Regulation of the plant alternative oxidase by pyruvate

In addition to a conventional cytochrome pathway, plant mitochondria contain a second, nonprotonmotive route of electron transfer, comprised of a single ubiquinol:oxygen oxidoreductase, the alternative oxidase (AOX). This enzyme is regulated by two interrelated posttranslational mechanisms. Firstly, reduction of an intermolecular disulphide bond results in an ‘activated’ noncovalently linked dimer. Secondly, the reduced protein is further stimulated by a-keto acids, most notably pyruvate. This is thought to occur via a-keto acid interaction with a well-conserved cysteine residue. Previously, we have established a system to functionally express the Sauromatum guttatum alternative oxidase (Sg-AOX) in the fission yeast Schizosaccharomyces pombe. Interestingly, the resulting antimycin-resistant respiratory activity in isolated yeast mitochondria, does not appear to be stimulated by pyruvate. Here, we report on the expression of both Sg-AOX as well as a second plant isozyme, the Arabidopsis thaliana AOX1a protein (At-AOX1a), in the same yeast system. In contrast to Sg-AOX, At-AOX1a-dependent activity can be stimulated by pyruvate f1.4-fold in isolated yeast mitochondria and f4.5-fold in isolated mitochondrial membranes depleted of endogenous pyruvate. Whilst Sg AOX activity is confirmed to be completely independent of pyruvate, its dependence upon the Q-redox poise would suggest that it is in a constitutively active state, comparable to the ‘pyruvate-activated’ kinetic dependence of At AOX1a. These data indicate that Sg AOX is the first example of a plant enzyme that appears to function without a dependence on organic acids for full activity. This finding is of particular interest, given that both Sg AOX and At AOX1a conserve the cysteine residue believed to interact directly with pyruvate. As both proteins exhibit a very similar primary structure, we have been able to identify structural components, additional to the regulatory cysteine, that may account for the differences in the regulatory behaviour reported here. The implications of such findings are discussed in terms of the proposed structure of AOX