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Sulfite-oxidizing enzymes from eukaryotes and prokaryotes have five-coordinate distorted square-pyramidal coordination about the molybdenum atom. The paramagnetic Mo(v) state is easily generated, and over the years four distinct CW EPR spectra have been identified, depending upon enzyme source and the reaction conditions, namely high and low pH (hpH and lpH), phosphate inhibited (Pi) and sulfite (or blocked). Extensive studies of these paramagnetic forms of sulfite-oxidizing enzymes using variable frequency pulsed electron spin echo (ESE) spectroscopy, isotopic labeling and density functional theory (DFT) calculations have led to the consensus structures that are described here. Errors in some of the previously proposed structures are corrected.

Sulfite-oxidizing enzymes (SOEs) are molybdenum enzymes that exist in almost all forms of life where they carry out important functions in protecting cells and organisms against sulfite-induced damage. Due to their nearly ubiquitous presence in living cells, these enzymes can be assumed to be evolutionarily ancient, and this is reflected in the fact that the basic domain architecture and fold structure of all sulfite-oxidizing enzymes studied so far are similar. The Mo centers of all SOEs have five-coordinate square pyramidal coordination geometry, which incorporates a pyranopterin dithiolene cofactor. However, significant differences exist in the quaternary structure of the enzymes, as well as in the kinetic properties and the nature of the electron acceptors used. In addition, some SOEs also contain an integral heme group that participates in the overall catalytic cycle. Catalytic turnover involves the paramagnetic Mo(V) oxidation state, and EPR spectroscopy, especially high-resolution pulsed EPR spectroscopy, provides detailed information about the molecular and electronic structure of the Mo center and the Mo-based sulfite oxidation reaction.

Protein film voltammetry of chicken liver sulfite oxidase (SO) bound at the pyrolytic graphite "edge" or modified gold electrodes shows that catalytic electron transport is controlled by the inherent electrochemical characteristics of the heme b domain and conformational changes that allow intramolecular electron transfer with the molybdenum active site. In the absence of sulfite, a single nonturnover electrochemical signal is observed at +90 mV (vs SHE) that is assigned to heme b. In the presence of sulfite, this signal transforms into a catalytic wave at similar potential. The shape and negligible pH dependence of this wave indicate that catalytic turnover is controlled by the one-electron transfers through heme b. The smaller turnover numbers obtained in this experiment (k(cat) approximately 2-4 s(-1), as compared to 100 s(-1) in solution) suggest that only a small fraction of SO is bound at the electrode in a manner that permits the conformational change necessary for fast interdomain electron transfer.

In our previous study of the fatal R160Q mutant of human sulfite oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc.2008, 130, 8471-8480), a new Mo(V) species, denoted "species 1", was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and (33)S and (17)O labeling. Here we report new results for species 1 of R160Q, based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in K(a)- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial sulfite as the only exchangeable ligand. The two types of (17)O signals that are observed arise from the coordinated and remote oxygen atoms of the sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the observed and calculated EPR parameters.

Sulfite oxidase (SO) is a molybdoheme enzyme that is important in sulfur catabolism, and mutations in the active site region are known to cause SO deficiency disorder in humans. This investigation probes the effects that mutating aromatic residues (Y273, W338, and H337) in the molybdenum-containing domain of human SO have on both the intramolecular electron transfer (IET) rate between the molybdenum and iron centers using laser flash photolysis and on catalytic turnover via steady-state kinetic analysis. The W338 and H337 mutants show large decreases in their IET rate constants (k (ET)) relative to the wild-type values, suggesting the importance of these residues for rapid IET. In contrast, these mutants are catalytically competent and exhibit higher k (cat) values than their corresponding k (ET), implying that these two processes involve different conformational states of the protein. Redox potential investigations using spectroelectrochemistry revealed that these aromatic residues close to the molybdenum center affect the potential of the presumably distant heme center in the resting state (as shown by the crystal structure of chicken SO), suggesting that the heme may be interacting with these residues during IET and/or catalytic turnover. These combined results suggest that in solution human SO may adopt different conformations for IET and for catalysis in the presence of the substrate. For IET the H337/W338 surface residues may serve as an alternative-docking site for the heme domain. The similarities between the mutant and wild-type EPR spectra indicate that the active site geometry around the Mo(V) center is not changed by the mutations studied here.

The electronic interactions between metals and dithiolenes are important in the biological processes of many metalloenzymes as well as in diverse chemical and material applications. Of special note is the ability of the dithiolene ligand to support metal centers in multiple coordination environments and oxidation states. To better understand the nature of metal-dithiolene electronic interactions, new capabilities in gas-phase core photoelectron spectroscopy for molecules with high sublimation temperatures have been developed and applied to a series of molecules of the type Cp(2)M(bdt) (Cp = η(5)-cyclopentadienyl, M = Ti, V, Mo, and bdt = benzenedithiolato). Comparison of the gas-phase core and valence ionization energy shifts provides a unique quantitative energy measure of valence orbital overlap interactions between the metal and the sulfur orbitals that is separated from the effects of charge redistribution. The results explain the large amount of sulfur character in the redox-active orbitals and the 'leveling' of oxidation state energies in metal-dithiolene systems. The experimentally determined orbital interaction energies reveal a previously unidentified overlap interaction of the predominantly sulfur HOMO of the bdt ligand with filled π orbitals of the Cp ligands, suggesting that direct dithiolene interactions with other ligands bound to the metal could be significant for other metal-dithiolene systems in chemistry and biology.

