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A combination of X-ray absorption and low-temperature electronic absorption spectroscopies has been used to probe the geometric and electronic structures of the human mitochondrial amidoxime reducing component enzyme (hmARC1) in the oxidized Mo(VI) and reduced Mo(IV) forms. Extended X-ray absorption fine structure analysis revealed that oxidized enzyme possesses a 5-coordinate [MoO 2 (S Cys )(PDT)] - (PDT = pyranopterin dithiolene) active site with a cysteine coordinated to Mo. A 5-coordinate geometry is retained in the reduced state, with the equatorial oxo being protonated. Low-temperature electronic absorption spectroscopy of hmARC1 reveals a spectrum for the oxidized enzyme that is significantly different from what has been reported for sulfite oxidase family enzymes. Time-dependent density functional theory computations on oxidized and reduced hmARC1, and a small molecule analogue for hmARC1 ox , have been used to assist us in making detailed band assignments and developing a greater understanding of enzyme electronic structure contributions to reactivity. Our understanding of the hmARC red HOMO and the LUMO of the benzamidoxime substrate reveal a potential π-bonding interaction between these redox orbitals, with two-electron occupation of the substrate LUMO along the reaction coordinate activating the O-N bond for cleavage and promoting oxygen atom transfer to the Mo site.

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.)

Oxo-molybdenum (dithiolene) complexes covalently linked individually to iron and zinc porphyrin have been synthesized to show an electron transfer between the two metal centres in relevance to electron transfer from Mo-cofactor to cytochrome b 5 domains in the oxidative half of the catalytic cycle of native sulfite oxidase. This association has been investigated by electrochemical, EPR measurement and X-ray absorbance spectroscopy techniques.

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.)

Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they carry out. Therefore, obtaining abundant quantities of active enzyme is necessary for exploring this family's biochemical capability. This mini-review summarizes the methods for overexpressing mononuclear molybdenum enzymes in the context of the challenges encountered in the process. Effective methods for molybdenum cofactor synthesis and incorporation, optimization of expression conditions, improving isolation of active vs. inactive enzyme, incorporation of additional prosthetic groups, and inclusion of redox enzyme maturation protein chaperones are discussed in relation to the current molybdenum enzyme literature. This article summarizes the heterologous and homologous expression studies providing underlying patterns and potential future directions.

Local surface plasmon resonance (LSPR)-enhanced catalysis has brought a substantial amount of opportunities across various disciplines such as photocatalysis, photodetection, and photothermal therapeutics. Plasmon-induced photothermal and hot carriers effects have also been utilized to activate the enzyme-like reactions. Compared with natural enzymes, the relatively low catalytic performance of nanozymes severely hampered the potential applications in the field of biomedicine. For these issues mentioned above, herein, we demonstrate a highly efficient sulfite oxidase (SuO x ) mimetic performance of plasmonic monolayer MoO x (ML-MoO x ) upon LSPR excitation. We also established that the considerable photothermal effect and the injection of hot carriers induced by LSPR are responsible for promoting the SuO x activity of ML-MoO x . The high transient local temperature on the surface of ML-MoO x generated by the photothermal effect facilitates to impact the reaction velocity and feed the SuO x -like activity, while the generation of hot carriers which are suggested as predominant effects catalyzes the oxidation of sulfite to sulfate through significantly decreasing the activation energy for the SuO x -like reaction. These investigations present a contribution to the basic understanding of plasmon-enhanced enzyme-like reaction and provided an insight into the optimization of the SuO x mimetic performance of nanomaterials.

