Reconstitution of the mitochondrial PrxIII antioxidant defence pathway: general properties and factors affecting PrxIII activity and oligomeric state.
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.
Cao
Zhenbo
Z
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Bhella
David
D
Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, Scotland, UK.
Lindsay
J Gordon
JG
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.
D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000975:Antioxidants; D024101:Mitochondrial Proteins; D016877:Oxidants; D011994:Recombinant Proteins; C467002:TXN2 protein, human; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase