Bovine mitochondrial SP-22 is a member of the peroxiredoxin family of peroxidases. It belongs to the peroxiredoxin 2-Cys subgroup containing three cysteines at positions 47, 66, and 168. The cloning and overexpression in Escherichia coli of recombinant wild type SP-22 and its three cysteine mutants (C47S, C66S, and C168S) are reported. Purified His-tagged SP-22 was fully active with Cys-47 being confirmed as the catalytic residue. The enzyme forms a stable decameric toroid consisting of five

more »

... ic dimeric units containing intermolecular disulfide bonds linking the catalytically active Cys-47 of one subunit and Cys-168 of the adjacent monomer. The disulfide bonds are not required for overall structural integrity. The toroidal units have average external and internal diameters of 15 and 7 nm, respectively, and can form stacks in a lateral arrangement of two or three rings. C47S had a pronounced tendency to stack in long tubular structures containing up to 60 rings. Further unusual structural features are the presence of radial spikes projecting from the external surface and ordered electron-dense material within the central cavity of the toroid. Oxidative stress arises as a consequence of an imbalance in the metabolism of redox-active species. The principal targets of oxidative damage are the key biomolecules of the cell, namely proteins, lipids, and nucleic acids. Accumulation of these damaged biomolecules is contributory to, or characteristic of, the aging process and several major pathologies, particularly neurodegenerative diseases (1, 2). All aerobic organisms generate reactive oxygen species (ROS), 1 as a consequence of normal cellular metabolism, principally via oxidative phosphorylation in the mitochondrion (1, . 1 The abbreviations used are: ROS, reactive oxygen species; AhpC, AhpD, and AhpF, alkyl hydroperoxide reductase subunits C, D and F; C47S, C66S, and C168S, bovine SP-22 mutant proteins, where cysteine 47, 66 and 168 were replaced with serine residues; DTT, dithiothreitol; E2, dihydrolipoyl acetyl transferase; E3, dihydrolipoamide dehydrogenase; FRS, free radical-generating system; PRDX5, human peroxiredoxin 5; SP-22, substrate protein 22; TEM, transmission electron microscopy; Trx, thioredoxin.