Sulfite oxidase (SO) is encoded by the nuclear SUOX gene and catalyzes the final step in cysteine catabolism thereby oxidizing sulfite to sulfate. Oxidation of sulfite is dependent on two cofactors within SO, a heme and the molybdenum cofactor (Moco), the latter forming the catalytic site of sulfite oxidation. SO localizes to the intermembrane space of mitochondria where both-pre-SO processing and cofactor insertion-are essential steps during SO maturation. Isolated SO deficiency (iSOD) is a rare inborn error of metabolism caused by mutations in the SUOX gene that lead to non-functional SO. ISOD is characterized by rapidly progressive neurodegeneration and death in early infancy. We diagnosed an iSOD patient with homozygous mutation of SUOX at c.1084G>A replacing Gly362 to serine. To understand the mechanism of disease, we expressed patient-derived G362S SO in Escherichia coli and surprisingly found full catalytic activity, while in patient fibroblasts no SO activity was detected, suggesting differences between bacterial and human expression. Moco reconstitution of apo-G362S SO was found to be approximately 90-fold reduced in comparison to apo-WT SO in vitro. In line, levels of SO-bound Moco in cells overexpressing G362S SO were significantly reduced compared to cells expressing WT SO providing evidence for compromised maturation of G362S SO in cellulo. Addition of molybdate to culture medium partially rescued impaired Moco binding of G362S SO and restored SO activity in patient fibroblasts. Thus, this study demonstrates the importance of the orchestrated maturation of SO and provides a first case of Moco-responsive iSOD., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Nitric oxide (NO) is an important gasotransmitter involved in numerous intra- and intercellular signaling events. In addition to the oxidative pathway of NO generation, which includes three NO synthase (NOS) isoforms in mammals, a reductive pathway contributes to NO generation. In this pathway, nitrite is reduced to NO by various metal-containing proteins. Among these, all members of the eukaryotic molybdenum (Mo)-dependent enzyme family were found to be able to reduce nitrite to NO. This Review focuses on the current state of research in the field of Mo-dependent nitrite reduction in eukaryotes. An overview on the five eukaryotic Mo-enzymes is given, and similarities as well as differences in their nitrite reduction mechanisms are presented and discussed in the context of physiological relevance., (© 2018 Federation of European Biochemical Societies.)

Many enzymes that produce or transform small molecules such as O 2 , H 2 , and CO 2 embed inorganic cofactors based on transition metals. Their active site, where the chemical reaction occurs, is buried in and protected by the protein matrix, and connected to the solvent in several ways: chains of redox cofactors mediate long-range electron transfer; static or dynamic tunnels guide the substrate, product and inhibitors; amino acids and water molecules transfer protons. The catalytic mechanism of these enzymes is therefore delocalized over the protein and involves many different steps, some of which determine the response of the enzyme under conditions of stress (extreme redox conditions, presence of inhibitors, light), the catalytic rates in the two directions of the reaction and their ratio (the "catalytic bias"). Understanding all the steps in the catalytic cycle, including those that occur on sites of the protein that are remote from the active site, requires a combination of biochemical, structural, spectroscopic, theoretical, and kinetic methods. Here we argue that kinetics should be used to the fullest extent, by extracting quantitative information from the comparison of data and kinetic models and by exploring the combination of experimental kinetics and theoretical chemistry. In studies of these catalytic mechanisms, direct electrochemistry, the technique which we use and contribute to develop, has become unescapable. It simply consists in monitoring the changes in activity of an enzyme that is wired to an electrode by recording an electric current. We have described kinetic models that can be used to make sense of these data and to learn about various aspects of the mechanism that are difficult to probe using more conventional methods: long-range electron transfer, diffusion along gas channels, redox-driven (in)activations, active site chemistry and photoreactivity under conditions of turnover. In this Account, we highlight a few results that illustrate our approach. We describe how electrochemistry can be used to monitor substrate and inhibitor diffusion along the gas channels of hydrogenases and we discuss how the kinetics of intramolecular diffusion relates to global properties such as resistance to oxygen and catalytic bias. The kinetics and/or thermodynamics of intramolecular electron transfer may also affect the catalytic bias, the catalytic potentials on either side of the equilibrium potential, and the overpotentials for catalysis (defined as the difference between the catalytic potentials and the open circuit potential). This is understood by modeling the shape of the steady-state catalytic response of the enzyme. Other determinants of the catalytic rate, such as domain motions, have been probed by examining the transient catalytic response recorded at fast scan rates. Last, we show that combining electrochemical investigations and MD, DFT, and TD-DFT calculations is an original way of probing the reactivity of the H-cluster of hydrogenase, in particular its reactions with CO, O 2 , and light. This approach contrasts with the usual strategy which aims at stabilizing species that are presumed to be catalytic intermediates, and determining their structure using spectroscopic or structural methods.

