Electron-mediating Cu(A) centers in proteins: a comparative high field (1)H ENDOR study

High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating Cu(A) center. These include nitrous oxide reductase (N(2)OR), the recombinant water-soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase (COX) ba(3) (M160T9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered "purple" azurin (purpAz). The three-dimensional (3-D) structures of these proteins, apart from the mutant, are known. The EPR spectra of all samples showed the presence of a mononuclear Cu(II) impurity with EPR characteristics of a type II copper. At W-band, the g( perpendicular) features of this center and of Cu(A) are well resolved, thus allowing us to obtain a clean Cu(A) ENDOR spectrum. The latter consists of two types of ENDOR signals. The first includes the signals of the four strongly coupled cysteine beta-protons, with isotropic hyperfine couplings, A(iso), in the 7-15 MHz range. The second group consists of weakly coupled protons with a primarily anisotropic character with A(zz) < 3 MHz. Orientation selective ENDOR spectra were collected for N(2)OR, M160QT0, and purpAz, and simulations of the cysteine beta-protons signals provided their isotropic and anisotropic hyperfine interactions. A linear correlation with a negative slope was found between the maximum A(iso) value of the beta-protons and the copper hyperfine interaction. Comparison of the best-fit anisotropic hyperfine parameters with those calculated from dipolar interactions extracted from the available 3-D structures sets limit to the sulfur spin densities. Similarly, the small coupling spectral region was simulated on the basis of the 3-D structures and compared with the experimental spectra. It was found that the width of the powder patterns of the weakly coupled protons recorded at g(perpendicular) is mainly determined by the histidine H(epsilon)(1) protons. Furthermore, the splitting in the outer wings of these powder patterns indicates differences in the positions of the imidazole rings relative to the Cu(2)S(2) core. Comparison of the spectral features of the weakly coupled protons of M160QT0 with those of the other investigated proteins shows that they are very similar to those of purpAz, where the Cu(A) center is the most symmetric, but the copper spin density and the H(epsilon)(1)-Cu distances are somewhat smaller. All proteins show the presence of a proton with a significantly negative A(iso) value which is assigned to an amide proton of one of the cysteines. The simulations of both strongly and weakly coupled protons, along with the known copper hyperfine couplings, were used to estimate and compare the spin density distribution in the various Cu(A) centers. The largest sulfur spin density was found in M160T9, and the lowest was found in purpAz. In addition, using the relation between the A(iso) values of the four cysteine beta-protons and the H-C-S-S dihedral angles, the relative contribution of the hyperconjugation mechanism to A(iso) was determined. The largest contribution was found for M160T9, and the lowest was found for purpAz. Possible correlations between the spin density distribution, structural features, and electron-transfer functionality are finally suggested.     

Structure of copper(II)-histidine based complexes in frozen aqueous solutions as determined from high-field pulsed electron nuclear double resonance

W-band (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) spectroscopic techniques were used to determine the hyperfine couplings of different protons of Cu(II)-histidine complexes in frozen solutions. The results were then used to obtain the coordination mode of the tridentate histidine molecule and to serve as a reference for Cu(II)-histidine complexation in other, more complex systems. Cu(II) complexes with L-histidine and DL-histidine-alpha-d,beta-d2 were prepared in H2O and in D2O, and orientation-selective W-band 1H and 2H pulsed ENDOR spectra of these complexes were recorded at 4.5 K. These measurements lead to the unambiguous assignment of the signals of the H alpha, H beta, imidazole H epsilon, and the exchangeable amino, Ham, protons. The 14N superhyperfine splitting observed in the X-band EPR spectrum and the presence of only one type of H alpha and H beta protons in the W-band ENDOR spectra show that the complex is a symmetric bis complex. Its g parallel is along the molecular symmetry axis, perpendicular to the equatorial plane that consists of four coordinated nitrogens in histamine-like coordinations (NNNN). Simulations of orientation-selective ENDOR spectra provided the principal components of the protons' hyperfine interaction and the orientation of their principal axes with respect to g parallel. From the anisotropic part of the hyperfine interaction of H alpha and H beta and applying the point-dipole approximation, a structural model was derived. An unexpectedly large isotropic hyperfine coupling, 10.9 MHz, was found for H alpha. In contrast, H alpha of the Cu(II)-1-methyl-histidine complex where only the amino nitrogen is coordinated, showed a much smaller coupling. Thus, the hyperfine coupling of H alpha can serve as a signature for a histamine coordination where both the amino and imino nitrogens of the same molecule bind to the Cu(II), forming a six-membered chelating ring. Unlike H alpha the hyperfine coupling of H epsilon is not as sensitive to the presence of a coordinated amino nitrogen of the same histidine molecule.     

