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This work was supported by a grant to the American Association for the Advancement of Science from the John D. and Catherine T. MacArthur Foundation, awarded to BNT. The structure was determined in the Molecular Structure Laboratory of the Department of Chemistry as a special class project in Chemistry 517.

Gas-phase photoelectron spectroscopy and density functional theory have been utilized to investigate the interactions between the p orbitals of dithiolate ligands and d orbitals of titanium in bent titanocene complexes as minimum molecular models of active site features of pyranopterin Mo/W enzymes. The compounds Cp(2)Ti(S-S) [where (S-S) is 1,2-ethenedithiolate (S(2)C(2)H(2)), 1, 1,2-benzenedithiolate (bdt), 2, or 1,3-propanedithiolate (pdt), 3, and Cp(-) is cyclopentadienyl] provide access to a formal 16-electron d(0) electronic configuration at the metal. A "dithiolate-folding-effect" involving an interaction of metal and sulfur orbitals is demonstrated in complexes with arene- and enedithiolates. This effect is not observed for the alkanedithiolate in complex 3.

The electronic structure of cis,trans-(L-N(2)S(2))MoO(X) (where L-N(2)S(2) = N,N'-dimethyl-N,N'-bis(2-mercaptophenyl)ethylenediamine and X = Cl, SCH(2)C(6)H(5), SC(6)H(4)-OCH(3), or SC(6)H(4)CF(3)) has been probed by electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies to determine the nature of oxomolybdenum-thiolate bonding in complexes possessing three equatorial sulfur ligands. One of the phenyl mercaptide sulfur donors of the tetradentate L-N(2)S(2) chelating ligand, denoted S(180), coordinates to molybdenum in the equatorial plane such that the OMo-S(180)-C(phenyl) dihedral angle is approximately 180 degrees, resulting in a highly covalent pi-bonding interaction between an S(180) p orbital and the molybdenum d(xy) orbital. This highly covalent bonding scheme is the origin of an intense low-energy S --Mo d(xy) bonding-to-antibonding LMCT transition (E(max) approximately 16000 cm(-)(1), epsilon approximately 4000 M(-)(1) cm(-)(1)). Spectroscopically calibrated bonding calculations performed at the DFT level of theory reveal that S(180) contributes approximately 22% to the HOMO, which is predominantly a pi antibonding molecular orbital between Mo d(xy) and the S(180) p orbital oriented in the same plane. The second sulfur donor of the L-N(2)S(2) ligand is essentially nonbonding with Mo d(xy) due to an OMo-S-C(phenyl) dihedral angle of approximately 90 degrees. Because the formal Mo d(xy) orbital is the electroactive or redox orbital, these Mo d(xy)-S 3p interactions are important with respect to defining key covalency contributions to the reduction potential in monooxomolybdenum thiolates, including the one- and two-electron reduced forms of sulfite oxidase. Interestingly, the highly covalent Mo-S(180) pi bonding interaction observed in these complexes is analogous to the well-known Cu-S(Cys) pi bond in type 1 blue copper proteins, which display electronic absorption and resonance Raman spectra that are remarkably similar to these monooxomolybdenum thiolate complexes. Finally, the presence of a covalent Mo-S pi interaction oriented orthogonal to the MOO bond is discussed with respect to electron-transfer regeneration in sulfite oxidase and Mo=S(sulfido) bonding in xanthine oxidase.

Sulfite dehydrogenase (SDH) from Starkeya novella, a sulfite-oxidizing molybdenum-containing enzyme, has a novel tightly bound alphabeta-heterodimeric structure in which the Mo cofactor and the c-type heme are located on different subunits. Flash photolysis studies of intramolecular electron transfer (IET) in SDH show that the process is first-order, independent of solution viscosity, and not inhibited by sulfate, which strongly indicates that IET in SDH proceeds directly through the protein medium and does not involve substantial movement of the two subunits relative to each other. The IET results for SDH contrast with those for chicken and human sulfite oxidase (SO) in which the molybdenum domain is linked to a b-type heme domain through a flexible loop, and IET shows a remarkable dependence on sulfate concentration and viscosity that has been ascribed to interdomain docking. The results for SDH provide additional support for the interdomain docking hypothesis in animal SO and clearly demonstrate that dependence of IET on viscosity and sulfate is not an inherent property of all sulfite-oxidizing molybdenum enzymes.

