Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4

This contribution describes the trapping of the hydroperoxyl radical at a pH of 4 during turnover of wild-type oxalate decarboxylase and its T165V mutant using the spin trap BMPO. Radicals were detected and identified by a combination of EPR and mass spectrometry. Superoxide, or its conjugate acid, the hydroperoxyl radical, is expected as an intermediate in the decarboxylation and oxidation reactions of the oxalate monoanion both of which are promoted by oxalate decarboxylase. Another intermediate, the carbon dioxide radical anion was also observed. The quantitative yields of superoxide trapping is similar in the wild type and the mutant while it is significantly different for the trapping of the carbon dioxide radical anion. This suggests that the two radicals are released from different sites of the protein

On the modeling of amplitude-sensitive electron spin resonance (ESR) detection using voltage-controlled oscillator (VCO)-based ESR-on-a-chip detectors

In this paper, we present an in-depth analysis of a voltage-controlled oscillator (VCO)-based sensing method for electron spin resonance (ESR) spectroscopy, which greatly simplifies the experimental setup compared to conventional detection schemes. In contrast to our previous oscillator-based ESR detectors, where the ESR signal was encoded in the oscillation frequency, in the amplitude-sensitive method, the ESR signal is sensed as a change of the oscillation amplitude of the VCO. Therefore, using VCO architecture with a built-in amplitude demodulation scheme, the experimental setup reduces to a single permanent magnet in combination with a few inexpensive electronic components. We present a theoretical analysis of the achievable limit of detection, which uses perturbation-theory-based VCO modeling for the signal and applies a stochastic averaging approach to obtain a closed-form expression for the noise floor. Additionally, the paper also introduces a numerical model suitable for simulating oscillator-based ESR experiments in a conventional circuit simulator environment. This model can be used to optimize sensor performance early on in the design phase. Finally, all presented models are verified against measured results from a prototype VCO operating at 14 GHz inside a 0.5 T magnetic field

Assigning the EPR fine structure parameters of the Mn(II) centers in bacillus subtilis oxalate decarboxylase by site-directed mutagenesis and DFT/MM calculations

Oxalate decarboxylase (OxDC) catalyzes the Mn-dependent conversion of the oxalate monoanion into CO2 and formate. EPR-based strategies for investigating the catalytic mechanism of decarboxylation are complicated by the difficulty of assigning the signals associated with the two Mn(II) centers located in the N- and C-terminal cupin domains of the enzyme. We now report a mutational strategy that has established the assignment of EPR fine structure parameters to each of these Mn(II) centers at pH 8.5. These experimental findings are also used to assess the performance of a multistep strategy for calculating the zero-field splitting parameters of protein-bound Mn(II) ions. Despite the known sensitivity of calculated D and E values to the computational approach, we demonstrate that good estimates of these parameters can be obtained using cluster models taken from carefully optimized DFT/MM structures. Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.This work was funded by the National Institute of General Medical Sciences (NIGMS) grant GM70641, by the National Institute of Environmental Health Sciences (NIEHS) grant ES002109, by the National Science Foundation (NSF) grant CHE-2002950, by the National Research Foundation of Singapore under the Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance Interdisciplinary Research Group, and by Stellate Therapeutics.Peer reviewe