Axial ligand modulation of the electronic structures of binuclear copper sites: analysis of paramagnetic 1H NMR spectra of Met160Gln Cu(A).

Cu(A) is an electron-transfer copper center present in heme-copper oxidases and N2O reductases. The center is a binuclear unit, with two cysteine ligands bridging the metal ions and two terminal histidine residues. A Met residue and a peptide carbonyl group are located on opposite sides of the Cu2S2 plane; these weaker ligands are fully conserved in all known Cu(A) sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba3 oxidase has been studied by NMR spectroscopy. In its oxidized form, the binuclear copper is a fully delocalized mixed-valence pair, as are all natural Cu(A) centers. The faster nuclear relaxation in this mutant suggests that a low-lying excited state has shifted to higher energies compared to that of the wild-type protein. The introduction of the Gln residue alters the coordination mode of His114 but does not affect His157, thereby confirming the proposal that the axial ligand-to-copper distances influence the copper-His interactions (Robinson, H.; Ang, M. C.; Gao, Y. G.; Hay, M. T.; Lu, Y.; Wang, A. H. Biochemistry 1999, 38, 5677). Changes in the hyperfine coupling constants of the Cys beta-CH2 groups are attributed to minor geometrical changes that affect the Cu-S-C(beta)-H(beta) dihedral angles. These changes, in addition, shift the thermally accessible excited states, thus influencing the spectral position of the Cys beta-CH2 resonances. The Cu-Cys bonds are not substantially altered by the Cu-Gln160 interaction, in contrast to the situation found in the evolutionarily related blue copper proteins. It is possible that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible Cu(A) excited states by tuning axial ligand interactions.     

Multifrequency pulsed electron paramagnetic resonance study of the S2 state of the photosystem II manganese cluster

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is employed to measure the strength of the hyperfine coupling of magnetic nuclei to the paramagnetic (S = 1/2) S2 form of photosystem II (PSII). Previous X-band-frequency ESEEM studies indicated that one or more histidine nitrogens are electronically coupled to the tetranuclear manganese cluster in the S2 state of PSII. However, the spectral resolution was relatively poor at the approximately 9 GHz excitation frequency, precluding any in-depth analysis of the corresponding bonding interaction between the detected histidine and the manganese cluster. Here we report ESEEM experiments using higher X-, P-, and Ka-band microwave frequencies to target PSII membranes isolated from spinach. The X- to P-band ESEEM spectra suffer from the same poor resolution as that observed in previous experiments, while the Ka-band spectra show remarkably well-resolved features that allow for the direct determination of the nuclear quadrupolar couplings for a single I = 1(14)N nucleus. The Ka-band results demonstrate that at an applied field of 1.1 T we are much closer to the exact cancellation limit (alpha iso = 2nu(14)N) that optimizes ESEEM spectra. These results reveal hyperfine (alpha iso = 7.3 +/- 0.20 MHz and alpha dip = 0.50 +/- 0.10 MHz) and nuclear quadrupolar (e(2)qQ = 1.98 +/- 0.05 MHz and eta = 0.84 +/- 0.06) couplings for a single (14)N nucleus magnetically coupled to the manganese cluster in the S 2 state of PSII. These values are compared to the histidine imidazole nitrogen hyperfine and nuclear quadrupolar couplings found in superoxidized manganese catalase as well as (14)N couplings in relevant manganese model complexes.     

