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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.

A multi-technique spectroscopic and theoretical study of the Cp(2)M(benzenedithiolato) (M=Ti,V,Mo; Cp = η(5)-C(5)H(5)) series provides deep insight into dithiolene electronic structure contributions to electron transfer reactivity and reduction potential modulation in pyranopterin molybdenum enzymes. This work explains the magnitude of the dithiolene folding distortion, and the concomitant changes in metal-ligand covalency, that are sensitive to electronic structure changes as a function of d-electron occupancy in the redox orbital. It is shown that the large fold angle differences correlate with covalency, and the fold angle distortion is due to a pseudo-Jahn-Teller (PJT) effect. The PJT effect in these and related transition metal dithiolene systems arise from the small energy differences between metal and sulfur valence molecular orbitals, which uniquely poise these systems for dramatic geometric and electronic structure changes as the oxidation state changes. Herein, we have used a combination of resonance Raman, magnetic circular dichroism, electron paramagnetic resonance, and UV photoelectron spectroscopies to explore the electronic states involved in the vibronic coupling mechanism. Comparison between the UV photoelectron spectroscopy (UPS) of the d(2) M=Mo complex and the resonance Raman spectra of the d(1) M=V complex reveals the power of this combined spectroscopic approach. Here, we observe that the UPS spectrum of Cp(2)Mo(bdt) contains an intriguing vibronic progession that is dominated by a “missing-mode” that is comprised of PJT active distortions. We discuss the relationship of the PJT distortions to facile electron transfer in molybdenum enzymes.

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.

Animal SOs possess two redox-active domains and are ideal for studying IET in relation to the overall conformation of the protein and the molecular dynamics of the motion of the two domains relative to one another. The procedures for preparing and purifying recombinant hSO [34, 44] have been further optimized in our laboratory [4, 5, 29], so that a wide range of mutant proteins in 50–100 mg quantities can be prepared for comprehensive kinetic, mechanistic, and spectroscopic studies. The ready accessibility of recombinant hSO has made it possible to investigate the effects of modifying amino acid residues of interest to better understand the long range IET process in hSO and to obtain insight into biochemical basis of pathological mutations of hSO. Experimental and theoretical studies agree that domain movement is necessary for SO to achieve a productive conformation during electron transfer, and that the length and composition of the interdomain tether is an important factor in the rates of IET. However, the interactions such as hydrogen bonding and hydrophobic effects involved in mediating the correct mutual orientation and the distance following the initial orientation are unknown. Studies of site-specific mutants reveal a great deal about the IET complex in hSO. Mutating several aromatic residues that extend from the pterin to the surface of the protein suggest that alternative ET pathways may be in operation in hSO. The profound effects of mutating residues surrounding the Mo active site (e. g. R160Q and Y343F) have been demonstrated [28, 45], and extension to mutations of other positively charged residues surrounding the active site, such as Lys200, is warranted. Finally, all of the discussions presented here and elsewhere for IET in hSO rest upon the single structure of intact native cSO. It is clear that X-ray crystal structures of hSO and various mutants are needed to better understand the unexpected properties of this fascinating biomedically important protein.

Intramolecular electron transfer (IET) between the molybdenum and heme centers of vertebrate sulfite oxidase (SO) is proposed to be a key step in the catalytic cycle of the enzyme. However, the X-ray crystallographic distance between these centers, R(MoFe) = 32.3 Å, appears to be too long for the rapid IET rates observed in liquid solution. The Mo and heme domains are linked by a flexible tether, and it has been proposed that dynamic interdomain motion brings the two metal centers closer together and thereby facilitates rapid IET. To date, there have been no direct distance measurements for SO in solution that would support or contradict this model. In this work, pulsed electron-electron double resonance (ELDOR) and relaxation induced dipolar modulation enhancement (RIDME) techniques were used to obtain information about R(MoFe) in the Mo(V)Fe(III) state of wild type recombinant human SO in frozen glassy solution. Surprisingly, the data obtained suggest a fixed structure with R(MoFe) = 32 Å, similar to that determined by X-ray crystallography for chicken SO, although the orientation of the R(MoFe) radius-vector with respect to the heme center was found to be somewhat different. The implications of these findings for the flexible tether model are discussed.

The construction and performance of a Ka-band pulsed electron paramagnetic resonance (EPR) cryogenic probehead that incorporates dielectric resonator (DR) is presented. We demonstrate that the use of DR allows one to optimize pulsed double electron–electron resonance (DEER) measurements utilizing large resonator bandwidth and large amplitude of the microwave field B1. In DEER measurements of Gd-based spin labels, use of this probe finally allows one to implement the potentials of Gd-based labels in distance measurements. Evidently, this DR is well suited to any applications requiring large B1-fields and resonator bandwidths, such as electron spin echo envelope modulation spectroscopy of nuclei having low magnetic moments and strong hyperfine interactions and double quantum coherence dipolar spectroscopy as was recently demonstrated in the application of a similar probe based on an loop-gap resonator and reported by Forrer et al. (J Magn Reson 190:280, 2008).