The Mo(V) state of the molybdoenzyme sulfite oxidase (SO) is paramagnetic and can be studied by electron paramagnetic resonance (EPR) spectroscopy. Vertebrate SO at pH 9 exhibits characteristic EPR spectra that correspond to two structurally different forms of the Mo(V) active center termed the low-pH (lpH) and high-pH (hpH) forms, respectively. Both EPR forms have an exchangeable equatorial OH ligand, but its orientation in the two forms is different. It has been hypothesized that the formation of the lpH species is dependent on the presence of chloride. In this work, we have prepared and purified samples of the wild type and various mutants of human SO that are depleted of chloride. These samples do not exhibit the typical lpH EPR spectrum at low pH but rather exhibit spectra that are characteristic of the blocked species that contains an exchangeable equatorial sulfate ligand. Addition of chloride to these samples results in the disappearance of the blocked species and the formation of the lpH ...

The tris(pyrazolyl)borate and related tripodal N-donor ligands originally developed by Trofimenko stabilize mononuclear compounds containing Mo(VI)O(2), Mo(VI)O, Mo(V)O, and Mo(IV)O units and effectively inhibit their polynucleation in organic solvents. Dioxo-Mo(VI) complexes of the type LMoO(2)(SPh), where L = hydrotris(3,5-dimethylpyrazol-1-yl)borate (Tp*), hydrotris(3-isopropylpyrazol-1-yl)borate (Tp(i) (Pr)), and hydrotris(3,5-dimethyl-1,2,4-triazol-1-yl)borate (Tz) and related derivatives are the only model systems that mimic the complete reaction sequence of sulfite oxidase, in which oxygen from water is ultimately incorporated into product. The quasi-reversible, one-electron reduction of Tp*MoO(2)(SPh) in acetonitrile exhibits a positive potential shift upon addition of a hydroxylic proton donor, and the magnitude of the shift correlates with the acidity of the proton donor. These reductions produce two Mo(V) species, [Tp*Mo(V)O(2)(SPh)](-) and Tp*Mo(V)O(OH)(SPh), that are related by protonation. Measurement of the relative amounts of these two Mo(V) species by EPR spectroscopy enabled the pK(a) of the Mo(V)(OH) unit in acetonitrile to be determined and showed it to be several pK(a) units smaller than that for water in acetonitrile. Similar electrochemical-EPR experiments for Tp(i) (Pr)MoO(2)(SPh) indicated that the pK(a) for its Mo(V)(OH) unit was ∼1.7 units smaller than that for Tp*Mo(V)O(OH)(SPh). Density functional theory calculations also predict a smaller pK(a) for (iPr)Mo(V)O(OH)(SPh) compared to Tp*Mo(V)O(OH)(SPh). Analysis of these results indicates that coupled electron-proton transfer (CEPT) is thermodynamically favored over the indirect process of metal reduction followed by protonation. The crystal structure of Tp(i) (Pr)MoO(2)(SPh) is also presented.

Molecules of the general form Tp*MoO(OR)(2) [where Tp* = hydrotris(3,5-dimethyl-1-pyrazolyl)borate and (OR)(2) = (OMe)(2), (OEt)(2), and (O(n)Pr)(2) for alkoxide ligands and (OR)(2) = O(CH(2))(3)O, O(CH(2))(4)O, and O[CH(CH(3))CH(2)CH(CH(3))]O for diolato ligands] were studied using gas-phase photoelectron spectroscopy, cyclic voltammetry, and density functional theory (DFT) calculations to examine the effect of increasing ligand size and structure on the oxomolybdenum core. Oxidation potentials and first ionization energies are shown to be sensitive to the character of the diolato and alkoxide ligands. A linear correlation between the solution-phase oxidation potentials and the gas-phase ionization energies resulted in an unexpected slope of greater than unity. DFT calculations indicated that this unique example of a system in which oxidation potentials are more sensitive to substitution than vertical ionization energies is due to the large differences in the cation reorganization energies, which range from 0.2 eV or less for the molecules with diolato ligands to around 0.5 eV for the molecules with alkoxide ligands.

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 pHor = 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.

The Cp(2)VCl(2) molecule is a prototype for bent metallocene complexes with a single electron in the metal d shell, but experimental measure of the binding energy of the d electron by photoelectron spectroscopy eluded early attempts due to apparent decomposition in the spectrometer to Cp(2)VCl. With improved instrumentation, the amount of decomposition is reduced and subtraction of ionization intensity due to Cp(2)VCl from the Cp(2)VCl(2)/Cp(2)VCl mixed spectrum yields the Cp(2)VCl(2) spectrum exclusively. The measured ionization energies provide well-defined benchmarks for electronic structure calculations. Density functional calculations support the spectral interpretations and agree well with the ionization energy of the d(1) electron and the energies of the higher positive ion states of Cp(2)VCl(2). The calculations also account well for the trends to the other Group V bent metallocene dichlorides Cp(2)NbCl(2) and Cp(2)TaCl(2). The first ionization energy of Cp(2)VCl(2) is considerably greater than the first ionization energies of the second- and third-row transition metal analogues.

