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

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

NH3 and O2 interaction with tetrahedral Ti3+ ions isomorphously substituted in the framework of TiAlPO-5. A combined pulse EPR, pulse ENDOR, UV-Vis and FT-IR study

Continuous Wave (CW), pulse Electron Paramagnetic Resonance (EPR) and pulse Electron Nuclear Double Resonance (ENDOR) spectroscopies, in conjunction with UV-Vis and Infrared (IR) spectroscopies, are used to investigate the chemical reactivity of tetrahedrally coordinated Ti(3+) ions isomorphously substituted in the framework of AlPO-5 towards NH(3) and O(2). The coordination of ammonia to Ti(3+) centres is followed in detail by complementary vibrational and electron magnetic resonance techniques. In particular HYSCORE spectra allow identifying the coordination of two ammonia molecules to Ti(3+) centres resolving the full hyperfine and quadrupole (14)N coupling tensors. The reactivity of the reduced TiAlPO sample towards molecular oxygen is detailed by means of CW-EPR and pulse ENDOR spectroscopy. (17)O(2) is employed, allowing to establish the formation of a "side-on" η(2) O(2)(-)-Ti(4+) electrostatic complex. Pulse ENDOR spectra provide detailed information on the local environment of the formed superoxide radical anion which acts as a paramagnetic probe, providing evidence for Ti-O-Ti oligomeric species.     

Electron paramagnetic resonance spectroscopic evidence for the interaction of HAlOH with water molecules

The complex HAlOH:(H(2)O) has been detected by matrix-isolation IR spectroscopy. This complex was speculated to be the species responsible for the chemiluminescent glow associated with the explosion of trimethyl aluminum or aluminum grenades in the upper atomosphere. Theoretical studies suggest that HAlOH:(H(2)O)(n) is a critical precursor in the formation of H(2) in the reaction of Al with liquid water. In our study, Al atoms were reacted with mixtures of D(2)O/He or H(2)(17)O/He in an adamantane matrix in a metal-atom reactor, known as a rotating cryostat, maintained at 77 K and at <10(-6) Torr. In addition to DAlOD and HAlOH, which formed from the reaction of Al atoms with adventitious water, EPR analysis of the Al-D(2)O/He reaction mixture from 77 to 290 K showed that HAlOH:(D(2)O) and DAlOD:(D(2)O) formed. The experimental nuclear hyperfine interactions (hfis) for these species were in close agreement with those calculated using the B3LYP density functional method and the 6-311+G(2df,p) basis set. The effect of complexation is to lower the Al hfi of HAlOH and DAlOD by ca. 8%, the H hfi of HAlOH by ca. 28%, and the D hfi of DAlOD by ca. 35%.     

Combined electron magnetic resonance and density functional theory study of 10 K X-irradiated beta-D-fructose single crystals

Primary free radical formations in fructose single crystals X-irradiated at 10 K were investigated at the same temperature using X-band Electron Paramagnetic Resonance (EPR), Electron Nuclear Double Resonance (ENDOR) and ENDOR induced EPR (EIE) techniques. ENDOR angular variations in the three principal crystallographic planes and a fourth skewed plane allowed the unambiguous determination of five proton hyperfine coupling tensors. From the EIE studies, these hyperfine interactions were assigned to three different radicals, labeled T1, T1* and T2. For the T1 and T1* radicals, the close similarity in hyperfine coupling tensors suggests that they are due to the same type of radical stabilized in two slightly different geometrical conformations. Periodic density functional theory calculations were used to aid the identification of the structure of the radiation-induced radicals. For the T1/T1* radicals a C3 centered hydroxyalkyl radical model formed by a net H abstraction is proposed. The T2 radical is proposed to be a C5 centered hydroxyalkyl radical, formed by a net hydrogen abstraction. For both radicals, a very good agreement between calculated and experimental hyperfine coupling tensors was obtained.     

2D-hyperfine sublevel correlation spectroscopy of tyrosyl radicals

Hyperfine sublevel correlation (HYSCORE) spectroscopy has been used to study the tyrosyl radicals in Photosystem II and bovine liver catalase. The HYSCORE data allow a complete resolution of all the 1H hyperfine tensors of these radicals. The present work shows that the proper analysis of the HYSCORE data allows the complete assignment of the 1H-hyperfine tensors in tyrosine radicals and this offers an alternative experimental tool relative to ENDOR.     