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.

This work demonstrates the feasibility of using Gd(III) tags for long-range Double Electron Electron Resonance (DEER) distance measurements in biomacromolecules. Double-stranded 14- base pair Gd(III)-DNA conjugates were synthesized and investigated at Ka band. For the longest Gd(III) tag the average distance and average deviation between Gd(III) ions determined from the DEER time domains was about 59 ± 12 A. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60 A, and analysis indicates that distance measurements up to 100 A are possible. Compared with commonly used nitroxide labels, Gd(III)-based labels will be most beneficial for the detection of distance variations in large biomacromolecules, with an emphasis on large scale changes in shape or distance. Tracking the folding/unfolding and domain interactions of proteins and the conformational changes in DNA are examples of such applications.

Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor-dependent enzymes that are found in plants, animals, and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO catalyzes the oxidation of toxic sulfite to sulfate as the final step in the catabolism of the sulfur-containing amino acids, methionine and cysteine. In humans, sulfite oxidase deficiency is an inherited recessive disorder that produces severe neonatal neurological problems that lead to early death. Plant SO (PSO) also plays an important role in sulfite detoxification and in addition serves as an intermediate enzyme in the assimilatory reduction of sulfate. In vertebrates, the proposed catalytic mechanism of SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme. A similar mechanism is proposed for SDH, involving its molybdenum cofactor and c-type heme. However, PSO, which lacks an integral heme cofactor, uses molecular oxygen as its electron acceptor. Here we review recent results for SOEs from kinetic measurements, computational studies, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements, and site-directed mutagenesis on active site residues of SOEs and of the flexible polypepetide tether that connects the heme and molybdenum domains of human SO. Rapid kinetic studies of PSO are also discussed.

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.

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2–3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (∼60 and 200 s–1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

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.

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

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.

Arginine 160 in human sulfite oxidase (SO) is conserved in all SO species sequenced to date. Previous steady-state kinetic studies of the R160Q human SO mutant showed a remarkable decrease in k(cat)/K(m)(sulfite) of nearly 1000-fold, which suggests that Arg 160 in human SO makes an important contribution to the binding of sulfite near the molybdenum cofactor [Garrett, R. M., Johnson, J. L., Graf, T. N., Feigenbaum, A., Rajagopalan, K. V. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6394-6398]. In the crystal structure of chicken SO, Arg 138, the equivalent of Arg 160 in human SO, is involved in the formation of a positively charged sulfite binding site [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garnett, R. M., Rajagopalan, K. V., Enemark, J. H., Rees, D. C. (1997) Cell 91, 973-983]. To further assess the role of Arg 160 in human SO, intramolecular electron transfer (IET) rates between the reduced heme [Fe(II)] and oxidized molybdenum [Mo(VI)] centers in the wild type, R160Q, and R160K human SO forms were investigated by laser flash photolysis. In the R160Q mutant, the IET rate constant at pH 6.0 was decreased by nearly 3 orders of magnitude relative to wild type, which indicates that the positive charge of Arg 160 is essential for efficient IET in human SO. Furthermore, the IET rate constant for the R160K mutant is about one-fourth that of the wild type enzyme, which strongly indicates that it is the loss of charge of Arg 160, and not its precise location, that is responsible for the much larger decrease in IET rates in the R160Q mutant. Steady-state kinetic measurements indicate that IET is rate-limiting in the catalytic cycle of the R160Q mutant. Thus, the large decrease in the IET rate constant rationalizes the fatal impact of this mutation in patients with this genetic disorder.

Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe(II)) and oxidized molybdenum (Mo(VI)) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k(cat). Steady-state kinetic analysis of the Y343F mutant showed an increase in K(m)(sulfite) and a decrease in k(cat) resulting in a 23-fold attenuation in the specificity constant k(cat)/K(m)(sulfite) at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.

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.

Several point mutations in the gene of human sulfite oxidase (hSO) result in isolated sulfite oxidase deficiency, an inherited metabolic disorder. Three conserved residues (H304, R309, K322) are hydrogen bonded to the phosphate group of the molybdenum cofactor, and the R309H and K322R mutations are responsible for isolated sulfite oxidase deficiency. The kinetic effects of the K322R mutation have been previously reported (Rajapakshe et al., Chem. Biodiversity, 2012, 9, 1621–1634); here we investigate several mutants of H304 and R309 by steady-state kinetics, laser flash photolysis studies of intramolecular electron transfer (IET), and spectroelectrochemistry. An unexpected result is that all of the mutants show decreased rates of IET but increased steady-state rates of catalysis. However, in all cases the rate of IET is greater than the overall turnover rate, showing that IET is not the rate determining step for any of the mutations.

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...