55Mn pulse ENDOR at 34 GHz of the S0 and S2 states of the oxygen-evolving complex in photosystem II

55Mn pulse ENDOR experiments at 34 GHz (Q-band) are reported for the S0 and S2 states of the oxygen-evolving complex of photosystem II. Their numerical analysis (i) shows that in both states all four Mn ions are magnetically coupled, (ii) allows a refinement of the hyperfine interaction (HFI) parameters obtained earlier for the S2 state at X-band (Peloquin, J. M.; Campbell, K. A.; Randall, D. W.; Evanchik, M. A.; Pecoraro, V. L.; Armstrong, W. H.; Britt, R. D. J. Am. Chem. Soc. 2000, 122, 10926-10942), (iii) provides the first reliable 55Mn HFI tensors for the S0 state, and (iv) leads to the suggestion that the Mn oxidation states in S0 and S2 are Mn4(III, III, III, IV) and Mn4(III, IV, IV, IV), respectively. In addition, a Q-band EPR spectrum is reported for the S0 state, and inversion-recovery experiments at 4.5 K directly show that the electron spin-lattice relaxation for the S0 state is about 2 orders of magnitude faster than that for the S2 state.     

Electronic structure of a binuclear nickel complex of relevance to [NiFe] hydrogenase

The binuclear complex [Ni(2)(L)(MeCN)(2)](3+) (L(2-) = compartmental macrocycle incorporating imine N and thiolate S donors) has a Ni(III) center bridged via two thiolate S-donors to a diamagnetic Ni(II) center. The ground-state has dominant 3d(z)(1)(2) character similar to that observed for [NiFe] hydrogenases in which Ni(III) is bridged via two thiolate donors to a diamagnetic center (Fe(II)). The system has been studied by X-ray crystallography and pulse EPR, ESEEM, and ENDOR spectroscopy in order to determine the extent of spin-delocalization onto the macrocycle L(2-). The hyperfine coupling constants of six nitrogen atoms have been identified and divided into three sets of two equivalent nitrogens. The most strongly coupled nitrogen atoms (a(iso) approximately 53 MHz) stem from axially bound solvent acetonitrile molecules. The two macrocycle nitrogens on the Ni(III) side have a coupling of a(iso) approximately 11 MHz, and those on the Ni(II) side have a coupling of a(iso) approximately 1-2 MHz. Density functional theory (DFT) calculations confirm this assignment, while comparison of the calculated and experimental (14)N hyperfine coupling constants yields a complete picture of the electron-spin density distribution. In total, 91% spin density is found at the Ni(III) of which 72% is in the 3d(z)(2) orbital and 16% in the 3d(xy) orbital. The Ni(II) contains -3.5% spin density, and 7.5% spin density is found at the axial MeCN ligands. In analogy to hydrogenases, it becomes apparent that binding of a substrate to Ni at the axial positions causes a redistribution of the electron charge and spin density, and this redistribution polarizes the chemical bonds of the axial ligand. For [NiFe] hydrogenases this implies that the H(2) bond becomes polarized upon binding of the substrate, which may facilitate its heterolytic splitting.     

