A biochemical and molecular characterisation of the alternative oxidases

The alternative oxidase (AOX) is a respiratory protein found in the mitochondrial electron transfer pathway throughout plants, sporadically in fungi and protozoa and within a small percentage of animals and proteobacteria. The AOX branches from the classical respiratory chain at the ubiquinone pool by coupling the oxidation of two molecules of ubiquinol to the complete 4-electron reduction of oxygen to water. The AOX can be found within several pathogenic organisms such as Trypanosoma bruceii and Cryptosporidium parvum and due to its absence in the mammalian host the AOX proves to be a potential therapeutic target in these systems. Recent crystallisation of the trypanosomal alternative oxidase (TAO) has enabled a more detailed examination of the structure of all AOXs, through homology modelling. Comparisons of sequence alignments of multiple AOXs and AOX homology models has revealed high conservation of residues predicted to be involved in ubiquinol binding and a proton coupled electron transport (PCET) network proposed to be involved in oxygen reduction. Recombinant wild type TAO and Sauromatum guttatum AOX (SgAOX) have been expressed in a haem deficient strain of E. coli, in addition to a number of mutants that contain mutations occuring within the ubiquinol binding pocket and the PCET. Over-expression and purification of recombinant wild type AOX and mutants has enabled the characterisation of residues within the ubiquinol binding site and PCET network, contained within the secondary ligation sphere of the AOX. The results of which confirmed previously proposed roles for these residues, drawn from the crystal structure of TAO and models of the redox cycle mechanisms. A novel AOX was identified within the fungus that causes Ash Dieback, Hymenoscyphus fraxineus. Protocols for the over expression and purification were established for the recombinant H. fraxineus AOX. This protein revealed significantly lower ubiquinol oxidase activities but a similar Km for oxygen and ubiquinol when compared to that previously seen with TAO and SgAOX. Typical potent AOX inhibitors, such as ascofuranone, were shown to be significantly less effective at inhibiting HfAOX. This led to testing inhibitors against multiple AOXs from different kingdoms. It was found that not only did the efficiency of inhibitors vary between AOXs but the ubiquinol oxidase rates varied as well. The results presented in this thesis suggest that the differences in inhibitor efficacy and ubiquinol oxidase activity are caused by the functional structure of the residues lining the inhibitor/substrate channel