Life Sciences Division

Following the protons in Superoxide radical Detoxification.

Researchers at the Laboratoire de Biophysique du Stress Oxydant (DBJC/SBE) and the Laboratoire de Chimie et Biochimie des Centres Redox Biologiques (DRDC) have identified a hydroxide species at the active site of Superoxide Reductase which sheds light on protonation events and the mechanism of toxic superoxide radical elimination in the cell.

Until recently, the only known enzyme to eliminate toxic superoxide was superoxide dismutase (SOD).  In 2000,  the Laboratoire de Chimie et Biochimie des Centres Redox Biologiques discovered that another enzyme called Superoxide reductase (SOR), is also involved in superoxide detoxification, but using a completely different mechanism. 

The active site of SOR consists of a unique non-heme iron atom binding site involving coordination with four histidine residues and a cystein residue, and where the iron is exposed to the aqueous environment. Superoxide O₂^•- binds to this iron atom and is very efficiently reduced to H₂O₂ at difusion-limited rates. Conserved residues near the active site such as lysine are expected to play important electrostatic roles in the SOR reaction such as attracting superoxide anion.

3-D X-ray crystal structure of the SuperOxide Reductase from Desulfoarculus  baarsii (from D. Bourgeois, V. Nivière et coll. Structure 12, 1729-1740 (2004)). Superoxide binds at the iron centre. Binding of Cl- results from crystallization conditions

Protonation events play crucial roles in the elimination of toxic cellular superoxide by SOR since two protons are required for the formation of H₂O₂ from O₂^•-. Researchers in the Laboratoire de Chimie et Biochimie des Centres Redox Biologiques have shown that a base (B^-) in its protonated form (BH) is involved in the protonation events during the SOR catalytic cycle.  Using Raman spectroscopy, researchers in the Laboratoire de Biophysique du Stress Oxydant have investigated pH-dependent structural changes at the active site of SOR and have identified B^- as a hydroxide HO^- ligand at the iron active site of the SOR from the D. baarsii, implying that BH is H₂O. 
Thus, although the HO- ligand was found to interact with the conserved Lys48 residue, the study indicates that the protons involved in superoxide elimination are donated directly from the aqueous environment of the active site and not from protein amino acids. These findings are essential in understanding how the cell combats oxidative stress by the efficient elimination of the toxic superoxide species.

Proposed mechanism for Superoxide Reductase from D. baarsii. The values in parentheses refer to the absorption maxima of the active site. The 644 nm species refers to the active site in its final oxidized state coordinated with residue glutamate E47, while the 560 nm species corresponds to the active site with HO- ligand. During the SOR reaction, protonation of the Fe3+-OOH intermediate comes from a water molecule whose acidity is increased by H-bonding interactions with Lys48.