ENDOR characterization of a synthetic diiron hydrazido complex as a model for nitrogenase intermediates

Molybdenum-dependent nitrogenase binds and reduces N2 at the [Fe7, Mo, S9, X, homocitrate] iron-molybdenum cofactor (FeMo-co). Kinetic and spectroscopic studies of nitrogenase variants indicate that a single Fe-S face is the most likely binding site. Recently, substantial progress has been made in determining the structures of nitrogenase intermediates formed during alkyne and N2 reduction through use of ENDOR spectroscopy. However, constraints derived from ENDOR studies of biomimetic complexes with known structure would powerfully contribute in turning experimentally derived ENDOR parameters into structures for species bound to FeMo-co during N2 reduction. The first report of a paramagnetic Fe-S compound that binds reduced forms of N2 involved Fe complexes stabilized by a bulky beta-diketiminate ligand (Vela, J.; Stoian, S.; Flaschenriem, C. J.; Münck, E.; Holland, P. L. J. Am. Chem. Soc. 2004, 126, 4522-4523). Treatment of a sulfidodiiron(II) complex with phenylhydrazine gave an isolable mixed-valence FeII-Fe(III) complex with a bridging phenylhydrazido (PhNNH2) ligand, and this species now has been characterized by ENDOR spectroscopy. Using both 15N, 2H labeled and unlabeled forms of the hydrazido ligand, the hyperfine and quadrupole parameters of the -N-NH2 moiety have been derived by a procedure that incorporates the (near-) mirror symmetry of the complex and involves a strategy which combines experiment with semiempirical and DFT computations. The results support the use of DFT computations in identifying nitrogenous species bound to FeMo-co of nitrogenase turnover intermediates and indicate that 14N quadrupole parameters from nitrogenase intermediates will provide a strong indication of the nature of the bound nitrogenous species. Comparison of the large 14N hyperfine couplings measured here with that of a hydrazine-derived species bound to FeMo-co of a trapped nitrogenase intermediate suggests that the ion(s) are not high spin and/or that the spin coupling coefficients of the coordinating cofactor iron ion(s) in the intermediate are exceptionally small.     

EPR-ENDOR of the Cu(I)NO complex of nitrite reductase

With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. (14)N- and (15)N-nitrite substrates gave rise to characteristic (14)NO and (15)NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with (63)Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated approximately 50% spin on the N of NO and approximately 17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cdelta1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cdelta1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 A from these protons. Such a distance positions the NO of Cu(I)NO as an axial ligand to Type 2 Cu(I).     

A multi-frequency pulse EPR and ENDOR approach to study strongly coupled nuclei in frozen solutions of high-spin ferric heme proteins

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.     

ENDOR investigation of the liganding environment of mixed-spin ferric cytochrome c'

The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) heme center in cytochrome c' was investigated by electron nuclear double resonance (ENDOR), a technique not previously applied to this mixed-spin system. Cytochrome c' was obtained from overexpressing variants of Rhodobacter sphaeroides 2.4.3. ENDOR for this study was done at the g(//) = 2.00 extremum where single-crystal-like, well-resolved spectra prevail. The heme meso protons of cytochrome c' showed a contact interaction that implied spin delocalization arising from the heme (d(z)(2)) orbital enhanced by iron out-of-planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 Ndelta hydrogen. This Ndelta hydrogen, which crystallographically has no hydrogen-bonding partner and thus belongs to a neutral imidazole, showed a larger hyperfine coupling than the corresponding hydrogen-bonded Ndelta proton from metmyoglobin. The unique residue Phe14 occludes binding of a sixth ligand in cytochrome c', and ENDOR from a proton of the functionally important Phe14 ring, approximately 3.3 A away from the heme iron, was detected. ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the sigma-antibonding (d(z)(2)) and (d(x)(2)-d(y)(2)) orbitals whose energies alter the relative stability and admixture of sextet and quartet states and whose electronic details were thus elucidated. ENDOR frequencies showed for cytochrome c' larger hyperfine couplings to the histidine nitrogen and smaller hyperfine couplings to the heme nitrogens than for high-spin ferric hemes. Both of these findings followed from the mixed-spin ground state, which has less (d(x)(2)-d(y)(2)) character than have fully high-spin ferric heme systems.     