The Mo(V) forms of the Tyr343Phe (Y343F) mutant of human sulfite oxidase (SO) have been investigated by continuous wave (CW) and variable frequency pulsed EPR spectroscopies as a function of pH. The CW EPR spectrum recorded at low pH (∼6.9) has g-values similar to those known for the low-pH form of the native vertebrate SO (original lpH form); however, unlike the spectrum of original lpH SO, it does not show any hyperfine splittings from a nearby exchangeable proton. The detailed electron spin echo (ESE) envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) experiments also did not reveal any nearby protons that could belong to an exchangeable ligand at the molybdenum center. These results suggest that under low-pH conditions the active site of Y343F SO is in the "blocked" form, with the Mo(V) center coordinated by sulfate. With increasing pH the EPR signal from the "blocked" form decreases, while a signal similar to that of the original lpH form appears and becomes the dominant signal at pH9. In addition, both the CW EPR and ESE-detected field sweep spectra reveal a considerable contribution from a signal similar to that usually detected for the high-pH form of native vertebrate SO (original hpH form). The nearby exchangeable protons in both of the component forms observed at high pH were studied by the ESEEM spectroscopy. These results indicate that the Y343F mutation increases the apparent pK(a) of the transition from the lpH to hpH forms by ∼2 pH units.

Two oxomolybdenum(V) complexes, (dttd)MoOCl and [(bdt)MoOCl 2 ] − (where dttd = 2,3:8,9-dibenzo-1,4,7,10-tetrathiadecane and bdt = 1,2-benzenedithiolate), which contain one or two equatorial chloro ligands, respectively, were studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the microwave K a -band (∼29 GHz). The ESEEM amplitude from the chloro ligands in both compounds is significantly greater than that tentatively attributed to chloride in the vicinity of the oxomolybdenum active site in the high chloride, low-pH ( lpH ) form of sulfite oxidase (SO). Thus, these ESEEM results rule out equatorial coordination of chloride in the enzyme, although the possibility for a weakly bound chloride in the trans axial position or nearby non-coordinated chloride(s) remains for lpH SO in solution.

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...

The electronic structures of the potassium salts of the homoscorpionates hydrotris(3,5-dimethylpyrazol-1-yl)borate (Tp*, 1), hydrotris(4-chloro-3,5-dimethylpyrazol-1-yl)borate (Tp*Cl, 2) and hydrotris(3,5-bis(trifluoromethyl)pyrazol-1-yl)borate (Tp(CF3)2, 3) are compared using gas-phase photoelectron spectroscopy and density functional theory (DFT). DFT calculations also are reported for the generic scorpionate potassium (hydrotris(pyrazol-1-yl)borate) (KTp). This is the first such experimental probe of the electronic structure of halogen containing scorpionate ligands and subtle differences in the ionizations from the frontier orbitals in the photoelectron spectra of 1 and 3 are observed that give insight into the influence of substituents upon metal–scorpionate bonding. Distinct assignments of the ionizations from the nitrogen σ-donor orbitals (σN) and σBH molecular orbitals are possible experimentally by the use of variable (He I and He II) excitation energies. The experimentally observed first ionization energy of 3 is stabilized by ∼2.0 eV relative to 1 due to the strong electron withdrawing effect of the trifluoromethyl groups. The photoelectron spectroscopic studies of NaTp(CF3)2 further confirm the assignments of ionizations from σN orbitals for 3 associated with the a and e sets in C3 symmetry. The X-ray crystal structure of 2 as the (μ-aqua)3(potassium hydrotris(4-chloro-3,5-dimethylpyrazol-1-yl)borate)2 dimer is also reported.

Electron spin echo envelope modulation (ESEEM) spectroscopy has been used to determine the hyperfine (hfi) and quadrupole (nqi) interactions of the exchangeable deuteron (proton) at the Mo(V) site of DMSO reductase. The data obtained have been translated into structure-related parameters. It was found that isotropic hfi constant of the proton is not unique, but is distributed within a range of 26–36 MHz. From this hfi distribution, a 30°-wide distribution of the OH bond orientations due to a rotation around the Mo-O bond was estimated. The angle between the axes of the nqi and anisotropic hfi tensors was found to be anomalously small in comparison with that expected from the Mo-O-D bond geometry. This peculiarity was attributed to the effect of spin density on the hydroxyl oxygen atom. The orientation of the Mo-OH fragment with respect to the g-frame was determined from the experimental orientations of the nqi and hfi tensor axes and a theoretical evaluation of the anisotropic hfi axis direction. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0393-8. On that page (frame on the left side), a link takes you directly to the supplementary material.

Solution redox potentials and heterogeneous electron transfer rate constants have been measured for mono-ene-1,2-dithiolate complexes of the type (Tp*)M(E)(S ∩ S) [Tp*=hydrotris(3,5-dimethyl-1-pyrazolyl)borate; M=Mo, W; E=O, NO]. The dithiolate ligands (S ∩ S) equatorially coordinated to the central metal include: 1,2-benzenedithiolate (bdt); 3,6-dichloro-1,2-benzenedithiolate (bdtCl 2 ); and 3,4-toluenedithiolate (tdt). Cyclic voltammograms reveal quasi-reversible one-electron reductions for all of the compounds; and, a one-electron quasi-reversible oxidation process is also observed for (Tp*)MoO(bdt), (Tp*)MoO(tdt), and (Tp*)WO(tdt). Electrochemical potentials are strongly dependent upon the nature of the substituents on the benzene ring of the dithiolate ligand. The nitrosyl–molybdenum complexes (E=NO) are more difficult to reduce than their corresponding oxo-molybdenum complexes. The tungsten compound, (Tp*)WO(tdt), has the most negative electrochemical potentials among the complexes investigated. Heterogeneous electron transfer rates are insensitive to the variations of the central metal and ligands. These results support a highly covalent bonding interaction between the metal and the dithiolate ligands that modulates electron transfer reactions within these compounds.

