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

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

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.     

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.     

Analogues for the molybdenum center of sulfite oxidase: oxomolybdenum(V) complexes with three thiolate sulfur donor atoms

cis,trans-(L-N2S2)Mo(V)O(SR) [L-N2S2H2 = N,N'-dimethyl-N,N'-bis(mercaptophenyl)ethylenediamine; R = CH2Ph, CH2CH3, and p-C6H4-Y (Y = CF3, Cl, Br, F, H, CH3, CH2CH3, and OCH3)] are the first structurally characterized mononuclear Mo compounds with three thiolate donors, as occurs at the Mo active site in sulfite oxidase. X-ray crystal structures of the cis,trans-(L-N2S2)Mo(V)O(SR) compounds, where R = CH2Ph, CH2CH3, p-C6H4-OCH3, and p-C6H4-CF3, show a similar coordination geometry about the Mo atom with all three sulfur thiolate donors in the equatorial plane. This coordination geometry places two adjacent S ppi orbitals parallel to the Mo=O bond, analogous to the orientation in the ene-dithiolate ligand in sulfite oxidase; the third S ppi orbital lies in the equatorial plane. Charge-transfer transitions from the S p to the Mo d orbitals occur at approximately 28,000 cm(-1) (epsilon: 4,400-6,900 L mol(-1)] cm(-1)) and 15,500 cm(-1) (epsilon: 3,200-4,900 L mol(-1) cm(-1)). The EPR parameters are nearly identical for all the cis,trans-(L-N2S2)Mo(V)O(SR) compounds (g1 approximately 2.022, g2 approximately 1.963, g3 approximately 1.956, Al approximately 58.4 x 10(-4) cm(-1), A2 approximately 23.7 x 10(-4) cm(-1), A3 approximately 22.3 x 10(-4) cm(-1)) and are typical of an oxo-Mo(V) center coordinated by multiple thiolate donors. The g and A tensors are related by a 24 degrees rotation about the coincident g2 and A2 tensor elements, reflecting the approximate Cs coordination symmetry. These EPR parameters more closely mimic those of the low pH form of sulfite oxidase and the "very rapid" species of xanthine oxidase than previous model compounds with two or four thiolate donors. The cis,trans-(L-N2S2)Mo(V)O(SR) compounds undergo a quasi-reversible, one-electron reduction and an irreversible oxidation that show a linear dependence upon the Hammett parameter, sigmap, of the Y group. The cis,trans-(L-N2S2)Mo(V)O(SR) compounds provide a well-defined platform for the systematic investigation of the electronic structures of the Mo(V)OS3 centers and their implications for molybdoenzymes.     

Coordination chemistry at the molybdenum site of sulfite oxidase: redox-induced structural changes in the cysteine 207 to serine mutant

The redox chemistry of the molybdenum site of the C207S mutant of recombinant human sulfite oxidase has been studied via potentiometric titrations employing both electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS) as probes of the active site structure. In earlier EXAFS studies, oxidized Cys207Ser enzyme has been shown to possess a novel tri-oxo active site, in which Ser207 does not appear to be a ligand to Mo [George, G. N.; Garrett, R. M.; Prince, R. C.; Rajagopalan, K. V. J. Am. Chem. Soc. 1996, 118, 8588-8592]. Redox titrations show that the active site is modified under reducing conditions to a mono-oxo Mo(IV) species, probably with Ser207 ligated to the metal. The Mo(IV) species can be reoxidized to a mono-oxo Mo(V) species still coordinated to Ser207, which in turn can be further reoxidized to yield the initial tri-oxo Mo(VI) structure with loss of Ser207 ligation.     

Structure of the molybdenum site in YedY, a sulfite oxidase homologue from Escherichia coli

YedY from Escherichia coli is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a Mo(IV) model complex. The K-edge energy was indicative of Mo(V) in YedY, in agreement with X- and Q-band electron paramagnetic resonance results, whereas the hSO protein contained Mo(VI). In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of ～2.4 ?), and one (axial) oxo ligand (Mo═O, ～1.7 ?). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (～2.1 ?) or possibly to a shorter Mo-O(-) bond. Yet another oxygen (or nitrogen) at a ～2.6 ? distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a ～2.6 ? distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism.     

