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Sulfite oxidase (SO) deficiency, an inherited disease that causes severe neonatal neurological problems and early death, arises from defects in the biosynthesis of the molybdenum cofactor (Moco) (general sulfite oxidase deficiency) or from inborn errors in the SUOX gene for SO (isolated sulfite oxidase deficiency, ISOD). The X-ray structure of the highly homologous homonuclear dimeric chicken sulfite oxidase (cSO) provides a template for locating ISOD mutation sites in human sulfite oxidase (hSO). Catalysis occurs within an individual subunit of hSO, but mutations that disrupt the hSO dimer are pathological. The catalytic cycle of SO involves five metal oxidation states (Mo VI , Mo V , Mo IV , Fe III , Fe II ), two intramolecular electron transfer (IET) steps, and couples a two-electron oxygen atom transfer reaction at the Mo center with two one-electron transfers from the integral b-type heme to exogenous cytochrome c, the physiological oxidant. Several ISOD examples are analyzed using steady-state, stopped-flow, and laser flash photolysis kinetics and physical measurements of recombinant variants of hSO and native cSO. In the structure of cSO, Mo … Fe = 32 Å, much too long for efficient IET through the protein. Interdomain motion that brings the Mo and heme centers closer together to facilitate IET is supported indirectly by decreasing the length of the interdomain tether, by changes in the charges of surface residues of the Mo and heme domains, as well as by preliminary molecular dynamics calculations. However, direct dynamic measurements of interdomain motion are in their infancy., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. John H. Enemark reports a relationship with The University of Arizona that includes: non-financial support. I was on the Editorial Board of the Journal of Inorganic Biochemistry about 10 years ago. None paying., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Over 50 molybdenum enzymes in three distinct families (sulfite oxidase, xanthine oxidase, DMSO reductase) are known, and representative X-ray crystal structures are available for all families. Structural analogues that replicate the coordination about the Mo atom in the absence of surrounding protein have been synthesized and characterized. The properties of metal complexes of non-innocent dithiolene ligands and their oxidized counter parts, dithiones, are summarized. Pulsed electron paramagnetic resonance (EPR) spectroscopy of the 33 S-labeled molybdenum domain of catalytically active bioengineered sulfite oxidase has clearly demonstrated delocalization of electron density from Mo V to the dithiolene component of the molybdenum cofactor (Moco) of the enzyme. Moco is highly covalent and has three redox active components: the Mo atom; the dithiolene; and the pterin. In principle, Moco can have a total of eight redox states, making it one of the most redox rich cofactors in biology. The {Moco} n formalism, developed here, gives the total number of electrons (n) associated with a particular redox state of Moco. This flexible notation eliminates the need to assign a specific oxidation state to each of the three components of Moco and allows for internal redistribution of electrons among the components upon substrate binding, changes in the surrounding network of hydrogen bonds, conformational changes, and catalysis. An unexpected result is that sulfite oxidase (an oxotransferase) is predicted to utilize the {Moco} 4-6 electron distributions during catalysis, whereas xanthine oxidase (a hydroxylase) is predicted to utilize the {Moco} 6-8 electron distributions during catalysis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Here we highlight past work on metal-dithiolene interactions and how the unique electronic structure of the metal-dithiolene unit contributes to both the oxidative and reductive half reactions in pyranopterin molybdenum and tungsten enzymes. The metallodithiolene electronic structures detailed here were interrogated using multiple ground and excited state spectroscopic probes on the enzymes and their small molecule analogs. The spectroscopic results have been interpreted in the context of bonding and spectroscopic calculations, and the pseudo-Jahn-Teller effect. The dithiolene is a unique ligand with respect to its redox active nature, electronic synergy with the pyranopterin component of the molybdenum cofactor, and the ability to undergo chelate ring distortions that control covalency, reduction potential, and reactivity in pyranopterin molybdenum and tungsten enzymes., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

A multitechnique 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 arises 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 composed of PJT-active distortions. We discuss the relationship of the PJT distortions to facile electron transfer in molybdenum enzymes.

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 (P i ) 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.

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).

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.