The synthesis and molecular structures of the (Tp*)Mo(NO)(SS) family of compounds (where (Tp*) is hydrotris(3,5-dimethyl-1-pyrazolyl)borate; (SS) is an ene-1,2-dithiolate) are reported. The compounds; (Tp*)Mo(NO)(bdt) (1), (Tp*)Mo(NO)(tdt) (2), and (Tp*)Mo(NO)(bdtCl2) (3) (bdt, 1,2-benzenedithiolate; tdt, 3,4-toluenedithiolate; bdtCl2, 3,6-dichlorobenzenedithiolate), are the first such structurally characterized {MoNO}4 compounds that contain a [(Tp*)Mo(NO)]2+ fragment ligated to an ene-1,2-dithiolate. The compounds crystallize in the space groups P21/c (1) (a=11.115(2) A, b=13.667(3) A, c=16.410(3) A; β=98.36(3)°, Volume=2466.4(8) A3, Z=4); P21/n (2) (a=15.579(3) A, b=9.942(2) A, c=16.527(3) A; β=95.79(3)°, Volume=2546.7(9) A3, Z=4); and Pnma (3) (a=17.9879(9) A, b=13.2277(7) A, c=11.2505(6) A; Volume=2676.9(2) A3, Z=4). The molecular structures show that the inner coordination sphere remains invariant within this family. A remarkable feature is the fold angle (θ) between the MoS2 plane and S2C2 plane of the ene-1, 2-dithiolate chelate ring. The fold angles (θ) of 42.1° in 1, 41.1° in 2 and 44.4° in 3 are substantially larger than in analogous compounds with a terminal oxo group. Additional insight into the chemistry and properties of complexes 1–3 has been obtained by cyclic voltammetry, IR, NMR, electronic absorption and He I photoelectron (PES) spectroscopies.

The titration of chicken liver sulfite oxidase (SO) with the one-electron reductant Ti(III) citrate, at pH 7.0, results in nearly quantitative selective reduction of the Mo(VI) center to Mo(V), while the b-type heme center remains in the fully oxidized Fe(III) state. The selective reduction of the Mo(VI/V) couple has been established from electronic and EPR spectra. The elec- tronic spectrum of the Fe(III) heme center is essentially unchanged during the titration, and the continuous wave (CW)-EPR spectrum shows the appearance of the well-known Mo(V) signal due to the low pH (lpH) form of SO. Further confirmation of the selective formation of the Mo(V)/Fe(III) form of SO is provided by the � 1:1 ratio of the integrated intensities of the Mo(V) and low- spin Fe(III) EPR signals after addition of one equivalent of Ti(III). The selective generation of the Mo(V)/Fe(III) form of SO is unexpected, considering that previous microcoulometry and flash photolysis investigations have indicated that the Mo(VI/V) and Fe(III/II) couples of SO have similar reduction potentials at pH 7. The nearly quantitative preparation of the one-electron re- duced Mo(V)/Fe(III) form of SO by reduction with Ti(III) has enabled the interaction between these two paramagnetic metalcenters, which are l inked by a flex- ible loop with no secondary structure, to be investigated for the first time by variable-frequency pulsed electron- electron double resonance (ELDOR) spectroscopy. The ELDOR kinetics were obtained from frozen solutions at 4.2 K at severalmicrowave frequencies by pumping on the narrow Mo(V) signaland observing the effect on the Fe(III) primary echo at both higher and lower fre- quencies within the microwave C-band region. The ELDOR data indicate that freezing the solution of one- electron reduced SO produces localized regions where the concentration of SO approaches that in the crystal structure, which results in the interpair interactions be- ing the dominant dipolar interaction. However, thor- ough analysis of the ELDOR decay curves and simulations suggests a distribution of intramolecular Mo ... Fe distances, consistent with the proposalof mul - tiple conformations in solution for the flexible loop that connects the Mo and heme domains of SO.

The oxomolybdenum mono-ene-1,2-dithiolate complex (Tp*)MoO(bdtCl2) (3) has been synthesized and characterized (Tp* is hydrotris(3,5-dimethyl-1-pyrazolyl)borate; bdtCl2 is 3,6-dichloro-1,2-benzenedithiolate). The X-ray structural data show that 3 crystallizes in the monoclinic space group, P21/c, where a=7.963 (3), b=26.272 (11), c=14.016 (6) A, β=105.352 (7). The (Tp*)MoO(bdtCl2) molecule exhibits a distorted pseudo-octahedral coordination geometry, with the Mo atom ligated by a terminal oxo atom, two sulfur donor atoms of the bdtCl2 ligand and three nitrogen atoms of the tridentate facially coordinated Tp* ligand. The coordination environment about the Mo atom is similar to that of (Tp*)MoO(bdt) (1) (bdt is 1,2-benzenedithiolate), but the fold angle between the MoS2 plane and S2C2 plane of the bdtCl2 ligand (θ=6.9°) is substantially smaller than the feature in 1 (θ=21.3°). The similar IR, EPR, and electronic absorption spectroscopic results for 1 and 3 indicate that the electron withdrawing nature of the chlorine substituents of 3 does not significantly perturb the electronic structure of the Mo(V) center. However, the solution redox potentials and the gas-phase ionization energies are sensitive to remote substituent effects.

Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d(1)) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant (32)S has no nuclear spin (I = 0). Here we describe direct incorporation of (33)S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from (33)S-labeled MPT in this catalytically active SO variant are dominated by the "interdoublet" transition arising from the strong nuclear quadrupole interaction, as also occurs for the (33)S-labeled exchangeable equatorial sulfite ligand [ Klein, E. L., et al. Inorg. Chem. 2012 , 51 , 1408 - 1418 ]. The estimated experimental hfi and nqi parameters for (33)S (aiso = 3 MHz and e(2)Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two (33)S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).

High-resolution pulsed EPR techniques are used to detect the benzylthiolate α protons and deuterons of cis,trans-(L-N2S2)MoVO(SCH2Ph) and cis,trans-(L-N2S2)MoVO(SCD2Ph), which mimic the β-CH2 protons of the coordinated cysteine residue at the Mo center of sulfite oxidase.

A refocused primary electron spin−echo envelope modulation (RP ESEEM) technique and an adjustable frequency S/C-band pulsed EPR spectrometer have been used to produce ESEEM spectra with the lines due to nearby protons being greatly enhanced relative to those due to distant matrix protons. Application of this technique to the high pH (hpH) form of the Mo(V) center of sulfite oxidase has enabled nearby protons to be directly detected for the first time. Simulation of the RP ESEEM spectrum of the hpH form suggests the presence of two nearby protons that have distributed hyperfine interactions (hfi); these protons are ascribed to a MoV−OH group with strong H-bonding interactions to other nearby proton donors or to the presence of a coordinated H2O ligand. The RP ESEEM technique promises to be widely applicable to the investigation of mutant forms of SO with altered Mo centers and paramagnetic centers in other metalloproteins where a nearby proton of interest is often masked by much more numerous distant proto...

The individual rate constants for intramolecular electron transfer (IET) between the Mo(VI)Fe(II) and Mo(V)Fe(III) forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high and low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO4(2-) and Cl-, which suggests that specific anion binding at the sterically constrained Mo active site is necessary for IET inhibition to occur.IET may require that SO adopt a conformation in which the Mo and Fe centers are held in close proximity by electrostatic interactions between the predominantly positively charged Mo active site, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disfavor IET. The rate constant for IET from Fe(II) to Mo(VI) decreases with increasing pH, both in the presence and absence of 50 mM SO4(2-) . However, the rate constant for the reverse process exhibits no significant pH dependence in the absence of SO4(2-), and increases with pH in the presence of 50 mM S04(2-). This behavior is consistent with a mechanism in which IET from Mo(V) to Fe(III) is coupled to proton transfer from Mo(V)-OH to OH-, and the reverse IET process is coupled to proton transfer from H2O to Mo(VI) = O. At high concentrations of small anions, direct access of H2O or OH- to the Mo-OH will be blocked, which provides a second possible mechanism for inhibition of IET by such anions. Inhibition by anions is not strictly competitive, however, and Tyr322 may play an important intermediary role in transferring the proton when an anion blocks direct access of H2O or OH- to the Mo-OH. Competing H-bonding interactions of the Mo-OH moiety with Tyr322 and with the anion occupying the active site may also be responsible for the well-known equilibrium between two EPR-distinct forms of SO that is observed for the two-electron reduced enzyme.

Solution and solid state electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies have been used to probe in detail the excited state electronic structure of LMoO(bdt) and LMoO(tdt) (L = hydrotris(3,5-dimethyl-1-pyrazolyl)borate; bdt = 1,2-benzenedithiolate; tdt = 3,4-toluenedithiolate). The observed energies, intensities, and MCD band patterns are found to be characteristic of LMoO(S-S) compounds, where (S-S) is a dithiolate ligand which forms a five-membered chelate ring with Mo. Ab initio calculations on the 1,2-ene-dithiolate ligand fragment, -SCCS-, show that the low-energy S → Mo charge transfer transitions result from one-electron promotions originating from an isolated set of four filled dithiolate orbitals that are primarily sulfur in character. Resonance Raman excitation profiles have allowed for the definitive assignment of the ene-dithiolate Sin-plane → Mo dxy charge transfer transition. This is a bonding-to-antibonding transition, and its intensity directly probe...

