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

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

Density Functional Calculations of 55Mn, 14N and 13C Electron Paramagnetic Resonance Parameters Support an Energetically Feasible Model System for the S2 State of the Oxygen‐Evolving Complex of Photosystem II

Metal and ligand hyperfine couplings of a previously suggested, energetically feasible Mn₄Ca model cluster ( SG2009^?1 ) for the S₂ state of the oxygen‐evolving complex (OEC) of photosystem II (PSII) have been studied by broken‐symmetry density functional methods and compared with other suggested structural and spectroscopic models. This was carried out explicitly for different spin‐coupling patterns of the S=1/2 ground state of the Mn^III(Mn^IV)₃ cluster. By applying spin‐projection techniques and a scaling of the manganese hyperfine couplings, computation of the hyperfine and nuclear quadrupole coupling parameters allows a direct evaluation of the proposed models in comparison with data obtained from the simulation of EPR, ENDOR, and ESEEM spectra. The computation of ⁵⁵Mn hyperfine couplings (HFCs) for SG2009^?1 gives excellent agreement with experiment. However, at the current level of spin projection, the ⁵⁵Mn HFCs do not appear sufficiently accurate to distinguish between different structural models. Yet, of all the models studied, SG2009^?1 is the only one with the Mn^III site at the Mn_C center, which is coordinated by histidine (D1‐His332). The computed histidine ¹⁴N HFC anisotropy for SG2009^?1 gives much better agreement with ESEEM data than the other models, in which Mn_C is an Mn^IV site, thus supporting the validity of the model. The ¹³C HFCs of various carboxylates have been compared with ¹³C ENDOR data for PSII preparations with ¹³C‐labelled alanine.     

Investigation of the calcium-binding site of the oxygen evolving complex of photosystem II using 87Sr ESEEM spectroscopy

The proximity of the calcium/strontium binding site of the oxygen evolving complex (OEC) of photosystem II (PSII) to the paramagnetic Mn cluster is explored with (87)Sr three-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy. CW-EPR spectra of Sr(2+)-substituted Ca(2+)-depleted PSII membranes show the modified g = 2 multiline EPR signal as previously reported. We performed three-pulse ESEEM on this modified multiline signal of the Mn cluster using natural abundance Sr and (87)Sr, respectively. Three-pulse ESEEM of the natural abundance Sr sample exhibits no detectable modulation by the 7% abundance (87)Sr. On the other hand, that of the (87)Sr enriched (93%) sample clearly reveals modulation arising from the I = (9)/(2) (87)Sr nucleus weakly magnetically coupled to the Mn cluster. Using a simple point dipole approximation for the electron spin, analysis of the (87)Sr ESEEM modulation depth via an analytic expression suggests a Mn-Ca (Sr) distance of 4.5 A. Simulation of three-pulse ESEEM with a numerical matrix diagonalization procedure gave good agreement with this analytical result. A more appropriate tetranuclear magnetic/structural model for the Mn cluster converts the 4.5 A point dipole distance to a 3.8-5.0 A range of distances. DFT calculations of (43)Ca and (87)Sr quadrupolar interactions on Ca (and Sr substituted) binding sites in various proteins suggest that the lack of the nuclear quadrupole induced splitting in the ESEEM spectrum of (87)Sr enriched PSII samples is related to a very high degree of symmetry of the ligands surrounding the Sr(2+) ion in the substituted Ca site. Numerical simulations show that moderate (87)Sr quadrupolar couplings decrease the envelope modulation relative to the zero quadrupole case, and therefore we consider that the 3.8-5.0 A range obtained without quadrupolar coupling included in the simulation represents an upper limit to the actual manganese-calcium distance. This (87)Sr pulsed EPR spectroscopy provides independent direct evidence that the calcium/strontium binding site is close to the Mn cluster in the OEC of PSII.     

