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J Mol Biol. 2007 Sep 28;372(4):1022-1033. doi: 10.1016/j.jmb.2007.07.018. Epub 2007 Jul 21.

Reconstitution of the mitochondrial PrxIII antioxidant defence pathway: general properties and factors affecting PrxIII activity and oligomeric state.

Author information

1
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
2
Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, Scotland, UK.
3
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. Electronic address: G.Lindsay@bio.gla.ac.uk.

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 H(2)O(2) levels with dual roles in antioxidant defence and H(2)O(2)-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 H(2)O(2) concentrations (<10 microM) and is progressively inhibited in the range 0.1 mM to 1.0 mM peroxide. Damage to PrxIII caused by over-oxidation is confirmed by the appearance of abnormal oxidised species of PrxIII on SDS-PAGE at elevated H(2)O(2) 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.

PMID:
17707404
DOI:
10.1016/j.jmb.2007.07.018
[Indexed for MEDLINE]

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