The nature of the catalytic centres of the oxomolybdoenzymes is considered with particular reference to the results of recent protein crystallographic studies. The different nature of these centres, with one or two molecules of a special pyranopterin (molybdopterin) ligating the metal through a dithiolene group, the presence or absence of a nucleotide appended to the phosphate of the molybdopterin AND the variation in the coordination chemistry at the metal render the term "THE molybdenum cofactor" meaningless and confusing. Rather, there is a series of such cofactors, related by the common denominators of a single molybdenum atom bound to the dithiolene group of the molybdopterin and, at some stage in the catalytic cycle, at least one terminal oxo group. This Mo(O)(molybdopterin) moiety is considered to be the metal-centred functional unit (McFU) of the oxomolybdoenzymes. Variations in the coordination chemistry and, therefore, the properties of the metal centre occur with the binding of other ligands, which can include: a terminal oxo or sulfido group, OH– and/or H2O group(s), a second pterin, and/or a serine, a cysteine or selenocysteine group from the polypeptide backbone of the protein. The role of molybdopterin is considered with particular reference to its potential involvement in the various redox processes necessary for the operation of the catalytic cycles of these enzymes; special attention is given to the possible cooperativity between formally metal-based and pterin-based redox processes.

The synthesis of a novel paramagnetic complex, (LMoO)2EA (where L = hydrotris(3,5-dimethyl-l-pyrazolyl)borate and EA = the tetra anion of 2,3,7,8-tetrahydroxy[1]benzopyrano[5,4,3-cde][1]benzopyran-5,10-dione, ellagic acid), containing two oxomolybdenum(V) ( S = 1 2 ) centers is described. The rigidity of the ellagic acid enforces co-planarity in the Mo2EA fragment which results in weak to moderate inter-metal interaction. The room temperature CW EPR spectrum of (LMoO)2EA shows an unusual hyperfine pattern due to the electronic spin coupling between the two S = 1 2 centers. The CV and DPV show two one-electron reductions separated by 97 mV.

The syntheses of mononuclear complexes cis-MoVOXL (X = Cl, Br, OMe, OEt, OPh, SPh, NCS, OSiMe3) and two binuclear complexes MoV2O3L2 of the title ligand LH2 are reported. Two forms of MoOClL, with cis oxo and chloro ligands, were crystallized, one in space group P21/n, with a = 10.440(2) A, b = 14.260(2) A, c = 12.041(2) A, β = 102.76(2)°, V = 1748(1) A3, and Z = 4, and the other in P21/c, with a = 13.564(4) A, b = 7.172(2) A, c = 18.242(6) A, β = 95.19(1)°, V = 1767(2) A3, and Z = 4. MoO(OSiMe3)L crystallizes in space group P21/c, with a = 15.923(3) A, b = 11.141(2) A, c = 14.186(2) A, β = 112.64(2)°, V = 2323(1) A3, and Z = 4, while MoO(NCS)L crystallizes in Pna21, with a = 22.471(2) A, b = 12.136(2) A, c = 7.138(1) A, V = 1947(1) A3, and Z = 4. The four structures reveal two possible conformations for ligand L: one with trans S atoms (cis,trans-MoOClL and -MoO(OSiMe3)L) and one with cis S atoms (cis,cis-MoOClL and -MoO(NCS)L). The cis,cis isomers are converted to the cis,trans forms under reflux in Me...

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 (MoOHn)-O-v 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.

Sulfite oxidizing enzymes (SOEs), including sulfite oxidase (SO) and bacterial sulfite dehydrogenase (SDH), catalyze the oxidation of sulfite (SO(3) (2-)) to sulfate (SO(4) (2-)). The active sites of SO and SDH are nearly identical, each having a 5-coordinate, pseudo-square-pyramidal Mo with an axial oxo ligand and three equatorial sulfur donor atoms. One sulfur is from a conserved Cys residue and two are from a pyranopterindithiolene (molybdopterin, MPT) cofactor. The identity of the remaining equatorial ligand, which is solvent-exposed, varies during the catalytic cycle. Numerous in vitro studies, particularly those involving electron paramagnetic resonance (EPR) spectroscopy of the Mo(V) states of SOEs, have shown that the identity and orientation of this exchangeable equatorial ligand depends on the buffer pH, the presence and concentration of certain anions in the buffer, as well as specific point mutations in the protein. Until very recently, however, EPR has not been a practical technique for directly probing specific structures in which the solvent-exposed, exchangeable ligand is an O, OH(-), H(2)O, SO(3) (2-), or SO(4) (2-) group, because the primary O and S isotopes ((16)O and (32)S) are magnetically silent (I = 0). This review focuses on the recent advances in the use of isotopic labeling, variable-frequency high resolution pulsed EPR spectroscopy, synthetic model compounds, and DFT calculations to elucidate the roles of various anions, point mutations, and steric factors in the formation, stabilization, and transformation of SOE active site structures.

The complexes LMoVIO2X [L = hydrotris(3,5-dimethylpyrazol-1-yl)borate; X = Cl, Br, NCS, OPh, SPh, SCH2Ph] are converted to air-stable complexes LMoVO(OSiMe3)X by one-electron coupled electron-electrophile transfer (CEET) reactions involving cobaltocene and the electrophilic reagent Me3SiCl. These complexes may also be obtained from LMoVO(OH)X by reaction with Me3SiCl in the presence of base. LMoVO(OSiMe3)(SCH2Ph) crystallises in space group P21/n, with a = 8.526 (1) A, b = 23.141 (3) A, c = 16.499 (2) A, β = 103.75 (12)° and Z = 4. The complex exhibits a distorted octahedral structure with a facially tridentate L ligand and mutually cis oxo [Mo=O = 1.675 (4) A], silyloxo [Mo–O = 1.932 (4) A] and thiolato [Mo–S = 2.398 (2) A] ligands. The detailed redox properties of LMoVO(OR)X (R = SiMe3, alkyl, aryl) differ from those of LMoVO(OH)X. Centres [MoVO(OR)] are candidates for the stable "inhibited" forms of certain molybdenum enzymes formed under conditions which apparently disfavour the catalytically active [MoVO(OH)] centres. In the coordinating solvent pyridine (py), both LMoVIO2(SPh) and LMoVO(OSiMe3)(SPh) are reduced in one-electron steps to stable LMoIVO(py)(SPh). LMoIVO(py)(SR) complexes are also obtained from LMoVIO2(SR) (R = Ph, CH2Ph, CHMe2) via a two-electron oxygen atom transfer reaction with tertiary phosphines in pyridine. Consequently, the Mo(IV) product is accessible via a concerted two-electron step or via two one-electron steps.

