Compelling EPR evidence that the alternative oxidase is a diiron carboxylate protein

The alternative oxidase is a respiratory chain protein found in plants, fungi and some parasites that still remains physically uncharacterised. In this report we present EPR evidence from parallel mode experiments which reveal signals at approximately g = 16 in both purified alternative oxidase protein (g = 16.9), isolated mitochondrial membranes (g = 16.1), and in trypanosomal AOX expressed in Escherichia coli membranes (g = 16.4). Such signals are indicative of a dicarboxylate diiron centre at the active site of the enzyme. To our knowledge these data represent the first EPR signals from AOX present in its native environment.