Pulsed EPR investigations of systems modeling molybdenum enzymes: hyperfine and quadrupole parameters of oxo-17O in [Mo 17O(SPh)4]-

Ka band ESEEM spectroscopy was used to determine the hyperfine (hfi) and nuclear quadrupole (nqi) interaction parameters for the oxo-17O ligand in [Mo 17O(SPh)4]-, a spectroscopic model of the oxo-Mo(V) centers of enzymes. The isotropic hfi constant of 6.5 MHz found for the oxo-17O is much smaller than the values of approximately 20-40 MHz typical for the 17O nucleus of an equatorial OH(2) ligand in molybdenum enzymes. The 17O nqi parameter (e2qQ/h = 1.45 MHz, eta approximately = 0) is the first to be obtained for an oxo group in a metal complex. The parameters of the oxo-17O ligand, as well as other magnetic resonance parameters of [Mo 17O(SPh)4]- predicted by quasi-relativistic DFT calculations, were in good agreement with those obtained in experiment. From the electronic structure of the complex revealed by DFT, it follows that the SOMO is almost entirely molybdenum d(xy) and sulfur p, while the spin density on the oxo-17O is negative, determined by spin polarization mechanisms. The results of this work will enable direct experimental identification of the oxo ligand in a variety of chemical and biological systems.     

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

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

The 17O hyperfine interaction in V17O(H217O)52+ and Mn(H217O)62+ determined by high field ENDOR aided by DFT calculations

The 17O hyperfine interaction of the water ligands and the V=O oxygen in the vanadyl aquo complex and of the water ligands in the Mn2+ aquo complex in a frozen solution were determined by W-band (95 GHz) electron-nuclear double resonance (ENDOR). Orientation selective ENDOR spectra of the vanadyl complex exhibited two distinct signals assigned to the vanadyl oxygen and the water ligands. The assignment of the signals was done based on the orientation of the principal axis system of the hyperfine interaction and through comparison with the hyperfine interaction predicted by DFT calculations. The latter showed good agreement with the experimental values thus providing clear evidence that the vanadyl oxygen is exchangeable. The interaction of the vanadyl oxygen, especially its anisotropic part, was significantly larger than that of the water oxygens due to a relatively large negative spin density on the oxygen p orbitals. The 17O hyperfine interaction of the water ligand in the Mn2+ complex was found to be similar to that of the water ligand in the vanadyl complex and was in good agreement with earlier single-crystal data. Here, due to the large thermal polarization, it was also possible to determine the absolute sign of the hyperfine coupling by selecting different EPR transitions.     

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.     

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.     

An ENDOR and HYSCORE investigation of a reaction intermediate in IspG (GcpE) catalysis

IspG is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we have specifically assigned the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique fourth Fe in the cluster and C2 and O3 of the ligand.     

Evaluating <Emphasis Type="Italic">π-π</Emphasis> stacking effects in macrocyclic transition metal complexes using EPR techniques

The HYSCORE spectra for two different macrocyclic transition metal complexes, namely cobalt tetraphenylporphyrin (CoTPP) and a copper Salen derivative ([Cu(1)]), were examined in relation to their interactions with pyridine (Py) and methylbenzyl amine (MBA), respectively. In both cases weak hyperfine interactions were detected by HYSCORE, but the origin of these interactions was found to originate from completely different effects. In the CoTPPpy adduct, with axial coordination of the substrate (pyridine) to the metal centre, weak couplings from the pyrrole nitrogens of a neighbouring porphyrin complex were identified, and confirmed through a series of isotopic labelling and dilution experiments. This result represents the first ever identification by HYSCORE of π-π interactions between such porphyrin complexes in solution. In the [Cu(1)]MBA adduct, again with axial coordination of the substrate (MBA) to the copper centre, weak couplings were also identified in the HYSCORE spectra, which could easily be misinterpreted as arising from intermolecular interactions between adjacent ligands. However, the origin of these couplings was clearly demonstrated to arise from intramolecular ¹³C-ligand interactions. These results demonstrate not only the sensitivity of the HYSCORE technique for detection of weak inter- and intra-molecular interactions in macrocyclic transition metal complexes, but additionally the need to consider dilution effects in the spectral assignments.     

