Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii

Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the al ternative oxidases (AOXs) from Chlamydomonas, reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open reading frames (ORFs) encoding putative proteins of 360 amino acids and 347 amino acids, respecti vely. For each of the ORFs, a potential mitochondrial-targeting sequence wa s found in the 5'-end regions. In comparison to AOX enzymes from plants and fungi, the predicted amino acid sequences of the ORFs showed their highest degree of identity with proteins from Aspergillus niger (38.1% and 37.2%) and Ajellomyces capsulatus (37% and 34.9%). Several residues supposed eithe r to be Fe ligands or to be involved in the ubiquinol-binding site were ful ly conserved in both C. reinhardtii putative AOX proteins. In contrast, a c ysteine residue conserved in the sequences of all higher plants and probabl y involved in the regulation of the enzyme activity was missing both from t he AOX1 and AOX2 amino acid sequences and from protein sequences from vario us other microorganisms. The transcriptional expression of the AOX1 and AOX 2 genes in wild-type cells and in mutant cells deficient in mitochondrial c omplex III activity was also investigated.