The alternative oxidase is an integral monotopic membrane protein that is tightly bound to the inner mitochondrial membrane from matrix side The enzyme 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 amino acid residues. The electron spin resonance study of Arabidopsis thaliana alternative oxidase AOX1a showed that the enzyme contains a hydroxo-bridged mixed-valent Fe(II)/Fe(III) binuclear iron center. A catalytic cycle has been proposed that involves this di-iron center and at least one transient protein-derived free radical, which is probably formed on a tyrosine residue.