In vitro characterization of a plastid terminal oxidase (PTOX).
The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.
Josse
Eve-Marie
EM
Université J. Fourier and CNRS, Grenoble, France.
Alcaraz
Jean-Pierre
JP
Labouré
Anne-Marie
AM
Kuntz
Marcel
M
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D019343:Citric Acid / Q000494:pharmacology; D016384:Consensus Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology; D008293:Malates / Q000494:pharmacology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology; D011809:Quinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D029681:Arabidopsis Proteins; D019343:Citric Acid; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D005707:Gallic Acid; D007501:Iron; D008293:Malates; D010088:Oxidoreductases; D011809:Quinones; D012965:Sodium Chloride; C016627:octyl gallate; C405742:IM protein, Arabidopsis