Pulsed EPR studies of a bacterial sulfite-oxidizing enzyme with pH-invariant hyperfine interactions from exchangeable protons

Variable-frequency pulsed electron paramagnetic resonance studies of the molybdenum(V) center of sulfite dehydrogenase (SDH) clearly show couplings from nearby exchangeable protons that are assigned to a Mo(V)OH(n) group. The hyperfine parameters for these exchangeable protons of SDH are the same at both low and high pH and similar to those for the high-pH forms of sulfite oxidases (SOs) from eukaryotes. The SDH proton parameters are distinctly different from the low-pH forms of chicken and human SO.     

Nature of halide binding to the molybdenum site of sulfite oxidase

Valuable information on the active sites of molybdenum enzymes has been provided from both Mo(V) electron paramagnetic resonance (EPR) spectroscopy and X-ray absorption spectroscopy (XAS). One of three major categories of Mo(V) EPR signals from the molybdenum enzyme sulfite oxidase is the low-pH signal, which forms in the presence of chloride. Two alternative structures for this species have been proposed, one in which the chloride is coordinated directly to Mo and a second in which chloride is held in the arginine-rich basic pocket some 5 ? from Mo. Here we present an independent assessment of the structure of this species by using XAS of the analogous bromide and iodide complexes. We show that there is no evidence of direct Mo-I coordination, and that the data are consistent with a structure in which the halide is bound at ～5 ? from Mo.     

MoV electron paramagnetic resonance of sulfite oxidase revisited: the low-pH chloride signal

Valuable information on the active sites of molybdenum enzymes has been provided by Mo(V) electron paramagnetic resonance (EPR) spectroscopy. In recent years, multiple resonance techniques have been extensively used to examine details of the active-site structure, but basic continuous-wave (CW) EPR has not been re-evaluated in several decades. Here, we present a re-examination of the CW EPR spectroscopy of the sulfite oxidase low-pH chloride species and provide evidence for direct coordination of molybdenum by chloride.     

Monodithiolene molybdenum(V, VI) complexes: a structural analogue of the oxidized active site of the sulfite oxidase enzyme family

The active sites of the xanthine oxidase and sulfite oxidase enzyme families contain one pterin-dithiolene cofactor ligand bound to a molybdenum atom. Consequently, monodithiolene molybdenum complexes have been sought by exploratory synthesis for structural and reactivity studies. Reaction of [MoO(S(2)C(2)Me(2))(2)](1-) or [MoO(bdt)(2)](1-) with PhSeCl results in removal of one dithiolate ligand and formation of [MoOCl(2)(S(2)C(2)Me(2))](1-) (1) or [MoOCl(2)(bdt)](1-) (2), which undergoes ligand substitution reactions to form other monodithiolene complexes [MoO(2-AdS)(2)(S(2)C(2)Me(2))](1-) (3), [MoO(SR)(2)(bdt)](1-) (R = 2-Ad (4), 2,4,6-Pr(i)(3)C(6)H(2) (5)), and [MoOCl(SC(6)H(2)-2,4,6-Pr(i)(3))(bdt)](1-) (6) (Ad = 2-adamantyl, bdt = benzene-1,2-dithiolate). These complexes have square pyramidal structures with apical oxo ligands, exhibit rhombic EPR spectra, and 3-5 are electrochemically reducible to Mo(IV)O species. Complexes 1-6 constitute the first examples of five-coordinate monodithiolene Mo(V)O complexes; 6 approaches the proposed structure of the high-pH form of sulfite oxidase. Treatment of [MoO(2)(OSiPh(3))(2)] with Li(2)(bdt) in THF affords [MoO(2)(OSiPh(3))(bdt)](1-) (8). Reaction of 8 with 2,4,6-Pr(i)(3)C(6)H(2)SH in acetonitrile gives [MoO(2)(SC(6)H(2)-2,4,6-Pr(i)(3))(bdt)](1-) (9, 55%). Complexes 8 and 9 are square pyramidal with apical and basal oxo ligands. With one dithiolene and one thiolate ligand of a square pyramidal Mo(VI)O(2)S(3) coordination unit, 9 closely resembles the oxidized sites in sulfite oxidase and assimilatory nitrate reductase as deduced from crystallography (sulfite oxidase) and Mo EXAFS. The complex is the first structural analogue of the active sites in fully oxidized members of the sulfite oxidase family. This work provides a starting point for the development of both structural and reactivity analogues of members of this family.     