In this work, the experimental conditions and parameters necessary to optimize the long-distance (≥ 60 Å) 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: (i) in optimized DEER measurements, the observation pulses have to be applied at the maximum of the EPR spectrum; (ii) the optimal temperature range for K a -band measurements is 14-17 K, while in W-band the optimal temperatures are between 6-9 K; (iii) W-band is preferable to K a -band for DEER measurements. Recent achievements and the conditions necessary for short-distance measurements (<15 Å) are also briefly discussed.

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.

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 construction and performance of a K a -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 B 1 . 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 B 1 -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).

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.

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.

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.

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., (© 2011 American Chemical Society)

Mitochondrial amidoxime reducing components (mARC-1 and mARC-2) represent a novel group of Mo-containing enzymes in eukaryotes. These proteins form the catalytic part of a three-component enzyme complex known to be responsible for the reductive activation of several N-hydroxylated prodrugs. No X-ray crystal structures are available for these enzymes as yet. A previous biochemical investigation [Wahl, B., et al. (2010) J. Biol. Chem., 285, 37847-37859 ] has revealed that two of the Mo coordination positions are occupied by sulfur atoms from a pyranopterindithiolate (molybdopterin, MPT) cofactor. In this work, we have used continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy and density functional theoretical (DFT) calculations to determine the nature of remaining ligands in the Mo(V) state of the active site of mARC-2. Experiments with samples in D(2)O have identified the exchangeable equatorial ligand as a hydroxyl group. Experiments on samples in H(2)(17)O-enriched buffer have shown the presence of a slowly exchangeable axial oxo ligand. Comparison of the experimental (1)H and (17)O hyperfine interactions with those calculated using DFT has shown that the remaining nonexchangeable equatorial ligand is, most likely, protein-derived and that the possibility of an equatorial oxo ligand can be excluded., (© 2011 American Chemical Society)

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 K(a) 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±12Å. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60Å, and analysis indicates that distance measurements up to 100Å 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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

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 <7 and >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 species. Similarly, if chloride is added before sulfite, the lpH species is formed instead of the blocked one. Qualitatively similar results were observed for samples of sulfite-oxidizing enzymes from other organisms that were previously reported to form a blocked species at low pH. However, the depletion of chloride has no effect upon the formation of the hpH species.

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 pH > or = 8 the high-pH form dominates for SDH(R55Q).

Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO [Kisker, C., et al. (1997) Cell 91, 973-983], which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 A and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into greater proximity have been proposed [Feng, C., et al. (2003) Biochemistry 42, 5816-5821] to explain the relatively rapid IET kinetics, which are much faster than those theoretically predicted from the crystal structure. To explore the proposed role(s) of the tether in facilitating this conformational change, we altered both its length and flexibility in HSO by site-specific mutagenesis, and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernible systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a 3-fold decrease in the IET rate constant. Deletions of nonconserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steady-state kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a 5-fold increase in the sulfite K(m) value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these proline to alanine and deletion mutants are identical to those of wild-type HSO, indicating no significant change in the Mo active site geometry.

Sulfite oxidases (SOs) are physiologically vital Mo-containing enzymes that occur in animals, plants, and bacteria and which catalyze the oxidation of sulfite to sulfate, the terminal reaction in the oxidative degradation of sulfur-containing compounds. X-ray structure determinations of SOs from several species show nearly identical coordination structures of the molybdenum active center, and a common catalytic mechanism has been proposed that involves the generation of a transient paramagnetic Mo(V) state through a series of coupled electron-proton transfer steps. This chapter describes the use of pulsed electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopic techniques to obtain information about the structure of this Mo(V) species from the hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of nearby magnetic nuclei. Variable frequency instrumentation is essential to optimize the experimental conditions for measuring the couplings of different types of nuclei (e.g., (1)H, (2)H, (31)P, and (17)O). The theoretical background necessary for understanding the ESEEM and ENDOR spectra of the Mo(V) centers of SOs is outlined, and examples of the use of advanced pulsed EPR methods (RP-ESEEM, HYSCORE, integrated four-pulse ESEEM) for structure determination are presented. The analysis of variable-frequency pulsed EPR data from SOs is aided by parallel studies of model compounds that contain key functional groups or that are isotopically labeled and thus provide benchmark data for enzymes. Enormous progress has been made on the use of high-resolution variable-frequency pulsed EPR methods to investigate the structures and mechanisms of SOs during the past ~15 years, and the future is bright for the continued development and application of this technology to SOs, other molybdenum enzymes, and other problems in metallobiochemistry.