Sulfite oxidase is a mononuclear molybdenum enzyme that oxidises sulfite to sulfate in many organisms, including man. Three different reaction mechanisms have been suggested, based on experimental and computational studies. Here, we study all three with combined quantum mechanical (QM) and molecular mechanical (QM/MM) methods, including calculations with large basis sets, very large QM regions (803 atoms) and QM/MM free-energy perturbations. Our results show that the enzyme is set up to follow a mechanism in which the sulfur atom of the sulfite substrate reacts directly with the equatorial oxo ligand of the Mo ion, forming a Mo-bound sulfate product, which dissociates in the second step. The first step is rate limiting, with a barrier of 39-49 kJ/mol. The low barrier is obtained by an intricate hydrogen-bond network around the substrate, which is preserved during the reaction. This network favours the deprotonated substrate and disfavours the other two reaction mechanisms. We have studied the reaction with both an oxidised and a reduced form of the molybdopterin ligand and quantum-refinement calculations indicate that it is in the normal reduced tetrahydro form in this protein.

Signaling by H 2 S is proposed to occur via persulfidation, a posttranslational modification of cysteine residues (RSH) to persulfides (RSSH). Persulfidation provides a framework for understanding the physiological and pharmacological effects of H 2 S. Due to the inherent instability of persulfides, their chemistry is understudied. In this review, we discuss the biologically relevant chemistry of H 2 S and the enzymatic routes for its production and oxidation. We cover the chemical biology of persulfides and the chemical probes for detecting them. We conclude by discussing the roles ascribed to protein persulfidation in cell signaling pathways.

Artificial light-driven signal chains are particularly important for the development of systems converting light into a current, into chemicals or for light-induced sensing. Here, we report on the construction of an all-protein, light-triggered, catalytic circuit based on photosystem I, cytochrome c (cyt c) and human sulfite oxidase (hSOX). The defined assembly of all components using a modular design results in an artificial biohybrid electrode architecture, combining the photophysical features of PSI with the biocatalytic properties of hSOX for advanced light-controlled bioelectronics. The working principle is based on a competitive switch between electron supply from the electrode or by enzymatic substrate conversion.

We have examined how the self-interaction error in density-functional theory (DFT) calculations affects energies calculated on large systems (600-1000 atoms) involving several charged groups. We employ 18 different quantum mechanical (QM) methods, including Hartree-Fock, as well as pure, hybrid, and range-separated DFT methods. They are used to calculate reaction and activation energies for three different protein models in vacuum, in a point-charge surrounding, or with a continuum-solvent model. We show that pure DFT functionals give rise to a significant delocalization of the charges in charged groups in the protein, typically by ∼0.1 e, as evidenced from the Mulliken charges. This has a clear effect on how the surroundings affect calculated reaction and activation energies, indicating that these methods should be avoided for DFT calculations on large systems. Fortunately, methods such as CAM-B3LYP, BHLYP, and M06-2X give results that agree within a few kilojoules per mole, especially when the calculations are performed in a point-charge surrounding. Therefore, we recommend these methods to estimate the effect of the surroundings with large QM systems (but other QM methods may be used to study the intrinsic reaction and activation energies).