Molybdenum site structure of Escherichia coli YedY, a novel bacterial oxidoreductase

We report a structural characterization using X-ray absorption spectroscopy of the molybdenum site of Escherichia coli YedY, a novel oxidoreductase related to be the sulfite oxidase family of molybdenum enzymes. We find that the enzyme can exist in Mo(V) and Mo(IV) oxidation states but cannot be readily oxidized to the Mo(VI) form. Mo(V) YedY has molybdenum coordination similar to that of sulfite oxidase, with one Mo═O at 1.71 ?, three Mo-S at 2.39 ?, and one Mo-OH at 2.09 ?, which elongates to 2.20 ? upon reduction to Mo(IV), indicating Mo-OH(2) coordination. The Mo(V) enzyme also possesses a long Mo-O coordination at 2.64 ?, which may be due to oxygen coordination by Asn-45 O(δ), with Mo-O(δ) approximately trans to the Mo═O group. A comparison with sulfite oxidase indicates that YedY possesses a much more uniform Mo-S coordination, with a maximum permitted deviation of less than 0.05 ?. Our results indicate that the YedY active site shows considerable similarity to but also important differences from that of reduced forms of sulfite oxidase.     

Synthetic, spectroscopic, and structural studies on organoimido molybdenum, tungsten, and rhenium phthalocyanines

Unprecedented imido phthalocyaninato complexes of pentavalent refractory metals [PcM(NR)Cl] (M = Mo, W, Re; R = tBu: 1, 3, 6, Mes: 2, 4, 7 or Ts: 5) have been synthesized by reductive cyclotetramerization of phthalonitrile in the presence of appropriate bis(imido) complexes of Mo, W and Re as templates. While d(1) Mo(V) and W(V) species 1-5 show distinctive EPR spectra corresponding to metal centered radicals with hyperfine coupling of two magnetically non-equivalent nitrogen atoms (4 equatorial and 1 axial N), corresponding d(2) Re(V) compounds 6 and 7 are diamagnetic. [PcMo(NtBu)Cl] 1 crystallizes from 1-chloronaphthalene in the tetragonal space group P4/n. The molecular structure reveals, that the metal center is located above the plane of the equatorial N4 and displaced towards the axial π-donor ligand. Due to the thermodynamic trans effect the Mo-Cl bond trans to the imido group is elongated to about 2.600(2) ?.     

Solvothermal Synthesis and Structure Studies of a Mixed-valence Oxochloromolybdenum Complex

Emerald green crystals of a new mixed-valence oxochloromolybdenum complex,Mo(OH)2(2,2′-bipy)Cl2.[MoO(2,2′-bipy)Cl3]2, were obtained in an attempt to synthesize the Mo-S cluster compounds under solvothermal conditions. The title compound was characterized by EPR and X-ray single-crystal diffraction techniques. Crystallographic data: orthorhombic, space group Fdd2, a = 12.684(1), b = 21.518(2), c = 29.096(3)A, Mr = 1103.97, V= 7941(1)A^3, Z= 8, Dc =1.843 g/cm^3,μ= 1.514 mm^-1, F(000) = 4304, R = 0.0654 and wR = 0.1544 for 1944 observed reflections with 1 ≥/ 2σ(I). The compound consists of two different neutral molecules, Mo^IV-(OH)2(2,2′-bipy)Cl2 and MoVO(2,2′-bipy)Cl3. Both Mo^IV and Mo^V ions are in a distorted octahedral environment. The Mo(1)^IV atom is surrounded by two Cl^- ions and two O atoms from OH^- and two N atoms from a 2,2′-bipy ligand. The Mo(2) atom is surrounded by three Cl^- ions, one O^2- anion and two N atoms from another 2,2“-bipy ligand. The bipyridine occupies two cis-equatorial sites, while the Cl^- ions are located at the axial and equatorial positions.     

