Poster: Photosynthesis

Abs # 374: Characterization of the active site of IMMUTANS, a plastid quinol oxidase with homology to AOX, by site-directed mutagenesis

Presenter:	Fu, Aigen , aigenfu@iastate.edu
Authors	Fu, Aigen  (A)   Barr, Jason  (A)   Rodermel, Steven R. (A)
Affiliations:	(A): Department of Genetics, Cellular and Development Biology, Iowa State University

IMMUTANS (IM) is an Arabidopsis chloroplast membrane protein with similarity to alternative oxidase (AOX), a mitochondrial inner membrane protein. It appears to serve as a quinol oxidase in carotenoid biosynthesis, as the terminal oxidase of chlororespiration, and as a novel electron sink during oxidative stress. Based on a revised structural model of AOX, we have conducted site-directed mutagenesis experiments to test a structural model of IM in which E136, E175, H178, E227, E296 and H299 are proposed to serve as Fe-binding sites in the reaction center. Four Glu residues were mutated to A, D, or H and two His residues were mutated to A, E, or N. In addition, H177, E224 and H298 were mutated to A as internal controls. We found that any change made to the 6 putative Fe-binding sites caused IM to lose its cyanide-resistant and propyl gallate-sensitive oxygen consumption activity. On the other hand, the proteins with mutations in H177, E224, and H298, which are adjacent to the Fe-ligands, exhibited wildtype activity. An allele of immutans missing exon-8 of the IM gene is able to produce normal levels of mRNA, but no IM protein is detected in the mutant plants. Sequence alignment showed that the exon-8 sequence is unique among plant plastid terminal oxidases and is not found in the mitochondrial AOX. After expression in E.coli, the mutant protein lacking exon-8 is produced but shows no activity, suggesting these sequences are important for IM function or folding. Finally, our results showed that plastoquinone, but not ubiquinone, is an efficient substrate of IM.