Reconstitution of the mitochondrial PrxII antioxidant defence pathway: General properties and factors affecting PrxII activity and oligomeric state

Cao, Z., Bhella, D. and Lindsay, J.G. (2007) Reconstitution of the mitochondrial PrxII antioxidant defence pathway: General properties and factors affecting PrxII activity and oligomeric state. Journal of Molecular Biology, 372, pp. 1022-1033. (doi:10.1016/j.jmb.2007.07.018)

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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 (<10 μM) 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 H2O2 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Cao, Dr Zhenbo and Bhella, Dr David and Lindsay, Professor Gordon
Authors: Cao, Z., Bhella, D., and Lindsay, J.G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Molecular Biology

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