The molybdenum(V) coordination environment of sulfite oxidase has been investigated by multifrequency ESEEM spectroscopy in approximately 70 mM phosphate buffer at pH = 6.5 in both H(2)O and D(2)O. The FT-ESEEM spectra in H(2)O typically consist of three lines. One of these lines is always close to twice the Larmor frequency of the P atom (2nu(P)) and is assigned to one or more coordinated phosphates, providing the first direct unambiguous detection of such coordination. Extensive simulations of this phosphate signal at the various operational frequencies indicated that the coordinated phosphate group(s) probably does (do) not adopt a fixed orientation, and as a result, a description of the Mo.P hyperfine interaction required the introduction of a distribution of such orientations, with Mo.P distance(s) of 3.2-3.3 Å. The other two lines in the FT-ESEEM spectra in H(2)O, located at nu(H) and 2nu(H), were assigned to matrix protons. In D(2)O buffer two additional lines, assigned to matrix deuterons, were also seen.

Interconversion of mononuclear cis-dioxo−Mo(VI) and oxo−Mo(V,IV) complexes of the hydrotris(3,5-dimethylpyrazol-1-yl)borate ligand (L) by one-electron and two-electron reactions is described. In the coordinating solvent pyridine (py), LMoVIO2(SPh) is reduced by cobaltocene in one-electron steps to stable LMoIVO(SPh)(py). The compound LMoIVO(SPh)(py)·0.6MeOH crystallizes in orthorhombic space group Pbca, with a = 13.790(2) A, b = 15.266(2) A, c = 27.807(5), V = 5853(3) A3, and Z = 8. The complex exhibits a distorted octahedral structure with a facially tridentate ligand L and mutually cis oxo [MoO = 1.667(5) A], pyridine [Mo−N = 2.184(5) A], and thiolate [Mo−S = 2.390(3) A] ligands. This and other LMoIVO(SR)(py) (R = Ph, CH2Ph, CHMe2) complexes are also obtained from LMoVIO2(SR) via two-electron oxygen atom transfer reactions involving tertiary phosphines in pyridine. In dry solvents, the oxo−Mo(IV) complexes are oxidized by ferrocenium ion to the EPR-active cations [LMoVO(SR)(py)]+ which are hydrolyzed ra...

Sulfite oxidase (SO) is a vital metabolic enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed mechanism of this molybdenum cofactor dependent enzyme involves two one-electron intramolecular electron transfer (IET) steps from the molybdenum center to the iron of the b 5-type heme and two one-electron intermolecular electron transfer steps from the heme to cytochrome c. This work focuses on how the electrostatic interaction between two conserved amino acid residues, R472 and D342, in human SO (hSO) affects catalysis. The hSO variants R472M, R472Q, R472K, R472D, and D342K were created to probe the effect of the position of the salt bridge charges, along with the interaction between these two residues. With the exception of R472K, these variants all showed a significant decrease in their IET rate constants, k et, relative to wild-type hSO, indicating that the salt bridge between residues 472 and 342 is important for rapid IET. Surprisingly, however, except for R472K and R472D, all of the variants show k cat values higher than their corresponding k et values. The turnover number for R472D is about the same as k et, which suggests that the change in this variant is rate-limiting in catalysis. Direct spectroelectrochemical determination of the Fe(III/II) reduction potentials of the heme and calculation of the Mo(VI/V) potentials revealed that all of the variants affected the redox potentials of both metal centers, probably due to changes in their environments. Thus, the position of the positive charge of R472 and that of the negative charge of D342 are both important in hSO, and changing either the position or the nature of these charges perturbs IET and catalysis.

Human sulfite oxidase (hSO), an essential molybdoheme enzyme, catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic cycle includes two, one-electron intramolecular electron transfers (IET) between the molybdenum (Mo) and the heme domains. Rapid IET rates are ascribed to conformational changes that bring the two domains into close proximity to one another. Previous studies of hSO have focused on the roles of conserved residues near the Mo active site and on the tether that links the two domains. Here four aromatic surface residues on the heme domain (phenylalanine 57 (F57), phenylalanine 79 (F79), tyrosine 83 (Y83), and histidine 90 (H90)) have been mutated, and their involvement in IET rates, the heme midpoint potential, and the catalytic activity of hSO have been investigated using laser flash photolysis, spectroelectrochemistry, and steady-state kinetics, respectively. The results indicate that the size and hydrophobicity of F57 play an important role in modulating the heme potential and that F57 also affects the IET rates. The data also suggest that important interactions of H90 with a heme propionate group destabilize the Fe(III) state of the heme. The positive charge on H90 at pH ≤ 7.0 may decrease the electrostatic interaction between the Mo and heme domains, thereby decreasing the IET rates of wt hSO at low pH. Lastly, mutations of F79 and Y83, which are located on the surface of the heme domain, but not in direct contact with the heme or the propionate groups, have little effect on either IET or the heme potential.