The Mn(2+)-bicarbonate complex in a frozen solution revisited by pulse W-band ENDOR

The coordination of bicarbonate to Mn (2+) is the simplest model system for the coordination of Mn (2+) to carboxylate residues in a protein. Recently, the structure of such a complex has been investigated by means of X-band pulse EPR (electron paramagnetic resonance) experiments ( Dasgupta, J. ; et al. J. Phys. Chem. B 2006, 110, 5099 ). Based on the EPR results, together with electrochemical titrations, it has been concluded that the Mn (2+) bicarbonate complex consists of two bicarbonate ligands, one of which is monodentate and other bidentate, but only the latter has been observed by the pulsed EPR techniques. The X-band measurements, however, suffer several drawbacks. (i) The zero-field splitting (ZFS) term of the spin Hamiltonian affects the nuclear frequencies. (ii) There are significant contributions from ENDOR (electron nuclear double resonance) lines of the M S not equal +/- (1)/ 2 manifolds. (iii) There are overlapping signals of (23)Na. All these reduce the uniqueness of the data interpretation. Here we present a high-field ENDOR investigation of Mn (2+)/NaH (13)CO 3 in a water/methanol solution that eliminates the above difficulties. Both Davies and Mims ENDOR measurements were carried out. The spectra show that a couple of slightly inequivalent (13)C nuclei are present, with isotropic and anisotropic hyperfine couplings of A iso1 = 1.2 MHz, T perpendicular1 = 0.7 MHz, A iso2 = 1.0 MHz, T perpendicular2 = 0.6 MHz, respectively. The sign of the hyperfine coupling was determined by variable mixing time (VMT) ENDOR measurements. These rather close hyperfine parameters suggest that there are either two distinct, slightly different, carbonate ligands or that there is some distribution in conformation in only one ligand. The distances extracted from T perpendicular1 and T perpendicular2 are consistent with a monodentate binding mode. The monodentate binding mode and the presence of two ligands were further supported by DFT calculations and (1)H ENDOR measurements. Additionally, (23)Na ENDOR resolved at least two types of (23)Na (+) in the Mn (2+)-bicarbonate complex, thus suggesting that the bicarbonate bridges two positively charged metal ions.     

EPR-ENDOR characterization of (17O, 1H, 2H) water in manganese catalase and its relevance to the oxygen-evolving complex of photosystem II

The synthesis of efficient water-oxidation catalysts demands insight into the only known, naturally occurring water-oxidation catalyst, the oxygen-evolving complex (OEC) of photosystem II (PSII). Understanding the water oxidation mechanism requires knowledge of where and when substrate water binds to the OEC. Mn catalase in its Mn(III)-Mn(IV) state is a protein model of the OEC's S(2) state. From (17)O-labeled water exchanged into the di-μ-oxo di-Mn(III,IV) coordination sphere of Mn catalase, CW Q-band ENDOR spectroscopy revealed two distinctly different (17)O signals incorporated in distinctly different time regimes. First, a signal appearing after 2 h of (17)O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the (17)O μ-oxo hyperfine coupling of the di-μ-oxo di-Mn(III,IV) bipyridine model (Usov, O. M.; Grigoryants, V. M.; Tagore, R.; Brudvig, G. W.; Scholes, C. P. J. Am. Chem. Soc. 2007, 129, 11886-11887), this signal was assigned to μ-oxo oxygen. EPR line broadening was obvious from this (17)O μ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-μ-oxo position, from which (17)O ENDOR showed a smaller 3.8 MHz hyperfine coupling and possible quadrupole splittings, indicating a terminal water of Mn(III). Exchangeable proton/deuteron hyperfine couplings, consistent with terminal water ligation to Mn(III), also appeared. Q-band CW ENDOR from the S(2) state of the OEC was obtained following multihour (17)O exchange, which showed a (17)O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as μ-oxo-(17)O by resemblance to the μ-oxo signals from Mn catalase and the di-μ-oxo di-Mn(III,IV) bipyridine model.     

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.     