Acetate binding at the photosystem II oxygen evolving complex: an S(2)-state multiline signal ESEEM study

Previously, using acetate deuterated in the methyl hydrogen positions, we showed that acetate binds in close proximity to the Mn cluster/Y(.)(z) tyrosine dual spin complex in acetate-inhibited photosystem II (PSII) preparations exhibiting the "split" EPR signal arising from the S(2)-Y(.)(z) interaction [Force, D. A.; Randall, D. W.; Britt, R. D. Biochemistry 1997, 36, 12062-12070]. By using paramagnetic NO to quench the paramagnetism of Y(.)(z), we are able to observe the ESEEM spectrum of deuterated acetate interacting with only the Mn cluster. A good fit of the ESEEM data indicates two (2)H dipolar hyperfine couplings of 0.097 MHz and one of 0.190 MHz. Modeling of these dipolar interactions, using our "dangler" 3 + 1 model for the S(2)-state of the Mn cluster, reveals distances consistent with direct ligation of acetate to the Mn cluster. As acetate inhibition is competitive with the essential cofactor Cl(-), this suggests that Cl(-) ligates directly to the Mn cluster. The effect of acetate binding on the structure of the Mn cluster is investigated by comparing the Mn-histidine coupling in NO/acetate-treated PSII and untreated PSII using ESEEM. We find that the addition of acetate and NO does not affect the histidine ligation to the Mn cluster. We also investigate the ability of acetate to access Y(.)(z) in Mn-depleted PSII, a PSII preparation expected to be more solvent accessible than intact PSII. We detect no coupling between Y(.)(z) and acetate. We have previously shown that small alcohols such as methanol can ligate to the Mn cluster with ease, while larger alcohols such as 2-propanol, as well as DMSO, are excluded [Force, D. A.; Randall, D. W.; Lorigan, G. A.; Clemens, K. L.; Britt, R. D. J. Am. Chem. Soc. 1998, 120, 13321-13333]. We probe the effect of acetate binding on the ability of methanol and DMSO to bind to the Mn cluster. We find that methanol is able to bind to the Mn cluster in the presence of acetate. We detect no DMSO binding in the presence of acetate. Thus, acetate binding does not increase the affinity or accessibility for DMSO binding at the Mn cluster. We also explore the possibility that the acetate binding site is also a binding site for substrate water. By comparing the ratioed three-pulse ESEEM spectra of a control, untreated PSII sample in 50% D(2)O to an NO/acetate-treated PSII sample in 50% D(2)O, we find that the binding of acetate to the oxygen evolving complex of photosystem II displaces deuterons bound very closely to the Mn cluster.     

Testing if the interstitial atom, X, of the nitrogenase molybdenum-iron cofactor is N or C: ENDOR, ESEEM, and DFT studies of the S = 3/2 resting state in multiple environments

A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, or C atom (denoted X) inside the central iron prismane ([6Fe]) of the [MoFe7S9:homocitrate] FeMo-cofactor (FeMo-co). We here extend earlier efforts to determine the identity of X through detailed tests of whether X = N or C by interlocking and mutually supportive 9 GHz electron spin echo envelope modulation (ESEEM) and 35 GHz electron-nuclear double resonance (ENDOR) measurements on 14/15N and 12/13C isotopomers of FeMo-co in three environments: (i) incorporated into the native MoFe protein environment; (ii) extracted into N-methyl formamide solution; and (iii) incorporated into the NifX protein, which acts as a chaperone during FeMo-co biosynthesis. These measurements provide powerful evidence that X not equal N/C, unless X in effect is magnetically decoupled from the S = 3/2 electron spin system of resting FeMo-co. They reveal no signals from FeMo-co in any of the three environments that can be assigned to X from either 14/15N or 13C: If X were either element, its maximum observed hyperfine coupling at all fields of measurement is estimated to be A(14/15NX) < 0.07/0.1 MHz, A(13CX) < 0.1 MHz, corresponding to intrinsic couplings of about half these values. In parallel, we have explicitly calculated the hyperfine tensors for X = 14/15N/13C/17O, nuclear quadrupole coupling constant e2qQ for X = 14N, and hyperfine constants for the Fe sites of S = 3/2 FeMo-co using density functional theory (DFT) in conjunction with the broken-symmetry (BS) approach for spin coupling. If X = C/N, then the decoupling required by experiment strongly supports the "BS7" spin coupling of the FeMo-co iron sites, in which a small X hyperfine coupling is the result of a precise balance of spin density contributions from three spin-up and three spin-down (3 upward arrow:3 downward arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 upward arrow:2 downward arrow for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g2 are sufficiently large that they should have been detected: we suggest that the experimental results are compatible with X = N only if aiso(14/15NX) < 0.03-0.07/0.05-0.1 MHz and aiso(13CX) < 0.05-0.1 MHz, compared with calculated values of aiso(14/15NX) = 0.3/0.4 MHz and aiso(13CX) = 1 MHz. However, the DFT uncertainties are large enough that the very small hyperfine couplings required by experiment do not necessarily rule out X = N/C.     