This review focuses on the synthesis, properties and electron paramagnetic resonance (EPR), electron spin echo envelope modulation (ESEEM) and electron-nuclear double resonance (ENDOR) spectroscopy of mononuclear d 1 oxo- and sulfido-Mo(V) complexes relevant to the understanding of the EPR-active Mo(V) forms of pterin-containing molybdenum enzymes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, and mARC exhibit nitrite reductase activity at low oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent changes in blood flow, leading to continued exploration for other candidate nitrite reductases. Another physiologically important molybdenum enzyme—sulfite oxidase (SO)—has not been extensively studied., Results: Using gas-phase nitric oxide (NO) detection and physiological concentrations of nitrite, SO functions as nitrite reductase in the presence of a one-electron donor, exhibiting redox coupling of substrate oxidation and nitrite reduction to form NO. With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Studies with recombinant heme and molybdenum domains of SO indicate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain, mediated by steric and redox effects of this domain. Using knockdown of all molybdopterin enzymes and SO in fibroblasts isolated from patients with genetic deficiencies of molybdenum cofactor and SO, respectively, SO was found to significantly contribute to hypoxic nitrite signaling as demonstrated by activation of the canonical NO-sGC-cGMP pathway., Innovation: Nitrite binds to and is reduced at the molybdenum site of mammalian SO, which may be allosterically regulated by heme and molybdenum domain interactions, and contributes to the mammalian nitrate-nitrite-NO signaling pathway in human fibroblasts., Conclusion: SO is a putative mammalian nitrite reductase, catalyzing nitrite reduction at the Mo(IV) center.

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.

In this review, we test the hypothesis that pyranopterin coordination plays a critical role in defining substrate reactivities in the four families of mononuclear molybdenum and tungsten enzymes (Mo/W-enzymes). Enzyme families containing a single pyranopterin dithiolene chelate have been demonstrated to have reactivity towards two (sulfite oxidase, SUOX-fold) and five (xanthine dehydrogenase, XDH-fold) types of substrate, whereas the major family of enzymes containing a bis-pyranopterin dithiolene chelate (dimethylsulfoxide reductase, DMSOR-fold) is reactive towards eight types of substrate. A second bis-pyranopterin enzyme (aldehyde oxidoreductase, AOR-fold) family catalyzes a single type of reaction. The diversity of reactions catalyzed by each family correlates with active site variability, and also with the number of pyranopterins and their coordination by the protein. In the case of the AOR-fold enzymes, inflexibility of pyranopterin coordination correlates with their limited substrate specificity (oxidation of aldehydes). In examples of the SUOX-fold and DMSOR-fold enzymes, we observe three types of histidine-containing charge-transfer relays that can: (1) connect the piperazine ring of the pyranopterin to the substrate-binding site (SUOX-fold enzymes); (2) provide inter-pyranopterin communication (DMSOR-fold enzymes); and (3) connect a pyran ring oxygen to deeply buried water molecules (the DMSOR-fold NarGHI-type nitrate reductases). Finally, sequence data mining reveals a number of bacterial species whose predicted proteomes contain large numbers (up to 64) of Mo/W-enzymes, with the DMSOR-fold enzymes being dominant. These analyses also reveal an inverse correlation between Mo/W-enzyme content and pathogenicity.

There are three families of mononuclear molybdenum enzymes that catalyze oxygen atom transfer (OAT) reactions, named after a typical example from each family, viz., dimethyl sulfoxide reductase (DMSOR), sulfite oxidase (SO), and xanthine oxidase (XO). These families differ in the construction of their active sites, with two molybdopterin groups in the DMSOR family, two oxy groups in the SO family, and a sulfido group in the XO family. We have employed density functional theory calculations on cluster models of the active sites to understand the selection of molybdenum ligands in the three enzyme families. Our calculations show that the DMSOR active site has a much stronger oxidative power than the other two sites, owing to the extra molybdopterin ligand. However, the active sites do not seem to have been constructed to make the OAT reaction as exergonic as possible, but instead to keep the reaction free energy close to zero (to avoid excessive loss of energy), thereby making the reoxidation (SO and XO) or rereduction of the active sites (DMSOR) after the OAT reaction facile. We also show that active-site models of the three enzyme families can all catalyze the reduction of DMSO and that the DMSOR model does not give the lowest activation barrier. Likewise, all three models can catalyze the oxidation of sulfite, provided that the Coulombic repulsion between the substrate and the enzyme model can be overcome, but for this harder reaction, the SO model gives the lowest activation barrier, although the differences are not large. However, only the XO model can catalyze the oxidation of xanthine, owing to its sulfido ligand.

