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

Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes

Dynamic nuclear polarization (DNP) enhanced solid-state NMR can provide orders of magnitude in signal enhancement. One of the most important aspects of obtaining efficient DNP enhancements is the optimization of the paramagnetic polarization agents used. To date, the most utilized polarization agents are nitroxide biradicals. However, the efficiency of these polarization agents is diminished when used with samples other than small molecule model compounds. We recently demonstrated the effectiveness of nitroxide labeled lipids as polarization agents for lipids and a membrane embedded peptide. Here, we systematically characterize, via electron paramagnetic (EPR), the dynamics of and the dipolar couplings between nitroxide labeled lipids under conditions relevant to DNP applications. Complemented by DNP enhanced solid-state NMR measurements at 600 MHz/395 GHz, a molecular rationale for the efficiency of nitroxide labeled lipids as DNP polarization agents is developed. Specifically, optimal DNP enhancements are obtained when the nitroxide moiety is attached to the lipid choline headgroup and local nitroxide concentrations yield an average e^––e^– dipolar coupling of 47 MHz. On the basis of these measurements, we propose a framework for development of DNP polarization agents optimal for membrane protein structure determination