functional analyses of variants of human SCO1, a mitochondrial metallochaperone

Abstract

Cytochrome c oxidase (COX) is a multimeric protein complex whose enzymatic activity contributes to the generation of an electrochemical potential required to synthesize adenosine triphosphate (ATP). Synthesis of Cytochrome c Oxidase 1 (SCO1) and SCO2 are two of the many accessory factors that are required to assemble individual structural subunits of COX into a functional holoenzyme complex. Mutations in either SCO gene cause severe, early onset forms of human disease. SCO1 and SCO2 are closely related paralogues localized to the inner mitochondrial membrane. Both proteins bind copper and exhibit a thiol disulphide oxidoreductase activity. Copper is bound by a highly conserved Cysteine x x x Cysteine motif and a histidine found within a thioredoxin fold, which is contained in the C-terminal half of the protein and projects into the mitochondrial intermembrane space. Mutations in either SCO1 or SCO2 affect their ability to deliver copper to COX II and metallate its CuA site, and also result in an increased rate of copper efflux from the cell. However, the relative importance of the ability to bind and transfer copper to SCO protein function remains poorly understood. Therefore, to investigate the significance of several cysteine residues and the conserved histidine to the copper-binding properties of SCO1, I functionally characterized a series of N- and C-terminal SCO1 mutant proteins by transducing them into control and patient fibroblasts, and quantifying their phenotypic effect on COX activity. I found that the two cysteines within the soluble, N-terminal matrix domain of SCO1 are not required for protein function. Overexpression of C-terminal SCO1 mutants only affected COX activity in SCO1-2 patient fibroblasts. To further characterize the copper-binding properties of these C-terminal mutants, soluble forms of each SCO1 variant were expressed and purified from bacteria, and the amount of total bound copper and the relative abundance of Cu(I) and Cu(II) were quantified. Although these analyses suggested that one mutant, SCO1 C169H, binds significantly more Cu(I) than the wild-type protein, none of the SCO1 variants exhibited properties that furthered our understanding of the precise role of SCO1 in the biogenesis of the CuA site of COX II.