Gas-phase photoelectron spectroscopy and density functional theory have been used to investigate the interactions between the sulfur π-orbitals of arene dithiolates and high-valent transition metals as minimum molecular models of the active site features of pyranopterin Mo/W enzymes. The compounds (Tp*)MoO(bdt) (compound 1), Cp 2 Mo(bdt) (compound 2), and Cp 2 Ti(bdt) (compound 3) [where Tp* is hydrotris(3,5-dimethyl-1-pyrazolyl)borate, bdt is 1,2-benzenedithiolate, and Cp is η 5 - cyclopentadienyl] provide access to three different electronic configurations of the metal, formally d 1 , d 2 , and d 0 , respectively. The gas-phase photoelectron spectra show that ionizations from occupied metal and sulfur based valence orbitals are more clearly observed in compounds 2 and 3 than in compound 1. The observed ionization energies and characters compare very well with those calculated by density functional theory. A “dithiolate-folding-effect” involving an interaction of the metal in-plane and sulfur-π orbitals is proposed to be a factor in the electron transfer reactions that regenerate the active sites of molybdenum and tungsten enzymes.

Reaction of VO(acac)(2) with 1,2-dithiols in the presence of triethylamine gives pentacoordinate oxovanadium complexes [HNEt(3)](2)[VO(bdt)(2)] (1), [HNEt(3)](2)[VO(tdt)(2)] (2), and [HNEt(3)](2)[VO(bdtCl(2))(2)] (3) (where H(2)bdt = 1,2-benzenedithiol, H(2)tdt = 3,4-toluenedithiol, and H(2)bdtCl(2) = 3,6-dichloro-1,2-benzenedithiol). Compounds 1-3 have been characterized by IR, UV/visible, EPR, and mass spectroscopies. The X-ray crystal stuctures of 1 and 2 show hydrogen-bonding interactions between the terminal oxo atom and triethylammonium counterions and between ligand sulfur atoms and the counterions. These interactions are comparable with those found at the active sites of mononuclear molybdenum enzymes.

Pulsed electron nuclear double resonance (ENDOR) spectra of nonexchangeable protons in the vicinity of the Mo(V) center of the high pH (hpH) and low pH (lpH) forms of native chicken liver sulfite oxidase (SO) and recombinant human SO have been obtained and analyzed for the first time. The close similarity of the spectra for the chicken and human enzymes indicates that the structures of their molybdenum centers are essentially identical. For lpH SO, the closest nonexchangeable proton is found to be approximately 2.8 A from the Mo atom. To more accurately determine the distance to this proton and facilitate its assignment, the C-band electron spin-echo envelope modulation (ESEEM) spectra of lpH SO were also analyzed. From the obtained distance and comparison with the X-ray structure, this closest nonexchangeable proton is assigned to the alpha-proton of the coordinated conserved cysteinyl residue (Cys185 in chicken, Cys207 in human). The closest Mo...H distance for the nonexchangeable protons of hpH SO is found to be approximately 3.3 A. For the cysteinyl alpha-proton, such an increase in the Mo...H distance only requires a very small change in torsional angles. This study demonstrates that details of the enzyme structural rearrangements with pH can be monitored by ENDOR spectroscopy and suggests that a similar approach may be routinely used to probe the orientation of the coordinated cysteinyl residue in mutant forms of SO that are catalytically compromised.

Complexes of the form (Tp * )MoOCl(p-OC 6 H 4 X) and (Tp * )MoO(p-OC 6 H 4 X) 2 (Tp * = hydrotris(3,5-dimethyl-1-pyrazolyl)-borate and X = OEt, OMe, Et, Me, H, F, Cl, Br, I, and CN) were examined by electrochemical techniques and gas-phase photoelectron spectroscopy to probe the effect of the remote substituent (X) on electron-transfer reactions at the oxomolybdenum core. Cyclic voltammetry revealed that all of these neutral Mo(V) compounds undergo a quasireversible one-electron oxidation (Mo V I /Mo V ) and a quasireversible one-electron reduction (Mo V /Mo I V ) at potentials that linearly depend on the electronic influence (Hammett σ p parameter) of X. The first ionization energies for (Tp * )MoO(p-OC 6 H 4 X) 2 (X = OEt, OMe, H, F, and CN) were determined by photoelectron spectroscopy. A nearly linear correlation was found for the Mo V I /Mo V oxidation potentials in solution and the gas-phase ionization energies. Calculated heterogeneous electron-transfer rate constants show a slight systematic dependence on the substituent.

Our previous studies have shown that the rate constant for intramolecular electron transfer (IET) between the heme and molybdenum centers of chicken liver sulfite oxidase varies from approximately 20 to 1400 s(-1) depending upon reaction conditions [Pacheco, A., Hazzard, J. T., Tollin, G., and Enemark, J. H. (1999) J. Biol. Inorg. Chem. 4, 390-401]. These two centers are linked by a flexible polypeptide loop, suggesting that conformational changes, which alter the Mo-Fe distance, may play an important role in the observed IET rates. In this study, we have investigated IET in sulfite oxidase using laser flash photolysis as a function of solution viscosity. The solution viscosity was varied over the range of 1.0-2.0 cP by addition of either polyethylene glycol 400 or sucrose. In the presence of either viscosogen, an appreciable decrease in the IET rate constant value is observed with an increase in the solvent viscosity. The IET rate constant exhibits a linear dependence on the negative 0.7th power of the viscosity. Steady-state kinetics and EPR experiments are consistent with the interpretation that viscosity, and not other properties of the added viscosogens, is responsible for the dependence of IET rates on the solvent composition. The results are consistent with the role of conformational changes on IET in sulfite oxidase, which helps to clarify the inconsistency between the large rate constant for IET between the Mo and Fe centers and the long distance (approximately 32 A) between these two metal centers observed in the crystal structure [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983].

