Superoxide reductase SOR was recently described as a new bacterial antioxidant system that eliminates the superoxide radical by reducing it to H2O2 without forming O2. A remarkable feature of its active site, a mononuclear non-haem iron centre, is that is has a cysteine ligand occupying an axial position.
This work shows that the mutation of a glutamate residue interacting with this cysteine induces a highly specific attenuation of the strength of the cysteine-iron bond (S-Fe). An investigation into the reactivity of this mutant with the superoxide revealed a direct correlation between the strength of the S-Fe bond and the ability of the active site to form the product of the H
2O
2 reaction. It seems that sulphur, when acting as an electron-donor to iron, promotes protonation of the iron-peroxo intermediate (Fe-O-O
-) formed during the catalytic cycle, resulting in the release of H
2O
2.
This data demonstrates the key role played by the second coordination sphere residues of the metal in SOR activity. The presence of glutamate makes it possible to finely modulate the potential of cysteine to act as an electron-donor to iron in order to optimise SOR activity.
This work presents the first experimental proof of the role played by this cysteine in the defence mechanism against superoxide radicals. Furthermore, the glutamate mutant described in this work has made it possible to trap the iron-peroxo reaction intermediate in SOR crystals and resolve its structure using X-ray diffraction (Science, 2007). These results provide a better understanding of the cellular mechanisms that provide an effective line of defence against oxygen-derived toxic species).