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.     

Pulsed EPR/ENDOR characterization of perturbations of the Cu(A) center ground state by axial methionine ligand mutations.

The effect of axial ligand mutation on the Cu(A) site in the recombinant water soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase ba(3) has been investigated. The weak methionine ligand was replaced by glutamate and glutamine which are stronger ligands. Two constructs, M160T0 and M160T9, that differ in the length of the peptide were prepared. M160T0 is the original soluble fragment construct of cytochrome ba(3) that encodes 135 amino acids of subunit II, omitting the transmembrane helix that anchors the domain in the membrane. In M160T9 nine C-terminal amino acids are missing, including one histidine. The latter has been used to reduce the amount of a secondary T2 copper which is most probably coordinated to a surface histidine in M160T0. The changes in the spin density in the Cu(A) site, as manifested by the hyperfine couplings of the weakly and strongly coupled nitrogens, and of the cysteine beta-protons, were followed using a combination of advanced EPR techniques. X-band ( approximately 9 GHz) electron-spin-echo envelope modulation (ESEEM) and two-dimensional (2D) hyperfine sublevel correlation (HYSCORE) spectroscopy were employed to measure the weakly coupled (14)N nuclei, and X- and W-band (95 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy for probing the strongly coupled (14)N nuclei and the beta-protons. The high field measurements were extremely useful as they allowed us to resolve the T2 and Cu(A) signals in the g( perpendicular) region and gave (1)H ENDOR spectra free of overlapping (14)N signals. The effects of the M160Q and M160E mutations were: (i) increase in A( parallel)((63,65)Cu), (ii) larger hyperfine coupling of the weakly coupled backbone nitrogen of C153, (iii) reduction in the isotropic hyperfine interaction, a(iso), of some of the beta-protons making them more similar, (iv) the a(iso) value of one of the remote nitrogens of the histidine residues is decreased, thus distinguishing the two histidines, and finally, (v) the symmetry of the g-tensor remained axial. These effects were associated with an increase in the Cu-Cu distance and subtle changes in the geometry of the Cu(2)S(2) core which are consistent with the electronic structural model of Gamelin et al. (Gamelin, D. R.; Randall, D. W.; Hay, M. T.; Houser, R. P.; Mulder, T. C.; Canters, G. W.; de Vries, S.; Tolman, W. B.; Lu, Y.; Solomon, E. I. J. Am. Chem. Soc. 1998, 120, 5246-5263).     

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

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

Hydrogen bonding affects the [NiFe] active site of Desulfovibrio vulgaris Miyazaki F hydrogenase: a hyperfine sublevel correlation spectroscopy and density functional theory study

Pulse electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy have been used to investigate nitrogen coordination of the active site of [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in its oxidized "ready" state. The obtained (14)N hyperfine (A = [+1.32, +1.32, +2.07] MHz) and nuclear quadrupole (e(2)qQ/h = -1.9 MHz, eta = 0.37) coupling constants were assigned to the N(epsilon) of a highly conserved histidine (His88) by studying a hydrogenase preparation in which the histidines were (15)N labeled. The histidine is hydrogen-bonded via its N(epsilon)-H to the nickel-coordinating sulfur of a cysteine (Cys549) that carries an appreciable amount of spin density. Through the hydrogen bond a small fraction of the spin density ( approximately 1%) is delocalized onto the histidine ring giving rise to an isotropic (14)N hyperfine coupling constant of about 1.6 MHz. These conclusions are supported by density functional calculations. The measured (14)N quadrupole coupling constants are related to the polarization of the N(epsilon)-H bond, and the respective hydrogen bond can be classified as being weak.     

