO) 3(PMe 3)(dppv)]BF 4, as seen previously for the ethanedithiolate [Fe 2(S 2C 2H 4)(CO) 3(PMe 3)(dppv)]BF 4. For [Fe 2(S 2C n H 2 n )(CO) 3(P i-Pr 3)(dppv)]BF 4; however, the spin is localized on the Fe(CO) 2(P i-Pr 3) center, although the Fe(CO)(dppv) site is rotated in the crystalline state. IR and EPR spectra, as well as redox potentials and density-functional theory (DFT) calculations, suggest that the Fe(CO) 2(P i-Pr 3) site is rotated in solution, driven by steric factors. Analysis of the DFT-computed partial atomic charges for the mixed-valence species shows that the Fe atom featuring a vacant apical coordination position is an electrophilic Fe(I) center. One-electron oxidation of [Fe 2(S 2C 2H 4)(CN)(CO) 3(dppv)] (-) resulted in 2e oxidation of 0.5 equiv to give the mu-cyano derivative [Fe (I) 2(S 2C 2H 4)(CO) 3(dppv)](mu-CN)[Fe (II) 2(S 2C 2H 4)(mu-CO)(CO) 2(CN)(dppv)], which was characterized spectroscopically.

Oxidation of the electron-rich (E1/2 = -175 vs Ag/AgCl) ethanedithiolato complex Fe2(S2C2H 4)-(CO)2(dppv)2 (1) under a CO atmosphere yielded [Fe2(S2C2H4)(μ-CO)(CO) 2(dppv)2]+ ([1(CO)]+), a model for the HOXCO state of the [FeFe]-hydrogenases. This complex exists as two isomers: a kinetically favored unsymmetrical derivative, unsym-[1(CO)]+, and a thermodynamically favored isomer, sym-[1(CO)]+, wherein both diphosphines span apical and basal sites. Crystallographic characterization of sym-[1(CO)]+ confirmed a C 2-symmetric structure with a bridging CO ligand and an elongated Fe-Fe bond of 2.7012(14) Å, as predicted previously. Oxidation of sym-[1(CO)]+ and unsym-[1(CO)]+ again by 1e- oxidation afforded the respective diamagnetic diferrous derivatives where the relative stabilities of the sym and unsym isomers are reversed. DFT calculations indicate that the stabilities of sym and unsym isomers are affected differently by the oxidation state of the diiron unit: the mutually trans CO ligands in the sym isomer are more destabilizing in the mixed-valence state than in the diferrous state. EPR analysis of mixed-valence complexes revealed that, for [1]+, the unpaired spin is localized on a single iron center, whereas for unsym/sym-[1(CO)]+, the unpaired spin was delocalized over both iron centers, as indicated by the magnitude of the hyperfine coupling to the phosphine ligands trans to the Fe-Fe vector. Oxidation of 1 by 2 equiv of acetylferrocenium afforded the dication [1]2+, which, on the basis of low-temperature IR spectrum, is structurally similar to [1]+. Treatment of [1]2+ with CO gives unsym-[1(CO)]2+. © 2008 American Chemical Society.

Helicases often achieve functional specificity through utilization of unique structural features incorporated into an otherwise conserved core. The archaeal Rad3 (xeroderma pigmentosum group D protein (XPD)) helicase is a prototypical member of the Rad3 family, distinct from other related (superfamily II) SF2 enzymes because of a unique insertion containing an iron-sulfur (FeS) cluster. This insertion may represent an auxiliary domain responsible for modifying helicase activity or for conferring specificity for selected DNA repair intermediates. The importance of the FeS cluster for the fine-tuning of Rad3-DNA interactions is illustrated by several clinically relevant point mutations in the FeS domain of human Bach1 (FancJ) and XPD helicases that result in distinct disease phenotypes. Here we analyzed the substrate specificity of the Rad3 (XPD) helicase from Ferroplasma acidarmanus (FacRad3) and probed the importance of the FeS cluster for Rad3-DNA interactions. We found that the FeS cluster stabilizes secondary structure of the auxiliary domain important for coupling of single-stranded (ss) DNA-dependent ATP hydrolysis to ssDNA translocation. Additionally, we observed specific quenching of the Cy5 fluorescent dye when the FeS cluster of a bound helicase is positioned in close proximity to a Cy5 fluorophore incorporated into the DNA molecule. Taking advantage of this Cy5 quenching, we developed an equilibrium assay for analysis of the Rad3 interactions with various DNA substrates. We determined that the FeS cluster-containing domain recognizes the ssDNA-double-stranded DNA junction and positions the helicase in an orientation consistent with duplex unwinding. Although it interacts specifically with the junction, the enzyme binds tightly to ssDNA, and the single-stranded regions of the substrate are the major contributors to the energetics of FacRad3-substrate interactions.

DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase alpha. Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase are lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified, and characterized. CD and NMR spectroscopy experiments demonstrated that p58C forms a well folded structure. The protein has a distinctive brownish color, and evidence from inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy revealed characteristics consistent with the presence of a [4Fe-4S] high potential iron protein cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wild-type protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first suggestion that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA-processing machinery.

The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.

Electron paramagnetic resonance (EPR) spectra are presented for iron complexes of catecholate, tironate, and a 3,4-dihydroxyphenylalanine (DOPA)-containing peptide of sequence Ac-Ala-DOPA-Thr-Pro-CONH2 ("AdopaTP"). This peptide was prepared to model potential metal-protein cross-links in the adhesive used by marine mussels, Mytilus edulis, for affixing themselves to surfaces. Spectra are shown for iron bound to each ligand in mono, bis, and tris coordination environments. For example, the catecholate complexes {Fe(cat)}, {Fe(cat)2}, and [Fe(cat)3]3- are provided. Detailed simulations are presented to describe the origin of spectra for the iron-catecholate and iron-peptide species, which show that the spectral features can be accounted for only with the inclusion of D- and E-strain. The spectroscopy of each compound is shown under both anaerobic and aerobic conditions. When exposed to air, the high-spin Fe3+ signal of [Fe(AdopaTP)3]3- decreases and an organic radical is formed. No other sample exhibited an appreciable radical signal. These data are discussed in light of the biomaterial synthesis carried out by marine mussels.

Sco1 is a mitochondrial membrane protein involved in the assembly of the CuA site of cytochrome c oxidase. The Bacillus subtilis genome contains a homologue of yeast Sco1, YpmQ (hereafter termed BSco), deletion of which leads to a phenotype lacking in caa3 (CuA-containing) oxidase activity but expressing normal levels of aa3 (quinol) oxidase activity. Here, we report the characterization of the metal binding site of BSco in its Cu(I)-, Cu(II)-, Zn(II)-, and Ni(II)-bound forms. Apo BSco was found to bind Cu(II), Zn(II), and Ni(II) at a 1:1 protein/metal ratio. The Cu(I) protein could be prepared by either dithionite reduction of the Cu(II) derivative or by reconstitution of the apo protein with Cu(I). X-ray absorption (XAS) spectroscopy showed that Cu(I) was coordinated by two cysteines at 2.22 +/- 0.01 A and by a weakly bound low-Z scatterer at 1.95 +/- 0.03 A. The Cu(II) derivative was reddish-orange and exhibited a strong type-2 thiolate to Cu(II) transition around 350 nm. Multifrequency electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) studies on the Cu(II) derivative provided evidence of one strongly coupled histidine residue, at least one strongly coupled cysteine, and coupling to an exchangeable proton. XAS spectroscopy indicated two cysteine ligands at 2.21 A and two O/N donor ligands at 1.95 A, at least one of which is derived from a coordinated histidine. The Zn(II) and Ni(II) derivatives were 4-coordinate with MS2N(His)X coordination. These results provide evidence that a copper chaperone can engage in redox chemistry at the metal center and may suggest interesting redox-based mechanisms for metalation of the mixed-valence CuA center of cytochrome c oxidase.

Azurin is a small electron-transfer protein belonging to the cupredoxin family. The Cu atom is located within a trigonal plane coordinated by two histidines (His46 and His117) and a cysteine (Cys112) with two more distant ligands (Gly45 and Met121) providing axial interactions. A Cys112SeCys derivative has been prepared by expressed protein ligation, and detailed UV/vis, EPR and EXAFS studies at the Cu and Se K-edges have been carried out. Marked changes are observed between the EPR parameters of the Cys112SeCys and WT azurin derivatives, which include a 2-fold increase in A(||), a decrease in g-values, and a large increase in rhombicity of the g-tensor. The Cu-Se and Se-Cu bond lengths obtained from analysis of the Cu and Se K-EXAFS of the oxidized protein were found to be 2.30 and 2.31 A, respectively, 0.14 A longer than the Cu-S distance of the WT protein. Unexpectedly, the Cu-Se bond lengths were found to undergo only minor changes during reduction, suggesting a very similar structure in both redox states and extending the "rack" hypothesis to the Se-substituted protein.

Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the bound ligand. Furthermore, it documents the complexity and uniqueness of the ligand-receptor structure, and highlights the fact that structural differences exist between the receptor bound with ligands of different pharmacological character that, nevertheless, produce similar crystal structures.