Two-pulse and four-pulse electron spin−echo envelope modulation spectroscopy (ESEEMS) at two operational frequencies and two-dimensional hyperfine sublevel correlation spectroscopy (HYSCORES), have been used to probe the MoV coordination environment of sulfite oxidase in H2O and D2O solutions, buffered at pH 9.5 and 7.0 with ∼100 mM Tris-type buffers. At pH 9.5 the ESEEM and HYSCORE results definitively reveal the presence of one solvent-exchangeable D(H) near the MoV center, probably in the form of a Mo−OH(D) moiety. The orientation of this group is not fixed (although it is substantially restricted) and thus gives rise to a distribution of hyperfine interaction (hfi) parameters. The resulting loss of amplitude makes direct observation of a proton-related line using ESEEM impossible. However, such a line is observable in ESEEM spectra of the comparable deuterated enzyme because the narrower distribution of hfi parameters leads to less line broadening of the ESEEM spectra. ESEEM and HYSCORE spectra of sul...

Protein X-ray crystallography has revealed the structures of the active sites of several molybdenum- and tungsten-containing enzymes that catalyze formal hydroxylation and oxygen atom transfer reactions. Each molybdenum (or tungsten) atom is coordinated by one (or two) ene-dithiolate groups of a novel pterin (molybdopterin), and the active sites are further differentiated from one another by the number of terminal oxo and/or sulfido groups and by coordinated amino acid residues. These active-site structures have no precedent in the coordination chemistry of molybdenum and tungsten.

The first ionization energy in the gas-phase photoelectron spectra (PES) of Tp*Mo(E)(tdt) complexes (where E = O, S, NO; Tp* = hydrotris(3,5-dimethyl-1-pyrazolyl)borate; tdt = 3,4-toluenedithiolate) is essentially independent of the nature of E, even though the formal oxidation state of the Mo center ranges from +2 to +5. The PES data for the tdt complexes contrast with the results for analogous complexes with alkoxide ligands, which show large variations in first ionization energy (Westcott, B. L.; Enemark, J. H. Inorg. Chem. 1997, 36, 5404−5405). For the tdt complexes the relative intensities of the two lowest energy ionizations do not substantially change as the excitation source is varied among Ne I, He I, and He II radiation, even though the atomic photoionization cross sections for Mo 4d and S 3p orbitals change dramatically over this energy region. These results all point to substantial covalency in the Mo−S bonds. It is proposed that the S atoms of the tdt ligand act as an “electronic buffer” to t...

Single crystals of barium samarium titanium oxide Ba6−3xSm8+2xTi18O54 (x = 0.27) have been synthesized and studied using x-ray diffraction. Superstructure reflections, which cause a doubling of the cell along the short axis, were taken into account and the refinement was conducted in the orthorhombic space group Pnma. Unit cell parameters from single crystal x-ray diffraction were a = 22.289(1), b = 7.642(1), and c = 12.133(1) Å. Refinement on F resulted in R1 = 5.37% for 1410 Fo > 4σ with the thermal parameters of the Sm and Ba atoms refined anisotropically and the thermal parameters of the Ti and O atoms refined isotropically. The structure is made up of a network of corner sharing TiO6−2 octahedra creating rhombic (perovskite-like) and pentagonal channels. The two pentagonal channels are fully occupied by Ba atoms. The refinement suggests that one rhombic channel is fully occupied by Sm atoms (Sm3/Sm4), one rhombic channel is partially occupied by Sm atoms (100% Sm1/86.25% Sm5), and one rhombic channel is shared by BaySm atoms (59.25% Ba3/40.75% Sm2), resulting in a formula of Ba10.38Sm17.08Ti36O108 with Z = 1. The above site occupancies differ from the site occupancies previously reported in the literature for refinements conducted with the short axis approximately equal to 3.8 Å.

The molybdenum-containing enzyme sulfite oxidase catalyzes the conversion of sulfite to sulfate, the terminal step in the oxidative degradation of cysteine and methionine. Deficiency of this enzyme in humans usually leads to major neurological abnormalities and early death. The crystal structure of chicken liver sulfite oxidase at 1.9 Å resolution reveals that each monomer of the dimeric enzyme consists of three domains. At the active site, the Mo is penta-coordinated by three sulfur ligands, one oxo group, and one water/hydroxo. A sulfate molecule adjacent to the Mo identifies the substrate binding pocket. Four variants associated with sulfite oxidase deficiency have been identified: two mutations are near the sulfate binding site, while the other mutations occur within the domain mediating dimerization.

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.

In this work, the experimental conditions and parameters necessary to optimize the long-distance (≥60 A) double electron–electron resonance (DEER) measurements of biomacromolecules labeled with Gd(III) tags are analyzed. The specific parameters discussed are the temperature, microwave band, the separation between the pumping and observation frequencies, pulse train repetition rate, pulse durations and pulse positioning in the electron paramagnetic resonance spectrum. It was found that: (1) in optimized DEER measurements, the observation pulses have to be applied at the maximum of the electron paramagnetic resonance spectrum; (2) the optimal temperature range for Ka-band measurements is 14–17 K, while in W-band the optimal temperatures are between 6 and 9 K; (iv) W-band is preferable to Ka-band for DEER measurements. Recent achievements and the conditions necessary for short-distance measurements (