Transient resonance Raman, fluorescence emission, and transient absorption spectroscopies show that photoinduced electron transfer occurs in the covalently linked porphyrin−Mo dyads ZnTTP(2,3-MoVO(pzb)) and ZnTTP(3,4-MoVO(pzb)). These compounds represent first generation models for observing real-time electron transfer dynamics in pterin-containing molybdenum enzymes. The results indicate that deactivation of the S1 state of the porphyrin occurs solely by electron transfer to Mo(V), with a quantum efficiency for the forward electron transfer process approaching unity.

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.

Magnetic circular dichroism (MCD) and absorption spectroscopies have been used to probe the electronic structure of [PPh4][MoO(p-SC6H4X)4] (X = H, Cl, OMe) and [PPh4][MoO(edt)2] complexes (edt = ethane-1,2-dithiolate). The results of density functional calculations (DFT) on [MoO(SMe)4]- and [MoO(edt)2]- model complexes were used to provide a framework for the interpretation of the spectra. Our analysis shows that the lowest energy transitions in [MoVOS4] chromophores (S4 = sulfur donor ligand) result from S-->Mo charge transfer transitions from S valence orbitals that lie close to the ligand field manifold. The energies of these transitions are strongly dependent on the orientation of the S lone-pair orbitals with respect to the Mo atom that is determined by the geometry of the ligand backbone. Thus, the lowest energy transition in the MCD spectrum of [PPh4][MoO(p-SC6H4X)4] (X = H) occurs at 14,800 cm-1, while that in [PPh4][MoO(edt)2] occurs at 11,900 cm-1. The identification of three bands in the absorption spectrum of [PPh4][MoO(edt)2] arising from LMCT from S pseudo-sigma combinations to the singly occupied Mo 4d orbital in the xy plane suggests that there is considerable covalency in the ground-state electronic structures of [MoOS4] complexes. DFT calculations on [MoO(SMe)4]- reveal that the singly occupied HOMO is 53% Mo 4dxy and 35% S p for the equilibrium C4 geometry. For [MoO(edt)2]- the steric constraints imposed by the edt ligands result in the S pi orbitals being of similar energy to the Mo 4d manifold. Significant S pseudo-sigma and pi donation may also weaken the Mo identical to O bond in [MoOS4] centers, a requirement for facile active site regeneration in the catalytic cycle of the DMSO reductases. The strong dependence of the energies of the bands in the absorption and MCD spectra of [PPh4][MoO(p-SC6H4X)4] (X = H, Cl, OMe) and [PPh4][MoO(edt)2] on the ligand geometry suggests that the structural features of the active sites of the DMSO reductases may result in an electronic structure that is optimized for facile oxygen atom transfer.

Temperature-dependent magnetic circular dichroism (MCD) spectroscopy has been used for the first time to probe the electronic structure of the Mo active site in sulfite oxidase (SO). The enzyme was poised in the catalytically relevant [Mo(V):Fe(II)] state by anaerobic reduction of the enzyme with the natural substrate, sulfite, in the absence of the physiological oxidant cytochrome c. The [Mo(V):Fe(II)] state is of particular importance, as it is proposed to be a catalytic intermediate in the oxidative half reaction, where SO is reoxidized to the resting [Mo(VI):Fe(III)] state by two sequential one-electron transfers to cytochrome c. The MCD spectrum of the enzyme shows no charge transfer transitions below approximately 17000 cm(-1). This has been interpreted to result from (1) a severe reduction in ene-1,2-dithiolate sulfur in-plane and out-of-plane p orbital mixing, (2) a decrease in the dithiolate sulfur out-of-plane p-Mo d(xy) orbital overlap, and (3) an orthogonal orientation between the vertical cysteine sulfur p (perpendicular to the Mo-Scys sigma-bond) and Mo d(xy) orbitals. The spectroscopically determined cysteine sulfur p-Mo d(xy) bonding scheme in the [Mo(V):Fe(II)] state is consistent with the crystallographically determined O-Mo-Scys-C dihedral angle of approximately 90 degrees and precludes a covalent interaction between the vertical cysteine sulfur p orbital and Mo d(xy), effectively decoupling the cysteine from an effective through-bond electron transfer pathway. We have tentatively assigned a 22250 cm(-1) positive C-term feature in the MCD as the cysteine S(sigma)-->Mo d(xy) charge transfer that becomes allowed by a combination of configuration interaction and low-symmetry; however, the orbital overlap is anticipated to be quite small due to the near orthogonality of these orbitals. Therefore, we propose that the primary role of the coordinated cysteine is to decrease the effective nuclear charge on Mo by charge donation to the metal, statically poising the active site at more negative reduction potentials during electron transfer (ET) regeneration. Finally, the results of this study are consistent with the pyranopterin ene-1,2-dithiolate acting to couple the Mo site into efficient superexchange pathways for ET regeneration following oxygen atom transfer to the substrate.

