Redox Cycling, pH Dependence, and Ligand Effects of Mn(III) in Oxalate Decarboxylase from <i>Bacillus subtilis</i>

This contribution describes electron paramagnetic resonance (EPR) experiments on Mn­(III) in oxalate decarboxylase of <i>Bacillus subtilis</i>, an interesting enzyme that catalyzes the redox-neutral dissociation of oxalate into formate and carbon dioxide. Chemical redox cycling provides strong evidence that both Mn centers can be oxidized, although the N-terminal Mn­(II) appears to have the lower reduction potential and is most likely the carrier of the +3 oxidation state under moderate oxidative conditions, in agreement with the general view that it represents the active site. Significantly, Mn­(III) was observed in untreated OxDC in succinate and acetate buffers, while it could not be directly observed in citrate buffer. Quantitative analysis showed that up to 16% of the EPR-visible Mn is in the +3 oxidation state at low pH in the presence of succinate buffer. The fine structure and hyperfine structure parameters of Mn­(III) are affected by small carboxylate ligands that can enter the active site and have been recorded for formate, acetate, and succinate. The results from a previous report [Zhu, W., et al. (2016) <i>Biochemistry</i> <i>55</i>, 429–434] could therefore be reinterpreted as evidence of formate-bound Mn­(III) after the enzyme is allowed to turn over oxalate. The pH dependence of the Mn­(III) EPR signal compares very well with that of enzymatic activity, providing strong evidence that the catalytic reaction of oxalate decarboxylase is driven by Mn­(III), which is generated in the presence of dioxygen

Heterobimetallic Complexes of Polypyridyl Ligands Containing Paramagnetic Centers: Synthesis and Characterization by IR and EPR

Ligands containing linked dipicolylamine (dpa) and bipyridine sites have been explored as platforms for the synthesis of heterometallic complexes containing the paramagnetic metals Cu²⁺ and Co²⁺. IR and EPR studies on the bimetallic complexes and simplified model compounds support dpa-selective binding by both of these metals. The IR spectra have also been compared to those of diamagnetic Rh⁺, Zn²⁺, and Pd²⁺ complexes whose metal binding sites had been independently determined through NMR techniques. The binding preferences have been used to control selective metalation in the synthesis of heterometallic Pt/Cu, Pd/Cu, and Rh/Cu complexes

EPR spectra of PBN radical adducts obtained from the incubation of 100

<p> <b>mM oxalate, 100 </b><b>mM KCl, 20 </b><b>mM PBN, and 50 </b><b>µM CsOxOx.</b> (A) PBN-oxalate derived radical adduct from the recombinant, wild type CsOxOx catalyzed reaction. (B) PBN-oxalate derived radical adduct from the CsOxOx A242E mutant catalyzed reaction showed no signal over background. (C) PBN-oxalate derived radical adduct from the CsOxOx D241A mutant catalyzed reaction. (D) All reaction components without enzyme.</p

CD spectra of recombinant, wild-type CsOxOx and the putative active site mutants.

<p>All samples were 938 ug/mL in phosphate buffer (pH 7.0): wild-type CsOxOx, black; D241A, dark blue; D241S, grey; A242E, orange; DASN241-244SENS, red; R169K, cyan; R349K, yellow.</p

The effect of pH on the affinity of recombinant CsOxOx D241A for oxalate.

<p>pH dependence of CsOxOx D241A mutant on kinetic parameters V_max/K_m (•) and V_max (▴) for the CsOxOx catalyzed reaction.</p

Model of free-radical mechanisms for oxalate oxidase and oxalate decarboxylase.

<p>Modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057933#pone.0057933-Escutia1" target="_blank">[23]</a>.</p

Manganese binding sites of the oxalate decarboxylase monomer and homology models of the manganese binding sites of CsOxOx.

<p>(A) OxDC (PDB ID 1UW8) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057933#pone.0057933-Just1" target="_blank">[32]</a> with manganese ions (purple), metal coordinating residues (atoms colored as follows: C, cyan; N, blue; O, red), conserved active site arginine residues (dark blue) and the N-terminal lid region (green) highlighted. (B) Homology model of the N-terminal CsOxOx Mn binding site metal coordinating residues and the DASN of the lid region. (C) Homology model of the C-terminal CsOxOx Mn binding site metal coordinating residues. The homology model of CsOxOx was constructed using its amino acid sequence and the experimentally solved structure of <i>Bacillus subtilis</i> OxDC (PDB ID 1UW8) using Swiss-Model (The Swiss Institute of Bioinformatics) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057933#pone.0057933-Arnold1" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057933#pone.0057933-Schwede1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057933#pone.0057933-Guex1" target="_blank">[50]</a>. Figure generated using Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).</p

The reaction catalyzed by <i>Ceriporiopsis subvermispora</i> oxalate oxidase.

<p>The reaction catalyzed by <i>Ceriporiopsis subvermispora</i> oxalate oxidase.</p

Steady state kinetic parameters and manganese content of recombinant, wild type CsOxOx and CsOxOx mutants.

a<p>K_M values were determined from assays in which oxalate concentration was varied over the range of 0.01 to 50 mM.</p>b<p>nd. Value was not determined.</p

pH dependence of recombinant, wild-type CsOxOx and A242E CsOxOx mutant at 416.0

<p> <b>GHz.</b> 8A: Recombinant, wild type CsOxOx A: Same sample as C after the addition of 100 mM glycine buffer, pH 3.0. B: Same sample as C after the addition of 100 mM acetate buffer, pH 4.0. C: in 25 mM Imidazole-Cl, pH 7.0. 8B: A242E CsOxOx mutant. A: Same sample as B after the addition of 100 mM acetate buffer, pH 4.0, B: in 25 mM Imidazole-Cl, pH 7.0.</p