Structure and electron paramagnetic resonance parameters of the manganese site of concanavalin A studied by density functional methods

The EPR parameters of the manganese site in the saccharide-binding protein concanavalin A have been studied by density functional methods, with an emphasis on metal (⁵⁵Mn) and ligand (¹H and ¹⁷O) hyperfine couplings, in comparison with high-field EPR and ENDOR data. Results for gradient-corrected and hybrid functionals with different exact-exchange admixture have been compared with experiment for the ⁵⁵Mn and the ¹H ligand hyperfine coupling and have been predicted for ¹⁷O hyperfine coupling based on comparison with experiment for the related [Mn(H₂O)₆]²⁺. Appreciable exact-exchange admixture in the hybrid functional is needed to obtain an adequate spin-density distribution and thus near-quantitative agreement with experimental EPR parameters. The common use of experimental proton hyperfine coupling tensors together with the point-dipole approximation for determination of bond lengths is evaluated by explicit calculations.     

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.     

Activation of C-O and C-C bonds and formation of novel HAlOH-ether complexes: an EPR study of the reaction of ground-state Al atoms with methylethyl ether and diethyl ether

Reaction mixtures, containing Al atoms and methylethyl ether (MEE) or diethyl ether (DEE) in an adamantane matrix, were prepared with the aid of a metal-atom reactor known as a rotating cryostat. The EPR spectra of the resulting products were recorded from 77-260 K, at 10 K intervals. Al atoms were found to insert into methyl-O, ethyl-O, and C-C bonds to form CH(3)AlOCH(2)CH(3), CH(3)OAlCH(2)CH(3), and CH(3)OCH(2)AlCH(3), respectively, in the case of MEE while DEE produced CH(3)CH(2)AlOCH(2)CH(3) and CH(3)AlCH(2)OCH(2)CH(3), respectively. From the intensity of the transition lines attributed to the Al atom C-O insertion products of MEE, insertion into the methyl-O bond is preferred. The Al hyperfine interaction (hfi) extracted from the EPR spectra of the C-O insertion products was greater than that of the C-C insertion products, that is, 5.4% greater for the DEE system and 7% greater for the MEE system. The increase in Al hfi is thought to arise from the increased electron-withdrawing ability of the substituents bonded to Al. Besides HAlOH, resulting from the reaction of Al atoms with adventitious water, novel mixed HAlOH:MEE and HAlOH:DEE complexes were identified with the aid of isotopic studies involving H(2)(17)O and D(2)O. The Al and H hfi of HAlOH were found to decrease upon complex formation. These findings are consistent with the nuclear hfi calculated using a density functional theory (DFT) method with close agreement between theory and experiment occurring at the B3LYP level using a 6-311+G(2df,p) basis set.     

Quantum chemical investigation of hyperfine coupling constants on first coordination sphere water molecule of gadolinium(III) aqua complexes

Hyperfine interactions (HFI) on the nuclei of the first coordination sphere water molecules in a model [Gd(H(2)O)(8)](3+) aqua complex and in the magnetic resonance imaging contrast agent [Gd(DOTA)(H(2)O)](-) were studied theoretically. Density functional theory (DFT) calculations combined with classical molecular dynamics (MD) simulations have been used in order to take into account dynamic effects in aqueous solution. DFT relativistic calculations show a strong spin-polarization of the first coordination sphere water molecules. This spin-polarization leads to a positive (17)O isotropic hyperfine coupling constant (A(iso)((17)O) = 0.58 +/- 0.11 MHz) and to a significant increase of the effective distance (r(eff)(Gd-O) = 2.72 +/- 0.06 A) of dipolar interaction compared to the mean internuclear distance (r(Gd-O) = 2.56 +/- 0.06 A) obtained from the MD trajectory of [Gd(DOTA)(H(2)O)](-) in aqueous solution. The point-dipole model for anisotropic hyperfine interaction overestimates therefore the longitudinal relaxation rate of the (17)O nucleus by approximately 45%. The (1)H isotropic hyperfine coupling constant of the bound water molecule is predicted to be very small (A(iso)((1)H) = 0.03 +/- 0.02 MHz), and the point-dipole approximation for first coordination sphere water protons holds. The calculated hyperfine parameters are in good agreement with available experimental data.     