The oxidation of sulfite to sulfate by two different models of the active site of sulfite oxidase has been studied. Both protonated and deprotonated substrates were tested. Geometries were optimized with density functional theory (TPSS/def2-SV(P)) and energies were calculated either with hybrid functionals and large basis sets (B3LYP/def2-TZVPD) including corrections for dispersion, solvation, and entropy, or with coupled-cluster theory (LCCSD(T0)) extrapolated toward a complete basis set. Three suggested reaction mechanisms have been compared and the results show that the lowest barriers are obtained for a mechanism where the substrate attacks a Mo-bound oxo ligand, directly forming a Mo-bound sulfate complex, which then dissociates into the products. Such a mechanism is more favorable than mechanisms involving a Mo-sulfite complex with the substrate coordinating either by the S or O atom. The activation energy is dominated by the Coulomb repulsion between the Mo complex and the substrate, which both have a negative charge of -1 or -2.

Sulfur K-edge X-ray absorption spectroscopy (XAS) and density functional theory (DFT) calculations have been used to determine the electronic structures of two complexes [Mo(IV)O(bdt)2](2-) and [Mo(VI)O2(bdt)2](2-) (bdt = benzene-1,2-dithiolate(2-)) that relate to the reduced and oxidized forms of sulfite oxidase (SO). These are compared with those of previously studied dimethyl sulfoxide reductase (DMSOr) models. DFT calculations supported by the data are extended to evaluate the reaction coordinate for oxo transfer to a phosphite ester substrate. Three possible transition states are found with the one at lowest energy, stabilized by a P-S interaction, in good agreement with experimental kinetics data. Comparison of both oxo transfer reactions shows that in DMSOr, where the oxo is transferred from the substrate to the metal ion, the oxo transfer induces electron transfer, while in SO, where the oxo transfer is from the metal site to the substrate, the electron transfer initiates oxo transfer. This difference in reactivity is related to the difference in frontier molecular orbitals (FMO) of the metal-oxo and substrate-oxo bonds. Finally, these experimentally related calculations are extended to oxo transfer by sulfite oxidase. The presence of only one dithiolene at the enzyme active site selectively activates the equatorial oxo for transfer, and allows facile structural reorganization during turnover.

Sulfite oxidase is a mitochondria-located molybdenum-containing enzyme catalyzing the oxidation of sulfite to sulfate in the amino acid and lipid metabolism. Therefore, it plays a major role in detoxification processes, where defects in the enzyme cause a severe infant disease leading to early death with no efficient or cost-effective therapy in sight. Here we report that molybdenum trioxide (MoO3) nanoparticles display an intrinsic biomimetic sulfite oxidase activity under physiological conditions, and, functionalized with a customized bifunctional ligand containing dopamine as anchor group and triphenylphosphonium ion as targeting agent, they selectively target the mitochondria while being highly dispersible in aqueous solutions. Chemically induced sulfite oxidase knockdown cells treated with MoO3 nanoparticles recovered their sulfite oxidase activity in vitro, which makes MoO3 nanoparticles a potential therapeutic for sulfite oxidase deficiency and opens new avenues for cost-effective therapies for gene-induced deficiencies.

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).