Na(+)-NQR is the entry point for electrons into the respiratory chain of Vibrio cholerae. It oxidizes NADH, reduces ubiquinone, and uses the free energy of this redox reaction to translocate sodium across the cell membrane. The enzyme is a membrane complex of six subunits that accommodates a 2Fe-2S center and several flavins. Both the oxidized and reduced forms of Na(+)-NQR exhibit a radical EPR signal. Here, we present EPR and ENDOR data that demonstrate that, in both forms of the enzyme, the radical is a flavin semiquinone. In the oxidized enzyme, the radical is a neutral flavin, but in the reduced enzyme the radical is an anionic flavin, where N(5) is deprotonated. By combining results of ENDOR and multifrequency continuous wave EPR, we have made an essentially complete determination of the g-matrix and all major nitrogen and proton hyperfine matrices. From careful analysis of the W-band data, the full g-matrix of a flavin radical has been determined. For the neutral radical, the g-matrix has significant rhombic character, but this is significantly decreased in the anionic radical. The out-of-plane component of the g-matrix and the nitrogen hyperfine matrices are found to be noncoincident as a result of puckering of the pyrazine ring. Two possible assignments of the radical signals are considered. The neutral and anionic forms of the radical may each arise from a different flavin cofactor, one of which is converted from semiquinone to flavohydroquinone, while the other goes from flavoquinone to semiquinone, at almost exactly the same redox potential, during reduction of the enzyme. Alternatively, both forms of the radical signal may arise from a single, extremely stable, flavin semiquinone, which becomes deprotonated upon reduction of the enzyme.

The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane.

We report the synthesis, crystal structure, physical properties and hydrolysis kinetics of [(O)(tmpa)V(IV)(mgr;-O)V(V)(tmpa)(O)](3+) (tmpa = tris(2-pyridylmethyl)amine), the first cation in its class for which both solid state and solution-phase characteristics are fully consistent with expectations for a Robin-Day type III mixed-valence complex. [V(2)(tmpa)(2)(O)(3)](ClO(4))(3).CH(3)CN crystallizes in the triclinic, Ponemacr; (No. 2) space group with a = 10.719(2) Å, b = 11.609(1) Å, c = 19.516(2) Å, alpha = 87.876(9) degrees, beta = 79.295(9) degrees, gamma = 68.743(9) degrees, and V = 2222.6(6) Å(3); Z = 2. The two vanadium centers are crystallographically equivalent in a linear, oxo-bridged cation with V-O(b) and V=O bond lengths of 1.800(1) and 1.597(4) Å, respectively. A 15-hyperfine line EPR spectrum (g = 1.9704, A = 46.4 x 10(-4) cm(-1)) was observed for [V(2)(tmpa)(2)(O)(3)](3+), consistent with coupling of a single electron to two equivalent (51)V atoms. The mixed-valence complex exhibits an intervalence transfer (IT) band at 1.05 x 10(4) cm(-1) (epsilon = 2.1 x 10(3) M(-1) cm(-1), CH(3)CN) whose energy is insensitive to variations in the solvent; d-d transitions are observed at 1.25 x 10(4) cm(-1) ((2)E-- (2)B(2)) and 1.44 x 10(4) cm(-1) ((2)B(1)-- (2)B(2)). [V(2)(tmpa)(2)(O)(3)](2+), [V(2)(tmpa)(2)(O)(3)](3+), and [V(2)(tmpa)(2)(O)(3)](4+) comprise a redox family related by vanadium (IV,V/IV,IV) and (V,V/IV,V) half-wave reduction potentials of 0.25 and 1.62 V vs NHE (25 degrees C, CH(3)CN, I = 0.1 M), respectively. The hydrolysis reaction: [V(2)(tmpa)(2)(O)(3)](3+) + H(2)O --[V(IV)(tmpa)(O)(OH)](+) + [V(V)(tmpa)(O)(2)](+) + H(+) proceeds with an acid-independent rate constant of 2.37 x 10(-3) s(-1) (25 degrees C; DeltaH() = 71 kJ/mol, DeltaS() = -59 J/mol K). [V(4)O(5)(tmpa)(4){Fe(CN)(6)}](ClO(4))(4).H(2)O was isolated as an unexpected product from the reaction of [V(2)(tmpa)(2)(O)(3)](ClO(4))(2).2H(2)O with equimolar K(3)[Fe(CN)(6)] in aqueous solution. It is proposed that pairs of V(IV)=O and V(V)=O units aggregate with [Fe(CN)(6)](4-) into a charge transfer complex. Hexacyanoferrate(II)-to-vanadium IT charge transfer bands are observed at 1.98 x 10(4) cm(-1) (epsilon = 5.3 x 10(3) M(-1) cm(-1)) and 2.72 x 10(4) cm(-1) (epsilon = 9.3 x 10(3) M(-1) cm(-1)); a 29-line EPR spectrum (g = 1.9737, A = 22.0 x 10(-4) cm(-1)) demonstrates equal delocalization of unpaired electron density over all four V atoms (CH(3)CN solution).

