Reconstitution of the Mitochondrial PrxIII Antioxidant Defence Pathway: General Properties and Factors Affecting PrxIII Activity and Oligomeric State
- 1 September 2007
- journal article
- Published by Elsevier BV in Journal of Molecular Biology
- Vol. 372 (4), 1022-1033
- https://doi.org/10.1016/j.jmb.2007.07.018
Abstract
The mitochondrial 2-Cys peroxiredoxin PrxIII serves as a thioredoxin-dependent peroxidase operating in tandem with its cognate partners, an organelle-specific thioredoxin (Trx2) and NADP-linked thioredoxin reductase (TRR2). This PrxIII pathway is emerging as a primary regulator of intracellular H2O2 levels with dual roles in antioxidant defence and H2O2-mediated signalling. Here we describe the reconstitution of the mammalian PrxIII pathway in vitro from its purified recombinant components and investigate some of its overall properties. Employing the site-directed PrxIII mutants C47S, C66S and C168S, the putative N and C-terminal catalytic cysteine residues are shown to be essential for function whereas the C66S mutant retains full activity. The pathway attains maximal capacity at low H2O2 concentrations (2O2 levels. The presence of an N-terminal His-tag on PrxIII markedly enhances dodecamer stability, particularly apparent in its oxidised state. Its removal promotes oxidised PrxIII dissociation into dimers and leads to a 3.0–3.5-fold stimulation in peroxidase activity. The unusual concatenated crystal structure of PrxIII consisting of two-interlocked dodecameric rings is also evident in dilute solution employing transmission electron microscopy; however, it represents only 3–5% of the population with most molecules present as single toroids. Moreover, concatenated PrxIII C168S reverts to single toroids on crystal dissolution indicating that these higher-order structures are produced dynamically during the crystallisation process.Keywords
This publication has 43 references indexed in Scilit:
- Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen SpeciesJournal of Neuroscience, 2004
- Knockout mice heterozygous forSod2show alterations in cardiac mitochondrial function and apoptosisAmerican Journal of Physiology-Heart and Circulatory Physiology, 2001
- Mice Deficient in Cellular Glutathione Peroxidase Develop Normally and Show No Increased Sensitivity to HyperoxiaPublished by Elsevier BV ,1997
- TheGpx1Gene Encodes Mitochondrial Glutathione Peroxidase in the Mouse LiverArchives of Biochemistry and Biophysics, 1997
- Superoxide Production by the Mitochondrial Respiratory ChainBioscience Reports, 1997
- Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice.Proceedings of the National Academy of Sciences of the United States of America, 1996
- Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutaseNature Genetics, 1995
- Distribution of glutathione peroxidases glutathione reductase in rat brain mitochondriaFEBS Letters, 1991
- Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondriaArchives of Biochemistry and Biophysics, 1977
- The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygenBiochemical Journal, 1973