A series of [Mo(IV)O(mnt)(SR)(N-N)](-) (mnt = maleonitriledithiolate; R = Ph, nap, p-Cl-Ph, p-CO2H-Ph, and p-NO2-Ph; N-N = 2,2'-bipyridine (bipy) and 1,10-phenanthroline (phen)) complexes analogous to the reduced active site of enzymes of the sulfite oxidase family has been synthesized and their participation in electron transfer reactions studied. Equatorial thiolate and dithiolene ligations have been used to closely simulate the three sulfur coordinations present in the native molybdenum active site. These synthetic analogues have been shown to participate in electron transfer via a pentavalent EPR-active Mo(V) intermediate with minimal structural change as observed electrochemically by reversible oxidative responses. The role of the redox-active dithiolene ligand as an electron transfer gate between external oxidants and the molybdenum center could be envisaged in one of the analogue systems where the initial transient EPR signal with = 2.008 is replaced by the appearance of a typical Mo(V)-centered EPR ( = 1.976) signal. The appearance of such a ligand-based transient radical at the initial stage has been supported by the ligand-centered frontier orbital from DFT calculation. A stepwise rationale has been provided by computational study to show that the coupled effects of the diimine bite angle and the thiolato dihedral angle determine the metal- or ligand-based frontier orbital occupancy. DFT calculation has further supported the similarity between the reduced, semireduced, and oxidized resting state of the molybdenum center in Moco of SO with the synthesized complexes and their corresponding one-electron and fully oxidized species.

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.

A perspective is provided of recent advances in our understanding of molybdenum-containing enzymes other than nitrogenase, a large and diverse group of enzymes that usually (but not always) catalyze oxygen atom transfer to or from a substrate, utilizing a Mo=O group as donor or acceptor. An emphasis is placed on the diversity of protein structure and reaction catalyzed by each of the three major families of these enzymes.

The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sulfite oxidase (SO) is an enzyme catalyzing the terminal step of the metabolism of sulfur-containing amino acids that is essential for almost all living organisms. The catalytic activity of SO in vertebrates strongly depends on the efficiency of the intramolecular electron transfer (IET) between the catalytic Moco domain and the cytochrome b5 (cyt b5) domain. The IET process is assumed to be mediated by large domain motions of the cyt b5 domains within the enzyme. Thus, the interaction of SO with charged surfaces may affect the mobility of the cyt b5 domain required for IET and consequently hinder SO activation. In this study, we present a molecular dynamics approach to investigating the ionic strength dependence of the initial surface adsorption of SO in two different conformations-the crystallographic structure and the model structure for an activated SO-onto mixed amino- and hydroxyl-terminated SAMs. The results show for both conformations at low ionic strengths a strong adsorption of the cyt b5 units onto the SAM, which inhibits the domain motion event required for IET. Under higher ion concentrations, however, the interaction with the surface is weakened by the negatively charged ions acting as a buffer and competing in adsorption with the cathodic cyt b5 domains. This competition prevents the immobilization of the cytochrome b5 units onto the surface, allowing the intramolecular domain motions favoring IET. Our predictions support the interpretation of recent experimental spectroelectrochemical studies on SO.

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.

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conserved in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 Å resolution, respectively.

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.

A sulfite oxidase (SO(X)) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto carboxylated gold coated magnetic nanoparticles (Fe(3)O(4)@GNPs) electrodeposited onto the surface of a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC)-N-hydroxy succinimide (NHS) chemistry. An amperometric sulfite biosensor was fabricated using SO(X)/Fe(3)O(4)@GNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode. The working electrode was characterized by Fourier Transform Infrared (FTIR) Spectroscopy, Cyclic Voltammetry (CV), Scanning Electron Microscopy (SEM) and Electrochemical Impedance Spectroscopy (EIS) before and after immobilization of SO(X). The biosensor showed optimum response within 2s when operated at 0.2V (vs. Ag/AgCl) in 0.1 M Tris-HCl buffer, pH 8.5 and at 35 °C. Linear range and detection limit were 0.50-1000 μM and 0.15 μM (S/N=3) respectively. Biosensor was evaluated with 96.46% recovery of added sulfite in red wine and 1.7% and 3.3% within and between batch coefficients of variation respectively. Biosensor measured sulfite level in red and white wines. There was good correlation (r=0.99) between red wines sulfite value by standard DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) method and the present method. Enzyme electrode was used 300 times over a period of 4 months, when stored at 4 °C. Biosensor has advantages over earlier biosensors that it has excellent electrocatalysis towards sulfite, lower detection limit, higher storage stability and no interference by ascorbate, cysteine, fructose and ethanol., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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