X-ray absorption spectra have been measured at the S K-, Cl K-, and Mo L-3- and L-2-edges for the d(0) dioxomolybdenum(VI) complexes LMoO2(SCH2Ph), LMoO2Cl, and LMoO2(OPh) (L = hydrotris(3,5-dimethyl-1-pyrazolyl)borate) to investigate ligand-metal covalency and its effects on oxo transfer reactivity. Two dominant peaks are observed at the S K-edge (2470.5 and 2472.5 eV) for LMoO2(SCH2Ph) and at the Cl K-edge (2821.9 and 2824.2 eV) for LMoO2Cl, demonstrating two major covalent contributions from S and Cl to the Mo d orbitals. Density functional calculations were performed on models of the three Mo complexes, and the energies and characters of the Mo 4d orbitals were interpreted in terms of the effects of two strong cis-oxo bonds and additional perturbations due to the thiolate, chloride, or alkoxide ligand. The major perturbation effects are for thiolate and Cl- pi mixed with the d(xz) orbital and sigma mixed with the d(z)(2) orbital. The calculated 4d orbital energy splittings for models of these two major contributions to the bonding of thiolate and Cl ligands (2.47 and 2.71 eV, respectively) correspond to the splittings observed experimentally for the two dominant ligand K-edge peaks for LMoO2(SCH2Ph) and LMoO2Cl (2.0 and 2.3 eV, respectively) after consideration of final state electronic relaxation. Quantification of the S and Cl covalencies in the d orbital manifold from the pre-edge intensity yields, similar to 42% and similar to 17% for LMoO2(SCH2Ph) and LMoO2Cl respectively. The Mo L-2-edge spectra provide a direct probe of metal 4d character for the three Mo complexes. The spectra contain a strong, broad peak and two additional sharp peaks at higher energy, which are assigned to 2p transitions to the overlapping t(2g) set and to the d(z)(2) and d(xy) levels, respectively. The total peak intensities of the Mo L-2-edges for LMoO2(OPh) and LMoO2Cl are similar to and larger than those for LMoO2(SCH2Ph), which agrees with the calculated trend in ligand-metal covalency. The theoretical and experimental description of bonding developed from these studies provides insight into the relationship of electronic structure to the oxo transfer chemistry observed for the LMoO2X complexes. These results imply that anisotropic covalency of the Mo-S-cys bond in sulfite oxidase may promote preferential transfer of one of the oxo groups during catalysis.

Adducts with MoO 4 2− tetrahedra coordinated to Cr(III) or Co(III) complexes have been synthesized and studied by IR and high resolution 95 Mo NMR spectroscopy. The 95 Mo chemical shifts of the adducts with cobalt(III) lie in the range −33.2 to + 49.4 ppm. This may be compared with an overall known chemical shift range in excess of 7000 ppm and implies a similarity in the molybdenum environment in all cases. For adducts with chelated cobalt(III) complexes several rather broad 95 Mo singnals are obtained with linewidths up to 260 Hz.