The coupling of an Mo VI O 2 →Mo IV O oxygen atom transfer reaction to a one-electron reoxidation of the resulting Mo(IV) center by an Fe(III) porphyrin center has been investigated in an attempt to model the first two redox steps in the catalytic cycle of the enzyme sulfite oxidase. Reaction of LMo VI O 2 Cl (L=hydrotris(3,5-dimethyl-1-pyrazolyl)borate) with excess tri-phenylphosphine in DMF rapidly and cleanly generates LMo IV OCl(DMF). This Mo(IV) species reacts more slowly with Fe III Cl(TTP) (TTP=tetratolylporphyrin) to give LMo V OCl 2 and Fe II (TTP) as the final products. The kinetics of this one-electron redox reaction have been followed by electronic spectroscopy and by 1 H NMR. The reaction is zero-order in Fe(III), first-order in Mo(IV), and independent of added chloride ion. The first-order rate constant determined by these methods ranged from 1.7−4.6×10 −5 s −1 . An inner-sphere (halogen transfer) mechanism is shown to be most consistent with the data.

Studies of the «green isomer of [LWOCl 2 ]PF 6 » (L=1,4,7-trimethyl-1,4,7-triazacyclononane) have revealed that this material is not a distortional isomer of blue [LWOCl 2 ]PF 6 but rather is a ternary mixture of blue [LWOCl 2 ]PF 6 , a tungsten(IV)-oxo species, and a tungsten(VI)-dioxo species characterized by a cis-WO 2 2+ unit. Tungsten 4f X-ray photoelectron spectroscopy (XPS) has shown the green material prepared according to literature methods to be a variable mixture of these three tungsten oxidation states

The catalytic mechanisms of sulfite oxidizing enzymes (SOEs) have been investigated by multi-frequency pulsed EPR measurements of “difficult” magnetic nuclei (35,37Cl, 33S, 17O) associated with the Mo(V) center. Extensive DFT calculations have been used to relate the experimental magnetic resonance parameters of these nuclei to specific active site structures. This combined spectroscopic and computational approach has provided new insights concerning the structure/function relationships of the active sites of SOEs, including: (i) the exchange of oxo ligands; (ii) the nature of the blocked forms; and (iii) the role of Cl− in low pH forms.

Bis(para-substituted phenolato)oxomolybdenum(V) complexes with the general formula LMoO(OC 6 H 4 X) 2 , where L=hydrotris(3,5-dimethyl-1-pyrazolyl)borate and X=Me, Et, OMe, OEt, OH, F, Cl, Br, I, and CN, have been prepared and characterized by elemental analyses, mass spectrometry, and IR, EPR, and UV-vis-near-IR spectrospy. The energies of both the ligand-field and the lowest-energy charge-transfer bands depend upon the σ p values of the para substituents (X) on the phenolato ligands

Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDH(R55M)) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053-2063, 2009). For SDH(R55M) only the high-pH form is observed, and samples prepared in H(2)(17)O-enriched buffer show essentially the same (17)O hyperfine interaction and nuclear quadrupole interaction parameters as SDH(WT) enzyme. However, the pH dependence of the EPR spectra of SDH(R55Q), in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDH(WT). For SDH(R55Q) the blocked form with bound sulfate is generated at low pH, as verified by (33)S couplings observed upon reduction with (33)S-labeled sulfite. This observation of bound sulfate for SDH(R55Q) supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156-2163, 2009). At pHor = 8 the high-pH form dominates for SDH(R55Q).

The final products of the reaction of NEt 4 [{HB(Me 2 pz) 3 }W(CO) 3 ] and tetraethylthiuram disulfide in refluxing acetonitrile the red, diamagnetic, air-stable thiotungsten (IV) complex {HB(Me 2 pz) 3 }WS(S 2 CNEt 2 ) (1) and known W 2 (μ-S) 2 (S 2 CNEt 2 ) 4 (4) and [W(S 2 CNEt 2 ) 4 ] + isolated as chloride salt 3)

Electron spin echo envelope modulation (ESEEM) investigations were carried out on samples of the low-pH (lpH) form of vertebrate sulfite oxidase (SO) prepared with (35)Cl- and (37)Cl-enriched buffers, as well as with buffer containing the natural abundance of Cl isotopes. The isotope-related changes observed in the ESEEM spectra provide direct and unequivocal evidence that Cl(-) is located in close proximity to the Mo(V) center of lpH SO. The measured isotropic hyperfine interaction constant of about 4 MHz ((35)Cl) suggests that the Cl(-) ion is either weakly coordinated to Mo(V) at its otherwise vacant axial position, trans to the oxo ligand, or is hydrogen-bonded to the equatorial exchangeable OH ligand. Scalar relativistic all-electron density functional theory (DFT) calculations of the hyperfine and nuclear quadrupole interaction parameters, along with steric and energetic arguments, strongly support the possibility that Cl(-) is hydrogen-bonded to the equatorial OH ligand rather than being directly coordinated to the Mo(V).