A HIGHLY CONSERVED GLUTAMATE RESIDUE (GLU-270) IS ESSENTIAL FOR PLANTALTERNATIVE OXIDASE ACTIVITY

We have previously demonstrated that expression of a Sauromatum guttat um alternative oxidase in Schizosaccharomyces pombe confers cyanide-re sistant respiratory activity on these cells (Albury, M. S., Dudley, P. , Watts, F. Z., and Moore, A. L. (1996) J. Biol. Chem. 271, 17062-1706 6). Using this functional expression system we have investigated the a ctive site of the plant alternative oxidase, which has been postulated to comprise a non-heme binuclear iron center. Mutation of a conserved glutamate (Glu-270), previously postulated to be a bridging ligand wi thin the active site, to asparagine abolishes catalytic activity becau se mitochondria containing the E270N mutant protein do not exhibit ant imycin A-resistant respiration. Western blot analysis, using antibodie s specific for the alternative oxidase, revealed that the E270N mutant protein was targeted to and processed by S. pombe mitochondria in a m anner similar to that of the wild type protein. It is possible that la ck of antimycin A-insensitive respiration observed in mitochondria con taining the E270N mutant protein is due to incorrect insertion of the mutant alternative oxidase into the inner mitochondrial membrane. Howe ver, Western blot analysis of subfractionated mitochondria shows that both wild-type and E270N alternative oxidase are specifically located in the inner mitochondrial membrane, suggesting that misfolding or lac k of insertion is unlikely. These results provide the first experiment al evidence to support the structural model in which the active site o f the alternative oxidase contains a coupled binuclear iron center.