Integrated paramagnetic resonance of high-spin Co(II) in axial symmetry: chemical separation of dipolar and contact electron-nuclear couplings

Integrated paramagnetic resonance, utilizing electron paramagnetic resonance (EPR), NMR, and electron-nuclear double resonance (ENDOR), of a series of cobalt bis-trispyrazolylborates, Co(Tp ( x )) 2, are reported. Systematic substitutions at the ring carbons and on the apical boron provide a unique opportunity to separate through-bond and through-space contributions to the NMR hyperfine shifts for the parent, unsubstituted Tp complex. A simple relationship between the chemical shift difference (delta H - delta Me) and the contact shift of the proton in that position is developed. This approach allows independent extraction of the isotropic hyperfine coupling, A iso, for each proton in the molecule. The Co..H contact coupling energies derived from the NMR, together with the known metrics of the compounds, were used to predict the ENDOR couplings at g perpendicular. Proton ENDOR data is presented that shows good agreement with the NMR-derived model. ENDOR signals from all other magnetic nuclei in the complex ( (14)N, coordinating and noncoordinating, (11)B and (13)C) are also reported.     

Proton positions in the Mn(2+) binding site of concanavalin A as determined by single-crystal high-field ENDOR spectroscopy

High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn(2+) ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H(2)O and in D(2)O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of approximately 3.4 T allowed us to assign the ENDOR signals to their respective M(S) manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn(2+) ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein (Deacon et al. J. Chem. Soc., Faraday Trans. 1997, 93(24), 4305-4312).     

Spectroscopic studies of metal binding and metal selectivity in Bacillus subtilis BSco, a Homologue of the Yeast Mitochondrial Protein Sco1p

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.     

Electron magnetic resonance and density functional theory study of room temperature X-irradiated β-D-fructose single crystals

Stable free radical formation in fructose single crystals X-irradiated at room temperature was investigated using Q-band electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR induced EPR (EIE) techniques. ENDOR angular variations in the three main crystallographic planes allowed an unambiguous determination of 12 proton HFC tensors. From the EIE studies, these hyperfine interactions were assigned to six different radical species, labeled F1-F6. Two of the radicals (F1 and F2) were studied previously by Vanhaelewyn et al. [Vanhaelewyn, G. C. A. M.; Pauwels, E.; Callens, F. J.; Waroquier, M.; Sagstuen, E.; Matthys, P. J. Phys. Chem. A 2006, 110, 2147.] and Tarpan et al. [Tarpan, M. A.; Vrielinck, H.; De Cooman, H.; Callens, F. J. J. Phys. Chem. A 2009, 113, 7994.]. The other four radicals are reported here for the first time and periodic density functional theory (DFT) calculations were used to aid their structural identification. For the radical F3 a C3 carbon centered radical with a carbonyl group at the C4 position is proposed. The close similarity in HFC tensors suggests that F4 and F5 originate from the same type of radical stabilized in two slightly different conformations. For these radicals a C2 carbon centered radical model with a carbonyl group situated at the C3 position is proposed. A rather exotic C2 centered radical model is proposed for F6.     

Copper chemistry of beta-diketiminate ligands: monomer/dimer equilibria and a new class of bis(mu-oxo)dicopper compounds