Advanced electronic magnetic resonance (EMR) as well as nuclear magnetic resonance (NMR) methods have been used to examine properties of coals, chars, and molecular species related to constituents of coal. During the span of this grant, progress was made on construction and applications to coals and chars of two high frequency EMR systems particularly appropriate for such studies--48 GHz and 95 GHz electron magnetic resonance spectrometer, on new low-frequency dynamic nuclear polarization (DNP) experiments to examine the interaction between water and the surfaces of suspended char particulates in slurries, and on a variety of proton nuclear magnetic resonance (NMR) techniques to measure characteristics of the water directly in contact with the surfaces and pore spaces of carbonaceous particulates.

Advanced electronic magnetic resonance (EMR) methods are used to examine properties of coals, chars, and molecular species related to constituents of coal. We have achieved substantial progress on upgrading the high field (HF) EMR (W-band, 95 GHz) spectrometers that are especially advantageous for such studies. Particularly, we have built a new second W-band instrument (Mark II) in addition to our Mark I. Briefly, Mark II features: (i) an Oxford custom-built 7 T superconducting magnet which is scannable from 0 to 7 T at up to 0.5 T/min; (ii) water-cooled coaxial solenoid with up to ±550 G scan under digital (15 bits resolution) computer control; (iii) custom-engineered precision feed-back circuit, which is used to drive this solenoid, is based on an Ultrastab 860R sensor that has linearity better than 5 ppm and resolution of 0.05 ppm; (iv) an Oxford CF 1200 cryostat for variable temperature studies from 1.8 to 340 K. During this grant period we have completed several key upgrades of both Mark I and II, particularly microwave bridge, W-band probehead, and computer interfaces. We utilize these improved instruments for HF EMR studies of spin-spin interaction and existence of different paramagnetic species in carbonaceous solids.

Myeloperoxidase (MPO) is increasingly being recognized as an important factor in many inflammatory diseases, particularly cardiovascular and neurological diseases. MPO-specific imaging agents would thus be highly useful to diagnose early disease, monitor disease progression, and quantify treatment effects. This study reports in vitro and in vivo characterizations of the mechanism of interaction between MPO and paramagnetic enzyme substrates based on physical and biological measurements. We show that these agents are activated through a radical mechanism, which can combine to form oligomers and, in the presence of tyrosine-containing peptide, bind to proteins. We further identified two new imaging agents, which represent the near extremes in either oligomerization (mono-5HT-DTPA-Gd) or protein-binding in their activation mechanism (bis-o-dianisidine-DTPA-Gd). On the other hand, we found that the agent bis-5HT-DTPA-Gd utilizes both mechanisms when activated. These properties yield distinct in vivo pharmacokinetics profiles for each of these agents that may be exploited for different applications. Specificity studies show that only MPO, but not eosinophil peroxidase, can highly activate these agents, and that MPO activity as low as 0.005 U/mg of tissue can be detected. Gd kinetic lability and cytotoxicity studies further confirm stability of the Gd ion and low toxicity for the 5HT-based agents, suggesting that these agents are suitable for translational in vivo studies.

Modulating the properties of proteins through de novo design or redesign of existing proteins has been a longstanding goal in protein chemistry. Over the past two decades, site-directed mutagenesis has been a powerful tool to probe the role of certain residues and to fine-tune the activity of proteins. A limitation of this approach has been the accessibility of only a restricted number of functional groups through the 20 amino acids in the genetic code. The more recent technique of expressed protein ligation (EPL) provides an alternative route that allows efficient incorporation of nonnatural residues into proteins. We report here the preparation and spectroscopic characterization of an azurin variant in which a cysteine ligand to the blue copper center has been replaced by EPL with selenocysteine (Sec). This reports marks the first time that selenocysteine is artificially incorporated into the active site of a metalloprotein. The variant displays a significantly increased A(parallel) (from 56 to 104 G) and red-shifted CT band (from 625 to 677 nm), while maintaining the general type 1 copper characteristics, including similarity in reduction potentials. This study illustrates that iso-structural substitution using EPL can fine-tune the structural and functional properties of a metal-binding site without loss of most of its characteristics. Further spectroscopic and X-ray crystallographic studies of this and other EPL variants will provide new insights into the fine-control of the structure and function of metalloproteins.

