EPR spin trapping of an oxalate-derived free radical in the oxalate decarboxylase reaction

Abstract

EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly 13C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution.

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

“…Table 1 shows the magnetic parameters used in the simulation together with their corresponding spectral weights. The production of a CO 2 •− adduct was expected and had been previously reported as being derived from oxalate [37]. Unexpectedly, the major contributor to the spectrum was the hydroperoxyl adduct with a combined spectral weight of 90.1% for the two diastereomers, while the carbon dioxide anion radical contributed only 9.9% according to the simulated spectra.…”

“…If superoxide and the CO 2 •− intermediate are generated in the same active site pocket, formation of a peroxycarbonate species is the logical next step and one would not expect to observe either CO 2 •− or hydroperoxyl in solution [27]. On the other hand, release of the carbon dioxide radical anion into solution, e.g ., through loss of control of this intermediate by the protein, may automatically lead to superoxide production in solution since this radical will react with oxygen [37]. However, this is not likely to happen under our experimental conditions since the high concentration of spin trap will outcompete oxygen for the reaction with CO 2 •− .…”

“…Current mechanistic proposals suggest that dioxygen is bound to one of two Mn ions and acts as a transient electron sink to destabilize the carbon-carbon bond in oxalate resulting in a bound superoxide radical. After decarboxylation has taken place superoxide acts as an electron source to reduce the resulting carbon dioxide anion radical (see Scheme 2) [37]. This mechanism requires oxygen to cycle through its 1-electron reduced state as superoxide which would likely be protonated in the pH range in which the enzyme is active [38].…”

“…Results from spin-trapping experiments on the T165V OxDC mutant suggested that the destabilization of the closed conformation of this lid region leads to increased loss of the intermediate carbon dioxide radical anion into solution where it reacts diffusion-controlled with dioxygen to produce superoxide and eventually hydrogen peroxide ( i.e., the same products expected for oxidase activity) [37]. Even in WT OxDC the CO 2 •− radical intermediate can be trapped by appropriate spin traps suggesting that the 0.2% oxidase activity might be simply due to loss of this intermediate from the active site [37, 41].…”

“…Even in WT OxDC the CO 2 •− radical intermediate can be trapped by appropriate spin traps suggesting that the 0.2% oxidase activity might be simply due to loss of this intermediate from the active site [37, 41]. Spin trapping of OxOx under turnover conditions also yields a CO 2 •− radical adduct with both PBN and DMPO spin traps [44, 45].…”