The mitochondrial F-ATP synthase synthesises ATP molecules, the energy currency of life, by utilising the proton transmembrane gradient force generated from the oxidation of nutrients in the electron transport chain. The bioenergetic cost (in protons) to make one ATP molecule is proportional to the size of the c-ring (i.e. number of the c-subunits) of the protein. Traditionally, this was thought to be 3.3-5 protons, until the recent discovery that it is more efficient in bovine ATP synthase. Compared with the c10-15 F-ATP synthase driving rings in yeast, eubacterial, and chloroplast ATP synthases, the bovine molecular machine has a c8-ring instead. Studies indicated that trimethylation of lysine 43 of the c-subunit is critical for the formation of the c8-ring. Since the amino acid sequences of c-subunits are identical in almost all vertebrates and are highly conserved across other Metazoans, it is suggested that all vertebrates and probably all, or most, invertebrates would contain c8-rings and the bioerengetic cost to produce one ATP molecule is 2.7 protons in all animals. In the present project, this hypothesis was examined by isolation of whole F-ATP synthase complex or c-subunits alone purified from selected animals by applying a GST tagged ligand purification method or by iodixanol gradient purification of mitochondria followed by chloroform/methanol extraction of the c-subunits. C-subunits were then separated on SDS-PAGE and several mass spectrometric techniques used to characterise any modifications of the c-subunits. C-subunits from possum, kina, sea cucumbers and sponge were isolated and characterised in this thesis and a total of eighteen vertebrate and fifteen invertebrate organisms were surveyed in the overall study. In every case, a high intensity peak of mass 1343 was detected when c-subunit chymotryptic digests were analysed by MALDI-TOF MS. Furthermore, peptides from chymotryptic digests of c-subunits analysed by ETD-MS with Orbitrap MS confirmed lysine 43 as the trimethylated residue in all species surveyed in these experiments. We conclude that all metazoan c subunits are trimethylated and their F-ATP synthases are driven by c8-rings. This evolutionary bioenergetics study was shared between Lincoln University and the Mitochondrial Biology Unit (MBU) Cambridge UK.[Show full abstract]
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