Biochemical and molecular characterization of the mitochondrial peroxiredoxin PsPrxII F from Pisum sativum.

The pea peroxiredoxin homologue PsPrxII F of the Arabidopsis thaliana mitochondrial AtPrxII F was isolated as cDNA and genomic DNA, and characterized in respect to its biochemical and molecular properties. The deduced amino acid sequence contains an N-terminal targeting address for mitochondrial import. Mitochondrial location of PsPrxII F was confirmed by immunocytochemistry. The mature enzyme, without the transit peptide, has a molecular mass of 18.75 kDa, and, at positions 59 and 84, carries the two catalytic cysteinyl residues which are characteristic for this particular Prx subgroup. Activity of site-directed mutagenized C84S-variant lacking the so-called resolving Cys dropped to about 12% of WT Prx while C59S lost its peroxidatic activity completely. Likewise, WT PsPrxII F and C84S-variant but not C59S protected plasmid DNA against strand breakage in a mixed function oxidation assay. WT PrxII F and the variant proteins aggregated to high mass oligomers not yet described for type II Prx. Upon oxidation with hydrogen peroxide PsPrxII F focussed in a series of spots of distinct pI but similar molecular masses in two-dimensional gels indicating different oxidation states of the protein. Using this technique, partial oxidation was also detected in leaf extracts and isolated mitochondria. PsPrxII F mRNA and protein accumulated in cold and heavy metals treated pea plants suggesting a particular function under stress.