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

Enzymes belonging to the Sulfite Oxidase (SO) enzyme family are found in virtually all forms of life, and are especially abundant in prokaryotes as shown by analysis of available genome data. Despite this fact, only a limited number of bacterial SO family enzymes has been characterized in detail to date, and these appear to be involved in very different metabolic processes such as energy generation from sulfur compounds, host colonization, sulfite detoxification and organosulfonate degradation. The few characterized bacterial SO family enzymes also show an intriguing range of structural conformations, including monomeric, dimeric and heterodimeric enzymes with varying numbers and types of redox centres. Some of the bacterial enzymes even catalyze novel reactions such as dimethylsulfoxide reduction that previously had been thought not to be catalyzed by SO family enzymes. Classification of the SO family enzymes based on the structure of their Mo domain clearly shows that three distinct groups of enzymes belong to this family, and that almost all SOEs characterized to date are representatives of the same group. The widespread occurrence and obvious structural and functional plasticity of the bacterial SO family enzymes make this an exciting field for further study, in particular the unraveling of the metabolic roles of the three enzyme groups, some of which appear to be associated almost exclusively with pathogenic microorganisms., (Copyright © 2010 Elsevier B.V. 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.

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b(5), and NADH/FAD-dependent cytochrome b(5) reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.

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.

A reliable and sensitive analysis of sulfites in food is essential in food monitoring. However, the established methods exhibit deficiencies in the very low concentration ranges (below 10 mg/L SO(2)), especially with more complex food matrices. With a focus on these challenges, an HPLC method with immobilized enzyme reactor (HPLC-IMER) for the analysis of sulfites in food was optimized and compared to a standard method. A modulated sample preparation procedure and the use of a novel sulfite oxidase from Arabidopsis thaliana were explored to make the method applicable for most food samples. The plant sulfite oxidase turned out to be superior to the commercially available animal sulfite oxidase in terms of detection limit (0.01 mg/L SO(2)), linear range (0.04-20 mg/L SO(2)) and stability. In a small scale comparison within our laboratory, as well as in a standardized proficiency testing, the HPLC-IMER was compared to an established distillative method. The enzyme-based method is not only more sensitive and specific, it also yields higher sulfite recoveries in almost all samples while exhibiting better statistic method parameters., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.

Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants at position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 A resolution and to 2.4 A resolution in the presence of sulfite, and the C185A variant to 2.8 A resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier extended X-ray absorption fine structure spectroscopy (EXAFS) work on the human C207S mutant. An unexpected result was that in the C185S variant, in the absence of sulfite, the active site residue Tyr322 became disordered as did the loop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue relocalized to the active site. The C185A variant structure also indicated the presence of a third oxygen ligand; however, Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized Mo(VI) state in both the C185S and C185A variants of chicken SO and show the expected trioxodithiolene active site. Density functional theory calculations of the trioxo form of the cofactor reasonably reproducd the Mo horizontal lineO distances of the complex; however, the calculated Mo-S distances were slightly longer than either crystallographic or EXAFS measurements. Taken together, these results indicate that the active sites of the C185S and C185A variants are essentially catalytically inactive, the crystal structures of C185S and C185A variants contain a fully oxidized, trioxo form of the cofactor, and Tyr322 can undergo a conformational change that is relevant to the reaction mechanism. Additional DFT calculations demonstrated that such methods can reasonably reproduce the geometry and bond lengths of the active site.

The physiologically vital enzyme sulfite oxidase employs rapid intramolecular electron transfer between a molybdenum ion in the C-terminal domain (the site of sulfite oxidation) and a heme moeity in the N-terminal domain to complete its catalytic cycle. Crystal structures of the enzyme show C- and N-terminal domain orientations that are not consistent with rapid intramolecular electron transfer. Domain motion has been postulated to explain this discrepancy. In the present work we employ molecular dynamics simulations to understand the large-scale domain motions of the enzyme. We observe motion of the N-terminal domain into an orientation similar to that postulated for rapid electron transfer. Our simulations also probe the dynamics of the active site and surrounding residues, adding a further level of structural and thermodynamic detail in understanding sulfite oxidase function.

