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.
Barranco-Medina
Sergio
S
Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008 Granada, Spain.
Krell
Tino
T
Finkemeier
Iris
I
Sevilla
Francisca
F
Lázaro
Juan-José
JJ
Dietz
Karl-Josef
KJ
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003080:Cold Temperature; D015180:Electrophoresis, Gel, Two-Dimensional; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D019216:Metals, Heavy / Q000494:pharmacology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D018532:Pisum sativum / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects
D019216:Metals, Heavy; D024101:Mitochondrial Proteins; D010940:Plant Proteins; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins