Abstract:

Cytochrome c oxidase (COX) is the primary determinant of cellular oxygen consumption and is thought to be important in regulating energy production. Understanding the regulation of COX is necessary to determine how respiration has evolved to accommodate metabolic adaptation. The COX holoenzyme is composed of three essential mitochondrially-encoded subunits, which form the catalytic core, and up to ten nuclear-encoded subunits whose functions may be primarily regulatory. I have taken two complementary approaches to exploring the role of these nuclear-encoded subunits in COX regulation.

I first compared sequences across taxa to investigate the evolution of the nuclear-encoded subunits. These subunits evolved subsequent to the origin of mitochondria but not all are shared between animals, yeasts, and plants. By mapping conserved amino acids onto the crystal structure of bovine COX, I showed that conserved residues are structurally organized into functional domains. These domains correspond both to known functional sites and to other uncharacterized regions. Amino acids important for structural stability are conserved at frequencies higher than expected within each taxon and groups of conserved residues cluster together more frequently than randomly selected residues. This suggests that selection is acting to maintain the structural foundation of COX across taxa, while active sites vary or coevolve within lineages.

The second approach employed experimental assays in Drosophila melanogaster to explore the functions of the nuclear-encoded subunits. The metabolic rate of male flies deficient for nuclear-encoded subunits is reduced while females are unaffected, suggesting that regulation of respiration may be sex-specific. To identify physiological cues that regulate COX activity, I tested two signaling pathways that are likely to interact with conserved elements of COX subunits identified in the comparative analysis. Insulin upregulates COX activity in Drosophila cell culture via an interaction with subunit Vb. Insulin signaling is not sufficient to rescue starvation-induced downregulation of COX. Also, as predicted by a conserved interaction site for thyroid hormone in subunit Va, 3,5-diiodothyronine (T2 upregulates COX in Drosophila . Juvenile hormone, the functional analog of T2 in insects, interacts with subunit Va to upregulate COX as well but appears to function through a different mechanism from T2 .