Publisher Summary This chapter explores multidimensional electron magnetic resonance of American coals. In a study described in the chapter, pure samples of dibenzothiophene and benzonaphthothiophene were purchased from Aldrich Chemical. Cation radicals of these materials, in addition to dibenzofuran, were prepared by two alternate routes: (1) formation of cation radicals by electron transfer on active silica–alumina surfaces, and (2) formation of cation radicals by ultraviolet irradiation in a boric-acid host glass. Each method was developed to produce stable populations of pure cation radicals in a powder or glassy matrix, thus duplicating the disordered state of chemical species in coal. Powdered boric acid and the desired thiophenic compound were mixed together in a 1:100 ratio by weight. The powder then was heated slowly to the melting point of boric acid. The glass was allowed to cool, was broken into small fragments, and was then loaded into a quartz sample tube, after which it was placed in a Ray-O-Net irradiation unit and subjected to ultraviolet irradiation for 30 min.

Rhenium-(V), -(VI) and -(VII) complexes of the [α2-P2W17O61]10– isomer, a mono-lacunary derivative of the [α-P2W18O62]6– (Wells–Dawson) ion have been prepared and characterized by multinuclear NMR spectroscopy, electrospray mass spectrometry and electron paramagnetic resonance spectroscopy, among other techniques. The molecules have the formulation [α2-ReOP2W17O61]n–, where n = 7,6,5 for ReV, ReVI and ReVII, respectively. 183W NMR spectroscopy for the ReV and ReVII analogs shows that the molecules have Cs symmetry, as expected for substitution in the α2 site. Simulations of the X-band and Q-band EPR spectra of the ReVI analog, using Cs symmetry, allow determination of the g, hyperfine and quadrupole coupling tensors. X-Band, Q-band and W-band EPR spectroscopy show extreme variations in linewidths due to random strains or distortions of the complex.

Successful preparation and electron paramagnetic resonance of 95 Mo- and 97 Mo-doped single crystals of tetraphenylarsonium oxopentaisothiocyanatoniobate(V) are described. The following nuclear quadrupole coupling constants were determined for 97 Mo: QD = 3.0± 0.2 MHz and QE = 0.1 ± 0.05 MHz. The principal axes of A (Mo) are coincident within error with those of P (Mo), but not with those of g . The principal values of g and the noncoincidence between the principal axes of g and A (Mo) vary for the MoO(SCN) 5 2− anion when doped into different host materials and dissolved in solution. Random misalignment of the guest impurity in the host material accounts for the differences in linewidths of the six primary molybdenum hyperfine transitions in single-crystal spectra, while 14 N nuclear quadrupole interaction accounts for the anisotropic line broadening. Anomalous orientation-dependent “frozen-solution” spectra of TPA 2 NbO(SCN) 5 in CHCl 3 are explained in terms of alignment of suspended crystallites in solution by the external magnetic field.

The preparation, single-crystal x-ray structures, variable-temperature magnetic susceptibility, and novel EPR data are presented for two mixed-valence Mn{sup II}Mn{sup III} complexes. (Mn{sub 2}(bpmp)({mu}-OAc){sub 2})(ClO{sub 4}){sub 2}{center dot}H{sub 2}O, where bpmp{sup {minus}} is the monoanion of 2,6-bis(bis(2-pyridylmethyl)aminomethyl)-4-methylphenol, crystallizes in the monoclinic space group P2{sub 1}/n with a = 12.493 (1) {angstrom}, b = 21.583 (2) {angstrom}, c = 16.631 (2) {angstrom}, {beta} = 95.31 (1){degree}, Z = 4; R{sub F} = 0.061 and R{sub wF} = 0.085 for 4298 unique reflections (I > 3{sigma}(I)) at 297 (1) K. (Mn{sub 2}(bcmp)({mu}-OAc){sub 2})(ClO{sup {minus}}{sub 4}){sub 2}{center dot}CH{sub 2}Cl{sub 2}, where bcmp{sup {minus}} is the monoanion of 2,6-bis(1,4,67-triazacyclonon-l-ylmethyl)-4-methylphenol, crystallizes in the orthorhombic space group Pca2{sub 1} with a = 19.273 (1) {angstrom}, b = 15.155 (1) {angstrom}, c = 13.749 (3) {angstrom}, Z = 4; R{sub F} = 0.074 and R{sub wF} = 0.085 for 2324 unique reflections (I > 3{sigma}(I)) at 296 (1) K.