The compounds {5,10,15-tri-p-tolyl-20-[[2,3-[((hydrotris(3,5-dimethylpyrazolyl)borato)oxomolybdenio)dioxy]phenyl]porphyrinato}bis(2-methylimidazole)iron(III) chloride, Fe(2,3-Mo-TTP)(2MeImH)(2)Cl (1), and {5,10,15-tri-p-tolyl-20-[3,4-[((hydrotris(3,5-dimethylpyrazolyl)borato)oxomolybdenio)dioxy]phenyl]porphyrinato}bis(2-methylimidazole)iron(III) chloride, Fe(3,4-Mo-TTP)(2MeImH)(2)Cl) (2), have been prepared in order to assess the effect of axial ligand plane orientation upon the stability, reduction potential, and NMR and EPR spectra of these novel (porphryinato)iron(III)-Mo(V) systems that possess two S = (1)/(2) metal centers. The proton NMR spectra of 1 and 2 are characteristic of perpendicular orientation of the planes of the axial 2MeImH ligands. These results contrast with those previously reported (Basu, P.; Shokirev, N. V.; Enemark, J. H.; Walker, F. A. J. Am. Chem. Soc. 1995, 117, 9042-9055) for the analogous compounds with NMeIm as the axial base (3, 4) whose (1)H NMR spectra are characteristic of one or both axial ligands in parallel planes. The equilibrium constants (beta(2)) for binding the bulky 2MeImH ligands of 1 and 2 are more than an order of magnitude smaller than those for NMeIm binding to 3 and 4. Three distinct pseudo-Nernstian one-electron couples are observed for 1 and 2 in DMF that can be assigned to the Fe(III/II), Mo(V/IV), and Fe(II/I) reductions, with the Fe(III/II) couple being most positive. The Fe(III/II) and Mo(V/IV) potentials are similar to those for 3 and 4 and only slightly perturbed from those of the individual isolated components. The EPR spectrum of 1 shows features due to Mo(V) and low-spin Fe(III) that are perturbed by weak exchange coupling (2.6 GHz, 0.078 cm(-)(1)) between the two metal centers which are separated by approximately 7.9 Å. The "large g(max)" feature characteristic of the 2MeImH adducts of Fe(III) tetraphenylporphyrinates is shifted toward the Mo(V) signal to 2.85; the anisotropy of the Mo(V) signal is lost, and no molybdenum hyperfine can be detected. The EPR spectrum of 2, which has a metal-metal separation of approximately 9.4 Å, shows an unperturbed "large g(max)" value of 3.41 for the Fe(III) center. The Mo(V) part of the spectrum is slightly perturbed from that of the precursor catecholate complex but is essentially identical to that of 4, which exhibits a rhombic Fe(III) signal.

A series of six-coordinate compounds containing a chelating dithiolate coordinated to the [LMo(V)O](2+) unit (L = hydrotris(3,5-dimethyl-1-pyrazolyl)borate) have been characterized by EPR spectroscopy as models for the molybdenum centers of pterin-containing molybdenum enzymes. The structure of LMoO(bdt) (1) (bdt = 1,2-benzenedithiolate) has been determined by X-ray crystallography; the space group is P2(1)/n with a = 10.727(1) Å, b = 14.673(2) Å, c = 15.887(2) Å, beta = 100.317(4) degrees and Z = 4. Compound 1 exhibits distorted octahedral stereochemistry; the terminal oxo group and the sulfur atoms are mutually cis to one another. The Mo=O distance is 1.678(4) Å, and the average Mo-S distance is 2.373(2) Å. The EPR parameters for 1, determined from simulation of the frozen-solution spectrum, are g(1) = 2.004, g(2) = 1.972, g(3) = 1.934 and A(1)((95,97)Mo) = 50.0 x 10(-)(4), A(2) = 11.4 x 10(-)(4), A(3) = 49.7 x 10(-)(4) cm(-)(1). The EPR parameters for several LMo(V)O{S(CH(2))(x)()S} compounds (x = 2-4) with saturated chelate skeletons are similar to those of 1, indicating that it is the coordinated S atoms and not unsaturation of the chelate skeleton that gives rise to the large g values for 1. The presence of g components larger than the free-electron value is ascribed to low-energy charge transfer transitions from the filled sulfur pi orbitals to half-filled Mo d orbitals. The EPR spectrum of [LMo(V)O{S(2)P(OEt)(2)}](+) shows an unusually large isotropic (31)P hyperfine splitting of 66.1 x 10(-)(4) cm(-)(1) from the noncoordinated phosphorus atom. The frozen-solution EPR spectra of the low-pH and high-pH forms of sulfite oxidase have been reinvestigated in D(2)O and the anisotropic g and A((95,97)Mo) parameters determined by simulation of the spectrum arising from the naturally abundant Mo isotopes (75% I = 0, 25% I = (5)/(2)). The EPR parameters for the low-pH form are g(1) = 2.007, g(2) = 1.974, g(3) = 1.968 and A(1) = 56.7 x 10(-)(4), A(2) = 25.0 x 10(-)(4), A(3) = 16.7 x 10(-)(4) cm(-)(1). The EPR parameters for the high-pH form are g(1) = 1.990, g(2) = 1.966, g(3) = 1.954 and A(1) = 54.4 x 10(-)(4), A(2) = 21.0 x 10(-)(4), A(3) = 11.3 x 10(-)(4) cm(-)(1). These are the first determinations of the complete A((95,97)Mo) hyperfine components for an enzyme that possesses an [Mo(VI)O(2)](2+) core in its fully oxidized state.

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.

