Abstract
The plastid terminal oxidase (PTOX) is distantly related to the mitochondrial alternative oxidase (AOX). Both are members of the diiron carboxylate quinol oxidase (DOX) class of proteins. PTOX and AOX contain 20 highly conserved amino acids, six of which are Fe-binding ligands. We have previously used in vitro and in planta activity assays to examine the functional importance of the Fe-binding sites. In this report, we conduct alanine-scanning mutagenesis on the 14 other conserved sites using our in vitro and in planta assay procedures. We found that the 14 sites fall into three classes: (i) Ala-139, Pro-142, Glu-171, Asn-174, Leu-179, Pro-216, Ala-230, Asp-287, and Arg-293 are dispensable for activity; (ii) Tyr-234 and Asp-295 are essential for activity; and (iii) Leu-135, His-151, and Tyr-212 are important but not essential for activity. Our data are consistent with the proposed role of some of these residues in active site conformation, substrate binding, and/or catalysis. Titration experiments showed that down-regulation of PTOX to approximately 3% of wild-type levels did not compromise plant growth, at least under ambient growth conditions. This suggests that PTOX is normally in excess, especially early in thylakoid membrane biogenesis.
Highlights
Sumption was monitored at 25 °C with a Clark O2 electrode alternative oxidase (AOX) and Plastid Terminal Oxidase (PTOX) have unique structural domains: AOX (Hansatech, Norfolk, England) using 100 –200 g of membrane contains a “dimerization domain” (D-domain) that has been protein in a 1.5-ml reaction mixture that contained a final con- implicated in AOX-AOX dimer formation [36], and PTOX
We determined the functional significance of 14 other amino acid residues that are highly conserved between AOX and PTOX, and we identified five additional amino acid residues (Leu-135, His-151, Tyr-212, Tyr-234, and Asp-295) that are essential for in vitro PTOX activity; two of these (Tyr-234 and Asp-295) are essential for activity in planta (Fig. 5)
These five amino acid residues reside in areas of AOX that have been proposed to be important for catalysis, protein stability, and/or substrate binding
Summary
Plant Growth—Plants in this report included wild-type Arabidopsis (Columbia ecotype), the spotty allele of im ( in Columbia) [10], and transgenic im plants (described below). The plants were germinated and grown at 22 °C under conditions of continuous illumination. The wild-type plants were maintained at 100 mol mϪ2 sϪ1. Spotty is light sensitive and was germinated under low light (15 mol mϪ2 sϪ1 for 5 days) before transfer to normal light (100 mol mϪ2 sϪ1). Site-directed Mutagenesis in Vitro—An IM cDNA lacking the N-terminal chloroplast-targeting sequence [10] was cloned into the BamH1 and Nhe sites of the Escherichia coli expression vector pET-11a (Novagen, Madison, WI). The QuikChangeTM site-directed mutagenesis protocol (Stratagene, La Jolla, CA) was used to generate mutations; the mutations were confirmed by sequencing. The mutant clones are designated by the amino acid that was altered, its location in the A. thaliana IM sequence, and the amino acid to which it was changed (e.g. L135A means Leu-135 was changed to Ala)
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