Abstract
The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.
Highlights
The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals
The four invariant glutamate residues and two histidines correspond in Ciona AOX with E200, E239, E290, E344, H242 and H347 (Fig. S1), numbered from the first methionine of the putative preprotein
We tested the functional significance of the conserved residues at the predicted diiron centre, by mutating four of them to alanine (E239A, H242A, E344A, H347A), in appropriate transgenic constructs for expression in mammalian cells and Drosophila (Fig. S2)
Summary
The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. The enzyme is inherently responsive to the metabolic signature of such stresses in different organisms It is activated by high levels of its reduced substrate, ubiquinol[4,5], which is assumed to reflect a lower affinity for the substrate than that exhibited by OXPHOS complex III, with which it competes. Expression of an inert transgene, such as GFP, in place of AOX, was unable to rescue the phenotypes produced by engineered deficiency of cytochrome oxidase[10,11]. This control cannot be unambiguously interpreted, since the expressed GFP was not targeted to mitochondria, and even if it were, does not possess other structural features of AOX that enable it to insert into the inner mitochondrial membrane in a specific fashion and interact with other components thereof
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