Aqueous spectroscopy and redox properties of carboxylate-bound titanium

The aqueous chemistry of Ti(III) and Ti(IV) in two different chemical environments is investigated given its relevance to environmental, materials, and biological chemistry. Complexes of titanium with the carboxylate ligands citrate and oxalate, found ubiquitously in Nature, were synthesized. The redox properties were studied by using cyclic voltammetry. All the titanium citrate redox couples are quasi-reversible. Electrospray mass spectrometry of the Ti(III) citrate solution shows the presence of a 1:2 Ti/cit complex in solution, in contrast to the predominant 1:3 Ti/cit complex with Ti(IV). The change in the coordination of the ligand to the metal on reduction may explain the quasi-reversible behavior of the electrochemistry. The redox potentials for Ti(IV) citrate in water vary with pH. At pH 7, the approximate E(1/2) is less than -800 mV. This stated change in redox properties is considered in light of the previously reported Ti(IV) citrate solution speciation. Analogous speciation behavior is suggested from the EPR spectroscopy of Ti(III) citrate aqueous solutions. The g tensors are deduced for several pH-dependent species from the simulated data. The X-ray crystal structure of a Ti(III)(2) oxalate dimer Ti(2)(mu-C(2)O(4))(C(2)O(4))(2)(H(2)O)(6).2H(2)O (3), which crystallizes from water below pH 2, is reported. Complex 3 crystallizes in a monoclinic P2(1)/c space group with a = 9.5088(19) Angstroms, b = 6.2382(12) Angstroms, c = 13.494(3) Angstroms, V = 797.8(3) Angstroms(3), and Z = 2. The infrared spectroscopy, EPR spectroscopy, and cyclic voltammetry on complex 3 are reported. The cyclic voltammetry shows an irreversible redox couple approximately -196 mV which likely corresponds to the Ti(IV)(2)/Ti(III)Ti(IV) couple. The EPR spectroscopy on solid complex 3 shows a typical S = 1 triplet-state spectrum. The solid follows non-Curie behavior, and the antiferromagnetic coupling between the two metal centers is determined to be -37.2 cm(-1). However, in solution the complex follows Curie behavior and supports a Ti(III)Ti(IV) oxidation state for the dimer.     

Hydrogen bonding and spin density distribution in the Qb semiquinone of bacterial reaction centers and comparison with the Qa site

In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (Q(A)) and secondary (Q(B)) electron acceptors are both ubiquinone-10, but with very different properties and functions. To investigate the protein environment that imparts these functional differences, we have applied X-band HYSCORE, a 2D pulsed EPR technique, to characterize the exchangeable protons around the semiquinone (SQ) in the Q(A) and Q(B) sites, using samples of (15)N-labeled reaction centers, with the native high spin Fe(2+) exchanged for diamagnetic Zn(2+), prepared in (1)H(2)O and (2)H(2)O solvent. The powder HYSCORE method is first validated against the orientation-selected Q-band ENDOR study of the Q(A) SQ by Flores et al. (Biophys. J.2007, 92, 671-682), with good agreement for two exchangeable protons with anisotropic hyperfine tensor components, T, both in the range 4.6-5.4 MHz. HYSCORE was then applied to the Q(B) SQ where we found proton lines corresponding to T ≈ 5.2, 3.7 MHz and T ≈ 1.9 MHz. Density functional-based quantum mechanics/molecular mechanics (QM/MM) calculations, employing a model of the Q(B) site, were used to assign the observed couplings to specific hydrogen bonding interactions with the Q(B) SQ. These calculations allow us to assign the T = 5.2 MHz proton to the His-L190 N(δ)H···O(4) (carbonyl) hydrogen bonding interaction. The T = 3.7 MHz spectral feature most likely results from hydrogen bonding interactions of O1 (carbonyl) with both Gly-L225 peptide NH and Ser-L223 hydroxyl OH, which possess calculated couplings very close to this value. The smaller 1.9 MHz coupling is assigned to a weakly bound peptide NH proton of Ile-L224. The calculations performed with this structural model of the Q(B) site show less asymmetric distribution of unpaired spin density over the SQ than seen for the Q(A) site, consistent with available experimental data for (13)C and (17)O carbonyl hyperfine couplings. The implications of these interactions for Q(B) function and comparisons with the Q(A) site are discussed.     

HYSCORE spectroscopy in the cytochrome b(559) of the photosystem II reaction center

A HYSCORE investigation of the heme center in the cytochrome b(559) is presented. To assign the observed signals to specific nuclei, bis-imidazol coordinated heme compounds that model the iron environment in cytochrome b(559) are also studied. In the model compounds selective isotopic substitution of nitrogen atoms has been performed. The HYSCORE spectra allow us to obtain the hyperfine and quadrupolar coupling tensors of heme and imidazol bonding nitrogen atoms. The results can be interpreted in terms of the structure and the electronic distribution of the active center. The hyperfine tensors indicate that the unpaired electron is confined in a nonbonding iron orbital with a negligible nitrogen p orbital contribution. Quadrupolar coupling tensors suggest that the orientation of the semioccupied orbital is driven by the orientation of the two parallel imidazol rings of the axial histidine side chains. The results are discussed in terms of the structure-function relationship of cytochromes.     

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

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