Probing the active site of the alternative oxidase by site-directed mutagenesis

In addition to a conventional cytochrome c oxidase, higher plants contain a cyanide-resistant alternative oxidase (AOX). Significant progress in the characterisation of this oxidase has included isolation of cDNAs encoding the AOX from a number of species. Analysis of the deduced amino acid sequences reveals a high degree of homology especially between the plant sequences and also shows the presence of three copies of an iron-binding motif (Glu-X-X-His). Lack of absorbance above 350 nm and the presence of these motifs, led to a hypothetical structure of the catalytic site of the AOX in which the active-site contains a coupled binuclear iron centre analogous to that found in methane monooxygenase and ribonucleotide reductase (Siedow et al., 1995, FEBS Lett. 362:10-14). Since this model was put forward a revised hypothetical structure has appeared (Andersson & Nordlund 1999, FEBS Lett. 449:17-22). Both the original model and the revised model classify the AOX as a member of the di-iron carboxylate family of proteins whilst the major difference concerns the topology of the enzyme. In order to probe the structure-function relationship of the plant AOX we have developed a functional yeast expression system and have previously demonstrated that Glu 270, proposed to be involved in binding iron, is essential for activity (Albury et al., 1998, J. Biol. Chem., 273:30301-30305). We are continuing to probe the structure of the AOX by mutating residues both within the active site and also in a putative quinone binding region. Data from respiratory analyses of these mutants, some of which result in a partially active AOX, will be presented and the structure of the AOX will be discussed in the light of these results