ESEEM studies of peptide nitrogen hyperfine coupling in tyrosyl radicals and model peptides

Tyrosyl radicals are important in long-range electron transfer in several enzymes, but the protein environmental factors that control midpoint potential and electron transfer rate are not well understood. To develop a more detailed understanding of the effect of protein sequence, we have performed 14N and 15N electron spin echo envelope modulation (ESEEM) measurements on tyrosyl radical, generated either in polycrystalline tyrosinate or in its 15N-labeled isotopomer, by UV photolysis. 14N-ESEEM was also performed on tyrosyl radical generated in tyrosine-containing pentapeptide samples. Simulation of the 14N- and 15N-tyrosyl radical ESEEM measurements yielded no significant isotropic hyperfine splitting to the amine or amide nitrogen; the amplitude of the anisotropic, nitrogen hyperfine coupling (0.21 MHz) was consistent with a dipole-dipole distance of 3.0 A. Density functional theory was used to calculate the isotropic and anisotropic hyperfine couplings to the amino nitrogen in four different tyrosyl radical conformers. Comparison with the simulated data suggested that the lowest energy radical conformer, generated in tyrosine at pH 11, has a 76 degrees Calpha-Cbeta-C1'-C2' ring and a -73 degrees C-Calpha-Cbeta-C1' backbone dihedral angle. In addition, the magnitude, orientation, and asymmetry of the nuclear quadrupole coupling tensor were derived from analysis of the tyrosyl radical 14N-ESEEM. The simulations showed differences in the coupling and orientation of the nuclear quadrupole tensor, when the tyrosinate and pentapeptide samples were compared. These results suggest sequence- or conformation-induced changes in the ionic character of the NH bond in different tyrosine-containing peptides.     

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

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

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

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

Ligand-bound S = 1/2 FeMo-cofactor of nitrogenase: hyperfine interaction analysis and implication for the central ligand X identity

Broken symmetry density functional theory (BS-DFT) has been used to address the hyperfine parameters of the single atom ligand X, proposed to be coordinated by six iron ions in the center of the paramagnetic FeMo-cofactor (FeMoco) of nitrogenase. Using the X = N alternative, we recently found that any hyperfine signal from X would be small (calculated A(iso)(X = (14)N) = 0.3 MHz) due to both structural and electronic symmetry properties of the [Mo-7Fe-9S- X] FeMoco core in its resting S = 3/2 state. Here, we extend our BS-DFT approach to the 2e(-) reduced S = 1/2 FeMoco state. Alternative substrates coordinated to this FeMoco state effectively perturb the electronic and/or structural symmetry properties of the cofactor. Using an example of an allyl alcohol (H2C=CH-CH2-OH) product ligand, we consider three different binding modes at single iron site and three different BS-DFT spin state structures and show that this binding would enhance the key hyperfine signal A(iso)(X) by at least 1 order of magnitude (3.8 < or = A(iso)(X = (14)N) < or = 14.7 MHz), and this result should not depend strongly on the exact identity of X (nitrogen, carbon, or oxygen). The interstitial atom, when the nucleus has a nonzero magnetic moment, should therefore be observable by ESR methods for some ligand-bound FeMoco states. In addition, our results illustrate structural details and likely spin-coupling patterns for models for early intermediates in the catalytic cycle.     

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.     

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.     

