EPR studies of the mitochondrial alternative oxidase. Evidence for a diiron carboxylate center.
The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.
Berthold
Deborah A
DA
Department of Biochemistry and Biophysics, Stockholm University Svante Arrhenius väg 16, Sweden. berthold@dbb.su.se
Voevodskaya
Nina
N
Stenmark
Pål
P
Gräslund
Astrid
A
Nordlund
Pär
P
D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000201:enzymology; D001669:Biochemical Phenomena; D001671:Biochemistry; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D007501:Iron / Q000737:chemistry*; D008024:Ligands; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D010940:Plant Proteins; D013997:Time Factors; D000099188:Alternative Oxidase
D007501:Iron; D008024:Ligands; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D000099188:Alternative Oxidase