Sequences required for the activity of PTOX (IMMUTANS), a plastid terminal oxidase - In vitro and in planta mutagenesis of iron-binding sites and a conserved sequence that corresponds to exon 8

The thylakoid membranes of most photosynthetic organisms contain a terminal oxidase (PTOX, the product of the Arabidopsis IMMUTANS gene) that functions in the oxidation of the plastoquinone pool. PTOX and AOX are diiron carboxylate proteins, and based on crystal structures of other members of this protein class, a structural model of PTOX has been proposed in which the ligation sphere of the diiron center is composed of six conserved histidine and glutamate residues. We tested the functional significance of these residues by site-directed mutagenesis of PTOX in vitro and in planta, taking advantage null immutans alleles for the latter studies. These experiments showed that the six iron-binding sites do not tolerate change, even conservative ones. We also examined the significance of a conserved sequence in (or near) the PTOX active site that corresponds precisely to Exon 8 of the IM gene. In vitro and in planta mutagenesis revealed that conserved amino acids within this domain can be altered but that deletion of all or part of the domain abolishes activity. Because protein accumulates normally in the deletion mutants, the data suggest that the conformation of the Exon 8 sequence is important for PTOX activity. An allele of immutans (designated 3639) was identified that lacks the Exon 8 sequence; it does not accumulate PTOX protein. Chloroplast import assays revealed that mutant enzymes lacking Exon 8 have enhanced turnover. We conclude that the Exon 8 domain is required not only for PTOX activity but also for its stability.