Density functional calculations on geometry-optimized oxidized (Mo(VI)) and reduced (Mo(IV)) analogues of the isolated active site of aldehyde oxidase (MOP), a member of the xanthine oxidase family of pyranopterindithiolate enzymes, show that fold angle changes of the dithiolate ligand modulate the relative metal and dithiolate contributions to the frontier redox orbitals. Proton abstraction from the equatorial aqua ligand of the oxidized Mo(VI) site also flattens the metal dithiolate fold angle. It is proposed that static and/or dynamic changes in the structure of the protein surrounding the active site can induce changes in the dithiolate fold angle and thereby provide a mechanism for electronic buffering of the redox orbital, for fine-tuning the nucleophilicity of the equatorial aqua/hydroxide ligand, and for modulating the electron-transfer regeneration of the active sites of molybdenum and tungsten enzymes via a "dithiolate folding effect".

Intramolecular electron transfer (ET) rates in sulfite oxidase (SO) were measured using flavin semiquinone reductants [5-deazariboflavin (dRFH.) and lumiflavin (LFH.)] generated by laser flash photolysis. Rapid bimolecular reduction of the heme by the dRF semiquinone occurred (k = 4 x 10(8) M-1 s-1 at pH 6; 1 x 10(8) M-1 s-1 at pH 9), followed by heme Fe(II) reoxidation due to intramolecular electron transfer to Mo(VI). Flash-induced difference spectra indicated that only spectral processes due to reduction and oxidation of the b-type heme prosthetic group were observed, with no detectable spectral contribution from the Mo cofactor. The extent of reoxidation decreased greatly from pH 6 to 9 (50% to 3%), as expected from the shifts in the redox potentials of the heme and Mo cofactor with pH, consistent with an electron transfer equilibrium between the two redox centers. The observed rate constant for the Fe(II) to Mo(VI) electron transfer decreased from 1650 s-1 at pH 6 to 60 s-1 at pH 9 and showed a maximum of 2400 s-1 at pH 7. Increases in salt concentration greatly decreased intramolecular ET rate constants (direct reduction by flavin semiquinone was unchanged), due to the binding of anions. Titration with the sodium salts of Cl-, SO4(2-), and H2PO4-/HPO4(2-) resulted in decreases in rate constants of intramolecular ET from 1500 s-1 to < 100 s-1 at pH 6 and 7. Similar dissociation constants were measured for the binding of these anions by flash photolysis and by steady-state enzyme kinetics using the inhibition of the sulfite/cytochrome c assay reaction for sulfite oxidase. A mechanism is proposed in which anion binding to the enzyme inhibits the rate of intramolecular electron transfer.

Publisher Summary This chapter presents a discussion on the bioinorganic chemistry of pterin-containing molybdenum and tungsten enzymes. The chapter focuses on the current results and the interplay of model and enzyme chemistry. Attention is directed to sulfite oxidase and xanthine oxidase, the archetypal examples of molybdenum enzymes, containing, respectively, dioxo-Mo(VI) and oxo-thio-Mo(VI) oxidized centers. Biochemical and model studies of molybdopterin, Mo-co, and related species are described in this chapter. A brief survey on the physical and spectroscopic techniques employed in the study of the enzymes is presented and their impact on the current understanding of the coordination about the molybdenum atom in sulfite oxidase and xanthine oxidase is discussed. Structural and spectroscopic models are also presented in the chapter. The chapter also explains the xanthine oxidase cycle and facets of intramolecular electron transfer in molybdenum enzymes. The pterin-containing tungsten enzymes and the evolving model chemistry are discussed. Current descriptions of the coordination environment of the molybdenum centers of the enzymes rest primarily upon the comparisons of the spectra of the enzymes with the spectra of well-characterized molybdenum complexes. The unexpected discovery that several hyperthermophilic organisms possess pterin-containing tungsten enzymes promises to stimulate the development of oxo-thio-tungsten chemistry.

Sulfite oxidase (SO) is a molybdenum-cofactor-dependent enzyme that catalyzes the oxidation of sulfite to sulfate, the final step in the catabolism of the sulfur-containing amino acids, cysteine and methionine. The catalytic mechanism of vertebrate SO involves intramolecular electron transfer (IET) from molybdenum to the integral b-type heme of SO and then to exogenous cytochrome c. However, the crystal structure of chicken sulfite oxidase (CSO) has shown that there is a 32 Å distance between the Fe and Mo atoms of the respective heme and molybdenum domains, which are connected by a flexible polypeptide tether. This distance is too long to be consistent with the measured IET rates. Previous studies have shown that IET is viscosity dependent (Feng et al., Biochemistry, 2002, 41, 5816) and also dependent upon the flexibility and length of the tether (Johnson-Winters et al., Biochemistry, 2010, 49, 1290). Since IET in CSO is more rapid than in human sulfite oxidase (HSO) (Feng et al., Biochemistry, 2003, 42, 12235) the tether sequence of HSO has been mutated into that of CSO, and the resultant chimeric HSO enzyme investigated by laser flash photolysis and steady-state kinetics in order to study the specificity of the tether sequence of SO on the kinetic properties. Surprisingly, the IET kinetics of the chimeric HSO protein with the CSO tether sequence are slower than wildtype HSO. This observation raises the possibility that the composition of the non-conserved tether sequence of animal SOs may be optimized for individual species.

[Mo(C15H22BN6)(C6H4ClO)2O], M(r) = 664.24, triclinic, P1, a = 10.585 (2), b = 12.068 (3), c = 13.728 (3) A, alpha = 88.01, beta = 69.94, gamma = 65.78 degrees, V = 1490.8 A3, Z = 2, Dx = 1.48 g cm-3, Mo K alpha, lambda = 0.71073 A, mu = 6.5 cm-1, F(000) = 678, T = 296 (1) K, R = 0.034, wR = 0.049 for 4634 observed independent reflections with F2 greater than 3 sigma (F2). This is the second structurally characterized mononuclear monooxo transition-metal complex containing the phenolato ligand. The molecule exhibits a distorted octahedral coordination geometry, and the Mo atom is ligated by a terminal O atom, two p-chloro-substituted phenolato groups, and a tridentate facially coordinated hydrotris(3,5-dimethylpyrazolyl)borate ligand (L).

The electron spin echo envelope modulation (ESEEM) investigation of the high-pH (hpH) form of sulfite oxidase (SO) and sulfite dehydrogenase (SDH) prepared in buffer enriched with H(2)(17)O reveals the presence of three types of exchangeable oxygen atoms at the molybdenum center. Two of these oxygen atoms belong to the equatorial OH ligand and the axial oxo ligand, and are characterized by (17)O hyperfine interaction (hfi) constants of about 37 MHz and 6 MHz, respectively. The third oxygen has an isotropic hfi constant of 3-4 MHz and likely belongs to a hydroxyl moiety hydrogen-bonded to the equatorial OH ligand. This exchangeable oxygen atom is not observed in the ESEEM spectra of the Y236F mutant of SDH, where the active site tyrosine has been replaced by phenylalanine.

A series of mononuclear (diolato)- or (dithiolato) oxomolybdenum(V) complexes, LMoO[E-(CH 2 ) n -E] (L=hydrotris(3,5-dimethyl-1-pyrazolyl) borate; E=O, S; n=2-4), and bis(alkoxo) complexes, LMoO(OR) 2 (R=Me, Et, n Pr), have been synthesized and characterized by elemental analysis, mass spectrometry, electron paramagnetic resonance spectroscopy (EPR), cyclic voltammetry, and IR and electronic absorption spectroscopy

He I gas-phase photoelectron spectra of LMo(E)(OR)2 [(L = hydrotris(3,5-dimethylpyrazolyl)borate), (E = O, NO)] complexes show that the nitrosyl complexes (formally Mo(II)) are 0.8 eV more difficult to ionize than the oxo analogues (formally Mo(V)). This counterintuitive result is ascribed to the differing π-interactions of the nitrosyl and oxo ligands.

The crystal structure of [SnLCl3] has been determined and shown to consist of discrete distorted fac-octahedral molecules [L = tris(3,5-dimethylpyrazolyl)hydroborate]. The single-crystal EPR spectra, at room temperature and Q-band frequencies, of [MoE(L)Cl2] and [MoO(L)(NCS)2], E = O or S, diluted in the structurally similar [SnLCl3] have been recorded. The data from the different possible diluent sites in [SnLCl3] are consistent with exact, or nearly exact, monoclinic EPR symmetry for each of the molybdenum compounds which involves a rotation of the g and A tensors about an axis perpendicular to the mirror plane in each molecule. The angles of rotation are in the range 30–38° and are in good agreement with those obtained via simulation of the frozen-solution X-band EPR spectra of the compounds. The relationships between the g and A tensors, the molecular geometries, and the electronic structures are discussed.

The reaction of (NEt4)2[MoNCl5] and K{HB(Me2pz)3}[HB(Me2pz)3- = tris (3,5- dimethylpyrazol-1-yl) hydroborate anion] produces both purple, diamagnetic {HB(Me2pz)3}MoNCl2 (1) and yellow, paramagnetic Net4 [{HB(Me2pz)3}MoNCl2] (2) (e.s.r.: {g} 1.961, {a}(95Mo) 57×10-4 cm-1), which have been separately isolated under different conditions. In contrast, the reaction of [ MoN (N3)4]- with Na{HB(Me2pz)3} results in the exclusive formation of red {HB(Me2pz)3} MoN (N3)2 (3). Crystals of (3) are monoclinic and belong to space group P21/n with a 16.440(2), b 8.787(2), c 16.754(2) Ǻ,β 112.47(1)°, V 2236.4 Ǻ3, and Z 4. The structure was solved by Patterson and Fourier methods, followed by least-squares refinement, using 3208 reflections, to a conventional R value of 0.032 ( Rw 0.047). In the distorted octahedral complex the molybdenum(VI) atom is coordinated by a facial HB(Me2pz)3- ligand , a terminal nitrido ligand with a Mo-N bond distance of 1.646(4)Ǻ, and two azide ligands. All complexes have been characterized by elemental analysis, infrared, 1H n.m.r. or e.s.r. spectroscopy, and mass spectrometry. Improved syntheses for the starting materials (NEt4)2 [MoNCl5] and (NEt4)2 [MoNCl4] are also reported.