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.

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.

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

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).

All reported sulfite-oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. To improve our understanding of the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions R55Q and R55M as well as the positively charged substitution R55K in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for improving our understanding of residue function in IET without the added complexity of the interdomain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) exhibited increased IET rate constants relative to that of the wild type (3-4-fold) when studied by laser flash photolysis. The gain in function observed in SDH(R55Q) and SDH(R55M) suggests that the reduction in the level of IET seen in HSO(R160Q) is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the interdomain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDH(WT), SDH(R55M), and SDH(R55Q) show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (K(eq)) and rate constants (k(et)) of both variants with respect to the SDH(WT) enzyme. SDH(WT) has a k(et) that is independent of pH and a K(eq) that increases as pH decreases; on the other hand, both SDH(R55M) and SDH(R55Q) have a k(et) that increases as pH decreases, and SDH(R55M) has a K(eq) that is pH-independent. IET in the SDH(R55Q) variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from that of SDH(WT), but which also occurs in HSO. IET in SDH(R55K) is slower than in SDH(WT). A new analysis of the possible mechanistic pathways for sulfite-oxidizing enzymes is presented and related to available kinetic and EPR results for these enzymes.

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 SDH R55M and SDHH57A are relatively insensitive to pH (approximately 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.

SOEs (sulfite-oxidizing enzymes) are physiologically vital and occur in all forms of life. During the catalytic cycle, the five-co-ordinate square pyramidal oxo-molybdenum active site passes through the Mo(V) state, and intimate details of the structure can be obtained from variable frequency pulsed EPR spectroscopy through the hyperfine and nuclear quadrupole interactions of nearby magnetic nuclei. By employing variable spectrometer operational frequencies, it is possible to optimize the measurement conditions for difficult quadrupolar nuclei of interest (e.g. (17)O, (33)S, (35)Cl and (37)Cl) and to simplify the interpretation of the spectra. Isotopically labelled model Mo(V) compounds provide further insight into the electronic and geometric structures and chemical reactions of the enzymes. Recently, blocked forms of SOEs having co-ordinated sulfate, the reaction product, were detected using (33)S (I=3/2) labelling. This blocking of product release is a possible contributor to fatal human sulfite oxidase deficiency in young children.

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

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 pH>9. 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.

Gas-phase photoelectron spectroscopy and density functional theory have been used to investigate the electronic structures of open-shell bent vanadocene compounds with chelating dithiolate ligands, which are minimum molecular models of the active sites of pyranopterin Mo/W enzymes. The compounds Cp2V(dithiolate) [where dithiolate is 1,2-ethenedithiolate (S2C2H2) or 1,2-benzenedithiolate (bdt), and Cp is cyclopentadienyl] provide access to a 17-electron, d1 electron configuration at the metal center. Comparison with previously studied Cp2M(dithiolate) complexes, where M is Ti and Mo (respectively d0 and d2 electron configurations), allows evaluation of d0, d1, and d2 electronic configurations of the metal center that are analogues for the metal oxidation states present throughout the catalytic cycle of these enzymes. A "dithiolate-folding effect" that involves an interaction between the vanadium d orbitals and sulfur p orbitals is shown to stabilize the d1 metal center, allowing the d1 electron configuration and geometry to act as a low-energy electron pathway intermediate between the d0 and d2 electron configurations of the enzyme.

Sulfite oxidase from Arabidopsis thaliana has been reduced at pH = 6 with sulfite labeled with 33S (nuclear spin I = 3/2), followed by reoxidation by ferricyanide to generate the Mo(V) state of the active center. To obtain information about the hyperfine interaction (hfi) of 33S with Mo(V), continuous-wave electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) experiments have been performed. The interpretation of the EPR and ESEEM spectra was facilitated by a theoretical analysis of the nuclear transition frequencies expected for the situation of the nuclear quadrupole interaction being much stronger than the Zeeman and hyperfine interactions. The isotropic hfi constant of 33S determined in these experiments was about 3 MHz, which demonstrates the presence of coordinated sulfate in the sulfite-reduced low-pH form of the plant enzyme.

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 (approximately 29GHz). 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.

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.