Abstract:Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves… Show more
“…In the first step (Scheme 4, step 1), the active, resting Mn(III) enzyme binds substrate (as the monoanion) to form a Michaelis complex. Substrate is shown with monodentate carboxylate coordination, consistent with recent x-ray structural studies on a substrate analog (glycolate) complex, which also identifies a role for Asn 75 and Asn 85 in hydrogen bond stabilization of the complex (16). Under anaerobic conditions, oxalate has been shown to reduce the Mn(III) form of the enzyme (10) (Scheme 4, step 2).…”
Section: Reactionsupporting
confidence: 78%
“…Recent advances in x-ray structures of oxalate oxidase (9,15,16) and the availability of recombinant enzyme provides a foundation for detailed mechanistic studies on this interesting enzyme. In the present work, we have observed unusual nonstoichiometric burst kinetics, which has led to a clearer understanding of the role of the manganese ion in the catalytic reaction.…”
Section: Discussionmentioning
confidence: 99%
“…Structural characterization of the glycolate (substrate analog) complex of OXO suggest that two asparagine residues (Asn 75 and Asn 85 ) may play a role in orienting and stabilizing complexes of substrate or intermediates. Site-directed mutagenesis of these two residues in recombinant barley OXO demonstrates that they are essential for activity (16).…”
Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O 2 -saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.
“…In the first step (Scheme 4, step 1), the active, resting Mn(III) enzyme binds substrate (as the monoanion) to form a Michaelis complex. Substrate is shown with monodentate carboxylate coordination, consistent with recent x-ray structural studies on a substrate analog (glycolate) complex, which also identifies a role for Asn 75 and Asn 85 in hydrogen bond stabilization of the complex (16). Under anaerobic conditions, oxalate has been shown to reduce the Mn(III) form of the enzyme (10) (Scheme 4, step 2).…”
Section: Reactionsupporting
confidence: 78%
“…Recent advances in x-ray structures of oxalate oxidase (9,15,16) and the availability of recombinant enzyme provides a foundation for detailed mechanistic studies on this interesting enzyme. In the present work, we have observed unusual nonstoichiometric burst kinetics, which has led to a clearer understanding of the role of the manganese ion in the catalytic reaction.…”
Section: Discussionmentioning
confidence: 99%
“…Structural characterization of the glycolate (substrate analog) complex of OXO suggest that two asparagine residues (Asn 75 and Asn 85 ) may play a role in orienting and stabilizing complexes of substrate or intermediates. Site-directed mutagenesis of these two residues in recombinant barley OXO demonstrates that they are essential for activity (16).…”
Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide and hydrogen peroxide. In this study, unusual nonstoichiometric burst kinetics of the steady state reaction were observed and analyzed in detail, revealing that a reversible inactivation process occurs during turnover, associated with a slow isomerization of the substrate complex. We have investigated the underlying molecular mechanism of this kinetic behavior by preparing recombinant barley oxalate oxidase in three distinct oxidation states (Mn(II), Mn(III), and Mn(IV)) and producing a nonglycosylated variant for detailed biochemical and spectroscopic characterization. Surprisingly, the fully reduced Mn(II) form, which represents the majority of the as-isolated native enzyme, lacks oxalate oxidase activity, but the activity is restored by oxidation of the metal center to either Mn(III) or Mn(IV) forms. All three oxidation states appear to interconvert under turnover conditions, and the steady state activity of the enzyme is determined by a balance between activation and inactivation processes. In O 2 -saturated buffer, a turnover-based redox modification of the enzyme forms a novel superoxidized mononuclear Mn(IV) biological complex. An oxalate activation role for the catalytic metal ion is proposed based on these results.
“…The involvement of Mn(II) and no flavin, iron or copper in the direct conversion of O 2 to H 2 O 2 makes oxalate oxidase unique [216]. Some mechanistic studies have also been recently performed [215,218] and allow first insights into the reaction pathways.…”
“…Manganese complexes are of current interest in studies of molecular magnetism and in applications as magnetic recording (Ako et al, 2006;Aromi & Brechin, 2006) and manganese-enhanced MRI (Koretsky& Silva, 2004), and they can serve as model compounds of the bioinorganic chemistry of manganese (Deeth, 2008;Dismukes, 2006) including O 2 -evolving center of photosystem II (Umena et al, 2011) and various manganese-containing redox enzymes as dioxygenases (Georgiev et al, 2006), oxalate oxidase (Opaleye et al, 2006), catalase (Whittaker, 2012), superoxid dismutase (Friedel et al, 2004) and peroxidase (Sundaramoorthy et al, 2010). More manganese complexes especially for catalase and/or superoxide dismutase mimetics contain chelating N,N-donor ligands as 1,10-phenanthroline or 2,2´-bipyridine (Devereux et al, 1996;Geraghty et al, 1998;Kani et al, 2008;McCann et al, 1998;Viossat et al, 2003).…”
The crystal structures of the title compounds, [Mn(phen) 2 Cl 2 ] (I) and [Mn(bipy) 2 Cl 2 ] (II), have been determined at 150 K. The manganese atoms in both compounds are coordinated by four pyridine nitrogen atoms from two 1,10-phenanthroline or 4,4´-bipyridine ligands and two chloride anions, resulting in a distorted cis-MnN 4 Cl 2 octahedral geometry. Both complexes are connected through C-H···Cl hydrogen bonds into frameworks. The π-π stacking interactions are observed in crystal structure of both ones.
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