Cristallisation dans le groupe P2 1 2 1 2 1 avec a=7,830, b=16,202 et c=5,861 A, Z=2. Affinement jusqu'a R=0,038. Les molecules ont la symetrie cristallographique C 2 et possedent une configuration octaedrique deformee. Spectres RMN et de dichroisme circulaire

The crystal and molecular structures of the two magnetically distinct forms of Fe(NO)(salen), C 16 H 14 N 3 O 3 Fe, have been determined by single crystal X-ray diffraction at 23 °C and at −175 °C. The complex crystallizes in the orthorhombic space group Pna2 1 with 4 molecules per unit cell. The unit cell dimensions are a = 26.377(2), b = 8.598(5) and c = 6.951(5) A at 23 °C and a = 26.18(2), b = 8.52(1) and c = 6.783(6) A at −175 °C. Full-matrix least-squares refinement of the 23 °C structure based on the 840 reflections with F 2 o ⩾ 3σ(F 2 o ) gave a conventional R-factor of 0.038. The structure consists of discrete Fe(NO)(salen) molecules with tetragonal pyramidal coordination about the iron atom. The disordered nitrosyl group occupies the axial coordination site in a bent geometry (average FeNO angle 147°). Full-matrix least-squares refinement of the −175°C structure based on the 406 reflections with F 2 o ⩾ 2σ(F 2 o ) gave a conventional R-factor of 0.087. This form of Fe(NO)(salen) also exhibits tetragonal pyramidal coordination geometry with a bent disordered nitrosyl group in the apical position. Marginally significant structural changes are observed: 1) the FeNO angle is 127°; 2) the Fe atom is 0.1 A closer to the mean coordination plane of the salen ligand are more nearly coplanar. These structural changes are consistent with a spin state of Fe(NO)(salen) from S = 3 2 to S = 1 2 upon cooling. The significant differences in th conformations of the salen ligand at the two temperatures are attributed to crystal packing. The infrared spectra of Fe(NO)(salen) at room temperature for various pressures ranging from ambient pressure up to 37 kbar are reported. The spectra suggest that Fe(NO)(salen) is converted to the S = 1 2 state at high pressure.

The methylation reaction of (C 5 Me 5 ) 2 Mo 2 (μ-S 2 )(μ-S) 2 (Ia) leads to two isomers of composition (C 5 Me 5 ) 2 Mo 2 (SMe) 2 S 2 (IV and IVb) which were separated by column chromatography. As an intermediate product of this reaction the iodine-containing adduct (C 5 Me 5 ) 2 Mo 2 S 4 I 2 is formed. All compounds were investigated by means of 1 H and 95 Mo NMR spectroscopy as well as by an X-ray structure analysis in the case of IVa. Thus, the SCH 3 ligands in IV are found to be arranged in a trans position. As a further result a cleavage of the η 2 -disulfur bridge, originally present in Ia, has been found to occur in the second step of the reaction sequence.

The compounds (HB(Me{sub 2}pz){sub 3})MoO(S{sub 2}P(OEt){sub 2}) (1a) and (HB(Me{sub 2}pz){sub 3})MoO{sub 2}(S{sub 2}P(OEt){sub 2}(2)) containing bidentate and monodentate S{sub 2}P (OEt){sub 2}-ligands, respectively, have been prepared by the reaction of Mo{sub 2}O{sub 3}(S{sub 2}P(OEt){sub 2}){sub 4} with K(HB(Me{sub 2}pz){sub 3}). (HB(Me{sub 2}pz){sub 3})MoO(S{sub 2}P(OEt){sub 2}) and its analog (HB(Me{sub 2}pz){sub 3})MoO(S{sub 2}P(OMe){sub 2}) have also been prepared in higher yield by the reaction of MoO{sub 2}O{sub 3}(S{sub 2}(OEt){sub 2}){sub 4} with Ph{sub 3}P followed by the reaction with K(HB(Me{sub 2}pz){sub 3}). Both 1a and 2 have been structurally characterized by x-ray crystallography, and the crystallographic data are presented. The molecular structures were also determined. Compound 1a reduces Me{sub 2}SO to Me{sub 2}S. The kinetic data and activation parameters are presented for this reaction. Compound 2 oxidizes Ph{sub 3}P to Ph{sub 3}PO. Both complexes catalyze the oxidation of Ph{sub 3}P by Me{sub 2}SO. 34 references, 4 figures, 5 tables.