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 oxidase [SO; EC 1.8.3.1] catalyses the physiologically vital oxidation of sulfite to sulfate, the terminal reaction in degradation of sulfur containing amino acids, cysteine and methionine. Sulfite oxidase from vertebrate sources is among the best studied molybdenum enzymes. Existence of SO in plants has been established recently by identification of a cDNA from Arabidopsis thaliana encoding a functional SO. The present study was undertaken to identify herbaceous and woody plants (viz., Azardirachta indica L., Cassia fistula L., Saraca indica L., Spinacea oleracea L., and Syzyzium cumini L.), a relatively less explored source, having significant SO activity and to characterize some of its immuno-biochemical properties. The Syzyzium cumini was chosen to characterize SO as it showed maximum enzyme activity in the crude extract as compared to other plants. Absorption spectra of SO revealed two peaks at 235 and 277 nm, but no distinct peak in the visible region could be observed. Crude extract of all the plants were taken into considerations for immuno-biochemical studies. Despite of significant protein structure-functional similarities between plant and animal SO, no cross-reactivity could be established between the two sources of SO. These data suggested that plants SO, however, differed with regards to their immunobiochemical properties.

Electronic paramagnetic resonance (EPR), electronic absorption, and magnetic circular dichroism spectroscopies have been performed on YedY, a SUOX fold protein with a Mo domain that is remarkably similar to that found in chicken sulfite oxidase, Arabidopsis thaliana plant sulfite oxidase, and the bacterial sulfite dehydrogenase from Starkeya novella. Low-energy dithiolene --> Mo and cysteine thiolate --> Mo charge-transfer bands have been assigned for the first time in a Mo(V) form of a SUOX fold protein, and the spectroscopic data have been used to interpret the results of bonding calculations. The analysis shows that second coordination sphere effects modulate dithiolene and cysteine sulfur covalency contributions to the Mo bonding scheme. In particular, a more acute O(oxo)-Mo-S(Cys)-C dihedral angle results in increased cysteine thiolate S --> Mo charge transfer and a large g(1) in the EPR spectrum. The spectrosocopic results, coupled with the available structural data, indicate that these second coordination sphere effects may play key roles in modulating the active-site redox potential, facilitating hole superexchange pathways for electron transfer regeneration, and affecting the type of reactions catalyzed by sulfite oxidase family enzymes.

The kinetics of the reaction of neutral [Mo(S2C6H4)3] with hydrogen sulfite to produce the anionic Mo(V) complex, [Mo(S2C6H4)3]-, and sulfate have been investigated. It has been shown that [Mo(S2C6H4)3] acts as the electron-proton sink in the oxygenation reaction of HSO3(-) by water. Reaction rates, monitored by UV/vis stopped-flow spectrometry, were studied in THF/water media as a function of the concentration of HSO3(-) and molybdenum complex, pH, ionic strength, and temperature. The reaction exhibits pH-dependent HSO3(-) saturation kinetics, and it is first-order in complex concentration. The kinetic data and MS-ESI spectra are consistent with the formation of [Mo O(S2C6H4)2(S2C6H5)]- (1) adduct as a crucial intermediate that transfers the oxygen atom to HSO3(-) yielding the Mo(V) species quantitatively.

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).

We present a theoretical study of the temperature-dependent magnetic circular dichroism (MCD) spectrum for complexes modeling the molybdoenzyme sulfite-oxidase (1) in its Mo(V) oxidation state. The theoretical study was based on a newly implemented time-dependent density functional method that takes into account first-order perturbations due to spin-orbit coupling and a constant magnetic field. It was possible, on the basis of the theoretical calculations, to give a full assignment of the MCD spectrum for 1 and interpret the C term of each band in the experimental MCD spectrum in terms of spin-orbit couplings between specific excited states and between excited states and the ground state.