Structural characterization of a high affinity mononuclear site in the copper(II)-α-synuclein complex

Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.     

Synthesis and EPR characterization of new models for the one-electron reduced molybdenum site of sulfite oxidase

Deconvoluting the different contributions of thiolate and ene-1,2-dithiolate donors to the underlying electronic structure of the Mo site in sulfite oxidase (SO) has proven to be a difficult task. One way in which these differences might be illuminated is by selectively substituting Se for S in model complexes which possess multiple sulfur donor ligand environments. Here we report the synthesis and structures of new oxo-Mo(V) complexes as effective models for the one-electron reduced active site of SO. We have used the tridentate heteroscorpionate ligand (2-dimethylethanethiol)bis(3,5-dimethylpyrazolyl)methane (L3SH) in order to model the constrained cysteinyl sulfur (S(Cys)) ligand environment observed in the crystal structure of the enzyme, and benzene-1,2-dithiol (bdt) as a mimic of the ene-1,2-dithiolate chelate. [(L3S)MoO(bdt)] and [(L3S)MoO(SPh)(2)] have been structurally characterized by X-ray crystallography, and as such, [(L3S)MoO(bdt)] is only the second known model compound that closely approximates the active site structure of reduced forms of SO. Additionally, benzenethiol (SPh) and benzeneselenol (SePh) have been used to perturb the equatorial ligand environment of [(L3S)MoO(bdt)].) This has provided much needed insight into the electronic structure of the one-electron reduced SO site and has allowed for increased understanding of the individual roles played by these different thiolate donors in the oxidative half-reaction of the enzyme. Interestingly, the EPR spectra of [(L3S)MoO(bdt)], [(L3S)MoO(SPh)(2)], and [(L3S)MoO(SePh)(2)] closely resemble that of both high pH (hpH) and low pH (lpH) SO, except for the fact that the magnitude of g(1) is found to be consistently higher in the model spectra compared to that of the enzyme. It is suggested that this derives from an increase in Mo-S covalency in the models relative to hpH and lpH SO.     

Polyoxomolybdenum(V/VI)-sulfite compounds: synthesis, structural, and physical studies

Reaction of Na(2)Mo(VI)O(4) x 2H(2)O with (NH(4))(2)SO(3) in the mixed-solvent system H(2)O/CH(3)CN (pH = 5) resulted in the formation of the tetranuclear cluster (NH(4))(4)[Mo(4)(VI)SO(16)] x H(2)O (1), while the same reaction in acidic aqueous solution (pH = 5) yielded (NH(4))(4)[Mo(5)(VI)S(2)O(21)] x 3H(2)O (2). Compound {(H(2)bipy)(2)[Mo(5)(VI)S(2)O(21)] x H(2)O}(x) (3) was obtained from the reaction of aqueous acidic solution of Na(2)Mo(VI)O(4) x 2H(2)O with (NH(4))(2)SO(3) (pH = 2.5) and 4,4'-bipyridine (4,4'-bipy). The mixed metal/sulfite species (NH(4))(7)[Co(III)(Mo(2)(V)O(4))(NH(3))(SO(3))(6)] x 4H(2)O (4) was synthesized by reacting Na(2)Mo(VI)O(4) x 2H(2)O with CoCl(2) x 6H(2)O and (NH(4))(2)SO(3) with precise control of pH (5.3) through a redox reaction. The X-ray crystal structures of compounds 1, 2, and 4 were determined. The structure of compound 1 consists of a ring of four alternately face- and edge-sharing Mo(VI)O(6) octahedra capped by the trigonal pyramidal sulfite anion, while at the base of the Mo(4) ring is an oxo group which is asymmetrically shared by all four molybdenum atoms. Compound 3 is based on the Strandberg-type heteropolyion [Mo(5)(VI)S(2)O(21)](4-), and these coordinatively saturated clusters are joined by diprotonated 4,4'-H(2)bipy(2+) through strong hydrogen bonds. Compound 3 crystallizes in the chiral space group C2. The structure of compound 4 consists of a novel trinuclear [Co(III)Mo(2)(V)SO(3)(2-)] cluster. The chiral compound 3 exhibits nonlinear optical (NLO) and photoluminescence properties. The assignment of the sulfite bands in the IR spectrum of 4 has been carried out by density functional calculations. The cobalt in 4 is a d(6) octahedral low-spin metal atom as it was evidenced by magnetic susceptibility measurements, cw EPR, BVS, and DFT calculations. The IR and solid-state UV-vis spectra as well as the thermogravimetric analyses of compounds 1-4 are also reported.     

