Biochemical and spectroscopic characterization of an aldehyde oxidoreductase isolated from Desulfovibrio aminophilus

Thapper, Anders; Rivas, Maria G.; Brondino, Carlos D.; Ollivier, Bernard; Fauque, Guy; Moura, Isabel; Moura, José J. G.

doi:10.1016/j.jinorgbio.2005.09.013

Cited by 14 publications

(18 citation statements)

References 28 publications

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“…Similar values were reported for other AORs from SRB with the same substrate. [21][22][23] Kinetic assays followed by dialysis were performed to determine whether the inhibition with EDO, GOL, cyanide, and arsenite is reversible or not. At the indicated inhibitor concentrations after 10 min incubation, the percentages of remnant activity for each inhibitor were 78.1 ( 0.4 for EDO, 48 ( 3 for GOL, and 69.1 ( 0.1 for cyanide; no activity was detected for arsenite.…”

Section: Resultsmentioning

confidence: 99%

“…20 In addition, the Mo(V) EPR signals associated with desulfo forms of XO, such as the "slow"-type signal and the signal obtained upon ethylene glycol (EDO) addition, were also observed. [20][21][22][23] On this basis, it was assumed that AORs from SRB and XO family enzymes have similar active sites and that both would experience the same changes upon reaction with substrates, inhibitors, and reducing agents. Because the structural data of DgAOR did not show the sulfido ligand coordinated to Mo, it was assumed that the enzyme crystallizes in the desulfo inactive form (Figure 1b).…”

Section: Introductionmentioning

confidence: 99%

“…7,8,23,27 These inhibited forms are important to understand changes in the coordination of the active site and the integrity of the electron transfer chain upon inhibition. In previous structural and EPR studies of arsenite-inhibited DgAOR, a correlation between structural and EPR properties of the Mo center was established.…”

Section: Introductionmentioning

confidence: 99%

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Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

et al. 2009

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Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme, where the catalytically labile OH/OH 2 ligand is lost and both alcohols coordinate the Mo site in a η 2 fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

et al. 2009

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“…FeS I, the proximal center, is closer to the Mo site and is buried inside the protein, whereas FeS II, the distal center, is situated near the surface of the protein. Both iron-sulfur centers and a fraction of the molybdenum ions are paramagnetic in the reduced state of the protein and, in addition, show intercenter magnetic couplings [25][26][27]. The currently accepted mechanism implies substrate interaction with the Mo center, which is then reduced from Mo(VI) to Mo(IV), followed by a two electron transfer to an external electron acceptor, in a process mediated by the two iron-sulfur centers.…”

Section: Introductionmentioning

confidence: 99%

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

et al. 2006

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Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both MoAs complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition.

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“…Aldehyde oxidoreductases from the SRB Desulfovibrio gigas (Dg), 11 Desulfovibrio desulfuricans (Dd), 12 Desulfovibrio alaskensis (Da), 13 and Desulfovibrio aminophilus (Dam) 14 are homodimeric proteins (∼100 kDa per monomer) that contain molybdenum at the active site. The structures of Dg Aor, the first structure reported for a mononuclear molybdenum-containing enzyme, 15,16 and Dd Aor 17 were solved at the resolution of 1.28 and 2.8 Å, respectively.…”

Section: Molecular Properties and Overall Folding Of Aldehyde Oxidorementioning

confidence: 99%

Structural and Electron Paramagnetic Resonance (EPR) Studies of Mononuclear Molybdenum Enzymes from Sulfate‐Reducing Bacteria

et al. 2007

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Molybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions. The metal atom (Mo or W) is coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. In addition, these proteins contain redox cofactors such as iron-sulfur clusters and heme groups. All of these metal cofactors are along an electron-transfer pathway that mediates the electron exchange between substrate and an external electron acceptor (for oxidative reactions) or donor (for reductive reactions). We describe in this Account a combination of structural and electronic paramagnetic resonance studies that were used to reveal distinct aspects of these enzymes. Mononuclear Molybdenum Enzymes: Classification and General PropertiesMolybdenum and tungsten are found in biological systems in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen-atom-transfer reactions.1,2 Mononuclear molybdenum-containing enzymes are ubiquitous and participate in several biological processes occurring in nature, such as denitrification, the greenhouse effect, and pollution of the water soil. 3,4,5 Mononuclear molybdenum enzymes have been divided into three groups called the xanthine oxidase (XO), dimethyl sulfoxide reductase (DMSOR), and sulfite oxidase (SO) families. 1,6 These three families include not only the enzymes that give the name to the different groups but also diverse enzymes, such as aldehyde oxidoreductases, nitrate reductases, and formate dehydrogenases, among others. The active site of these enzymes (Figure 1a) includes the metal atom coordinated to one or two pyranopterin molecules and to a variable number of ligands such as oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteines), and selenium (selenocysteines) atoms. The pyranopterin molecule is an organic ligand that can be either in the monophosphate form or have a nucleotide molecule attached by a pyrophosphate link (Figure 1b). 7 In addition, these proteins may also have other redox cofactors such as iron-sulfur (FeS) centers, hemes, and flavin groups, which are involved in intra-and intermolecular electron-transfer processes. A tungstencontaining aldehyde oxidoreductase from Pyrococcus furiosus presents a similar active-site structure and has been classified in a different family. 2 However, other tungsten enzymes such as the formate dehydrogenase from Desulfovibrio gigas belong to the DMSOR family, being closely related to the corresponding molybdenum enzymes. 8With a few exceptions, these enzymes catalyze the transfer of an oxygen atom from water to the product (or vice versa) in reactions that imply a net exchange of two electrons between the enzyme and substrate and in which the metal ion cycles between the redox states IV and VI. Figure 2 shows two representative exampl...

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Biochemical and spectroscopic characterization of an aldehyde oxidoreductase isolated from Desulfovibrio aminophilus

Cited by 14 publications

References 28 publications

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

Structural and Electron Paramagnetic Resonance (EPR) Studies of Mononuclear Molybdenum Enzymes from Sulfate‐Reducing Bacteria

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