EPR and ESEEM study of the plastoquinone anion radical QA in photosystem II treated at high pH

The effect of treatment at pH = 11 on the photosystem II was studied by EPR and electron spin echo envelope modulation (ESEEM). The magnetic interaction between the semiquinone Q_A^−. and the non-heme Fe²⁺ (S = 2) was absent. ESEEM showed that the Q_A^−. interacts magnetically with two ¹⁴N nuclei. The first interaction has a hyperfine coupling tensor (A_XX, A_YY, A_ZZ)=(2.0, 1.7, 2.3 MHz) and nuclear quadrupole interaction parameters e²qQ/h=3.24 MHz and η = 0.45 while those of the second are (A_XX, A_YY, A_ZZ)=(1.2, 1.5, 2.3 MHz), e²qQ/h = 1.56 MHz and η = 0.71. These are assigned to an amide nitrogen of the peptide backbone and the amino nitrogen of an imidazole respectively. By analogy to the bacterial reaction centre, these nitrogens are attributed to the Ala 261 and His 215 of the D2 protein. It was shown earlier that the imidazole coupling is absent in cyanide-treated PSII, its presence here is attributed to a difference in the position of the imidazole group itself.     

A new mechanism for electron spin echo envelope modulation

Electron spin echo envelope modulation (ESEEM) has been observed for the first time from a coupled heterospin pair of electron and nucleus in liquid solution. Previously, modulation effects in spin-echo experiments have only been described in liquid solutions for a coupled pair of homonuclear spins in nuclear magnetic resonance or a pair of resonant electron spins in electron paramagnetic resonance. We observe low-frequency ESEEM (26 and 52 kHz) due to a new mechanism present for any electron spin with S > 12 that is hyperfine coupled to a nuclear spin. In our case these are electron spin (S = 32) and nuclear spin (I = 1) in the endohedral fullerene N@C(60). The modulation is shown to arise from second-order effects in the isotropic hyperfine coupling of an electron and (14)N nucleus.     

An EPR, ESEEM, structural NMR, and DFT study of a synthetic model for the covalently ring-linked tyrosine-histidine structure in the heme-copper oxidases

We report CW-EPR, ESEEM, and structural NMR results, as well as DFT calculations, on model compounds relevant to the unusual cross-linked Tyr-His (YH) moiety at the active site of the heme-copper oxidases. CW-EPR spectra of an (15)N isotopically labeled 4-methyl-2-(4-methyl-imidazole-1-yl)-phenol radical are nearly identical to those of the natural abundance (14)N compound. We obtain good simulations of these EPR spectra without including hyperfine couplings to the nitrogen nuclei. This implies that the electron distribution of the radical is largely localized on the phenol ring with only a small amount of spin delocalized onto the nitrogens of the imidazole. Using three-pulse ESEEM spectroscopy, we have successfully detected the two imidazole ring nitrogens, one near the "exact cancellation" ESEEM condition and the other more weakly coupled. We assign these to the imino and amino nitrogens, respectively, based on DFT calculations performed on this radical species. The experimental results and the supporting density functional calculations clearly show that the imidazole substituent has only a minor effect on the electronic structure of the substituted phenol radical.     

g-Anisotropy of the S2-state manganese cluster in single crystals of cyanobacterial photosystem II studied by W-band electron paramagnetic resonance spectroscopy

The multiline signal from the S2-state manganese cluster in the oxygen evolving complex of photosystem II (PSII) was observed in single crystals of a thermophilic cyanobacterium Thermosynechococcus vulcanus for the first time by W-band (94 GHz) electron paramagnetic resonance (EPR). At W-band, spectra were characterized by the g-anisotropy, which enabled the precise determination of the tensor. Distinct hyperfine splittings (hfs's) as seen in frozen solutions of PSII at X-band (9.5 GHz) were detected in most of the crystal orientations relative to the magnetic field. In some orientations, however, the hfs's disappeared due to overlapping of a large number of EPR lines from eight crystallographic symmetry-related sites of the manganese cluster within the unit cell of the crystal. Analysis of the orientation-dependent spectral features yielded the following g-tensor components: g(x) = 1.988, g(y) = 1.981, g(z) = 1.965. The principal values suggested an approximate axial symmetry around the Mn(III) ion in the cluster.     

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.