DFT investigation of the molybdenum cofactor in periplasmic nitrate reductases: structure of the Mo(V) EPR-active species

The periplasmic nitrate reductase NAP belongs to the DMSO reductase family that regroups molybdoenzymes housing a bis-molybdopterin cofactor as the active site. Several forms of the Mo(V) state, an intermediate redox state in the catalytic cycle of the enzyme, have been evidenced by EPR spectroscopy under various conditions, but their structure and catalytic relevance are not fully understood. On the basis of structural data available from the literature, we built several models that reproduce the first coordination sphere of the molybdenum cofactor and used DFT methods to make magneto-structural correlations on EPR-detected species. "High-g" states, which are the most abundant Mo(V) species, are characterized by a low-anisotropy g tensor and a high g(min) value. We assign this signature to a six-sulfur coordination sphere in a pseudotrigonal prismatic geometry with a partial disulfide bond. The "very high-g" species is well described with a sulfido ion as the sixth ligand. The "low-g" signal can be successfully associated to a Mo(V) sulfite-oxidase-type active site with only one pterin moiety coordinated to the molybdenum ion with an oxo or sulfido axial ligand. For all these species we investigate their catalytic activity using a thermodynamic point of view on the molybdenum coordination sphere. Beyond the periplasmic nitrate reductase case, this work provides useful magneto-structural correlations to characterize EPR-detected species in mononuclear molybdoenzymes.     

Determination of the g-tensors and their orientations for cis,trans-(L-N2S2)Mo(V)OX (X = Cl, SCH2Ph) by single-crystal EPR spectroscopy and molecular orbital calculations

A single-crystal study of cis,trans-(L-N2S2)MoVOCl (1) doped into cis,trans-(N2S2)MoVIO2 (3) has enabled the g-tensor of 1 and its orientation with respect to the molecular structure to be determined. The EPR parameters (g1, 2.004; g2, 1.960; g3, 1.946; A1, 71.7 x 10(-4) cm(-1); A2, 11.7 x 10(-4) cm(-1); A3, 32.0 x 10(-4) cm(-1)) of cis,trans-(L-N2S2)MoVOCl [L-N2S2H2 = N,N'-dimethyl-N,N'-bis(mercaptophenyl)ethylenediamine] mimic those of the low-pH form of sulfite oxidase and the "very rapid" species of xanthine oxidase. The principal axis that corresponds to g1 is rotated approximately 10 degrees from the Mo[triple bond]O vector, while the principal axis that corresponds to g3 is located in the equatorial plane and approximately 38 degrees from the Mo-Cl vector. Independent theoretical calculations of the g-tensor of 1 were performed using two types of techniques: (1) the spectroscopically parametrized intermediate neglect of differential overlap technique (INDO/S) combined with single-excitation configuration interaction (CIS); (2) a scalar relativistic DFT (BP86 and B3LYP functionals) treatment using the zeroth order regular approximation to relativistic effects (ZORA) in combination with recently developed accurate multicenter mean field spin-orbit operators (RI-SOMF) and the estimation of solvent effects using dielectric continuum theory at the conductor-like screening model (COSMO) level. The excellent agreement between experiment and theory, as well as the high consistency between the INDO/S and BP86/ZORA results, provides a sound basis for analysis of the calculated orientation of the g-tensor for cis,trans-(L-N2S2)MoVO(SCH2Ph) (2), for which single-crystal EPR data are not available but which contains three equatorial sulfur donor atoms, as occurs in sulfite oxidase and xanthine oxidase. The implications of these results for the EPR spectra of the Mo(V) centers of enzymes are discussed.     

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.