A series of Cu(I) and Cu(II) complexes of a variety of beta-diketiminate ligands (L(-)) with a range of substitution patterns were prepared and characterized by spectroscopic, electrochemical, and, in several cases, X-ray crystallographic methods. Specifically, complexes of the general formula [LCuCl](2) were structurally characterized and their magnetic properties assessed through EPR spectroscopy of solutions and, in one instance, by variable-temperature SQUID magnetization measurements on a powder sample. UV-vis spectra indicated reversible dissociation to 3-coordinate monomers LCuCl in solution at temperatures above -55 degrees C. The Cu(I) complexes LCu(MeCN) exhibited reversible Cu(I)/Cu(II) redox couples with E(1/2) values between +300 and +520 mV versus NHE (cyclic voltammetry, MeCN solutions). These complexes were highly reactive with O(2), yielding intermediates that were identified as rare examples of neutral bis(mu-oxo)dicopper complexes on the basis of their EPR silence, diagnostic UV-vis absorption data, and O-isotope-sensitive resonance Raman spectroscopic features. The structural features of the compounds [LCuCl](2) and LCu(MeCN) as well as the proclivity to form bis(mu-oxo)dicopper products upon oxygenation of the Cu(I) complexes are compared to data previously reported for complexes of more sterically hindered beta-diketiminate ligands (Aboelella, N. W.; Lewis, E. A.; Reynolds, A. M.; Brennessel, W. W.; Cramer, C. J.; Tolman, W. B. J. Am. Chem. Soc. 2002, 124, 10600. Spencer, D. J. E.; Aboelella, N. W.; Reynolds, A. M.; Holland, P. L.; Tolman, W. B. J. Am. Chem. Soc. 2002, 124, 2108. Holland, P. L.; Tolman, W. B. J. Am. Chem. Soc. 1999, 121, 7270). The observed structural and reactivity differences are rationalized by considering the steric influences of both the substituents on the flanking aromatic rings and those present on the beta-diketiminate backbone.     

Electronic structure of azidomyoglobin and associated magnetic and hyperfine properties

The electronic structure of azidomyoglobin has been investigated for understanding the observed magnetic and hyperfine properties of this system. The results of our investigation show that a configuration with five electrons in d-like molecular orbital states, as in the case of ferricytochrome c but unlike nitrosylhemoglobin, provides a satisfactory explanation of the observed strongly rhombic g-tensor, the ^57m Fe quadrupole splitting from Mössbauer measurements and the porphyrin ¹⁴N quadrupole interactions. For the magnetic hyperfine interactions of the ^57m Fe and porphyrin ¹⁴N nuclei, there are significant differences between theory and experiment. For the ^57m Fe nucleus, after incorporating the influence of spin-orbit effects, which leads to unquenching of the orbital angular momentum through admixture of excited state configurations to the ground state one, very good agreement is found with single crystal Mössbauer data. For ¹⁴N hyperfine interactions associated with the pyrrole group however, where spin-orbit effects are expected to be much less pronounced, the theoretical values of the hyperfine constants are found to be less than a fifth of those derived from ENDOR measurements. It is suggested that the difference between theory and experiment could be bridged through incorporation of exchange polarization contribution to the ¹⁴N hyperfine interaction from the sizeable valence electron spin density (about 65 per cent of the total) on the iron atom. The need for additional experimental measurements is pointed out, among them ENDOR measurements to determine the hyperfine properties of the azide nitrogens for which the end nitrogens are predicted from the present work to have sizeable magnetic hyperfine constants (about –10 MHz).This work was supported by National Institute of Health Grant HL15196     

Cu3(mu-S)2]3+ clusters supported by N-donor ligands: progress toward a synthetic model of the catalytic site of nitrous oxide reductase

By treating Cu(I) complexes of neutral, bidentate N-donor ligands with S8, clusters with novel delocalized mixed-valence [Cu3(mu-S)2]3+ cores have been isolated. X-ray crystal structures and UV-vis and resonance Raman spectral features of these clusters reveal similarities to the tetracopper-sulfide "CuZ" site in nitrous oxide reductase. A delocalized S = 1 ground state for the mixed-valent CuIIICu2II cores is supported by the observation of high symmetry in the X-ray structures and 10-line hyperfine features arising from coupling to three equivalent Cu ions in EPR spectra obtained at room temperature (shown) and 10 K. The delocalization we observe contrasts with the localization reported previously for a [Cu3(mu-O)2]3+ analogue (Root, D. E.; Henson, M. J.; Machonkin, T.; Mukherjee, P.; Stack, T. D. P.; Solomon, E. I. J. Am. Chem. Soc. 1998, 120, 4982), which we rationalized through DFT calculations.