The affinity of yeast and bacterial SCO proteins for CU(I) and CU(II). A capture and release strategy for copper transfer

Abstract

SCO (Synthesis of Cytochrome c Oxidase) proteins are present in prokaryotic and eukaryotic cells, and are often required for efficient synthesis of the respiratory enzyme cytochrome c oxidase. The Bacillus subtilis version of SCO (i.e., BsSCO) has much greater affinity for Cu(II) than it does for Cu(I) (Davidson and Hill, 2009), and this has been contrasted to mitochondrial SCO proteins that are characterized as being specific for Cu(I) (Nittis, George and Winge, 2001). This differential affinity has been proposed to reflect the different physiological environments in which these two members of the SCO protein family reside. In this study the affinity of mitochondrial SCO1 from yeast is compared directly to that of BsSCO in vitro. We find that the yeast SCO1 protein has similar preference for Cu(II) over Cu(I), as does BsSCO. We propose a mechanism for SCO function which would involve high-affinity binding to capture Cu(II), and relatively weak binding of Cu(I) to facilitate copper transfer.

Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis

“…SCO could act as a thiol–disulfide exchange protein specifically targeted to cytochrome c oxidase subunit II to keep the Cu A -associated cysteine residues in a reduced state . Copper binding studies show that SCO proteins form 1:1 complexes with both Cu­(I) and Cu­(II) ions, and a role for SCO in the delivery of copper to the Cu A site has been supported. , Building on the copper transfer model of SCO functionality, we have observed for Bs SCO and for SCO1 from yeast a strong preferential affinity for Cu­(II) over Cu­(I). , For Bs SCO, we have reported a Cu­(II) binding constant of 3.5 pM and a Cu­(I) binding constant of 10 μM. This extreme preference for Cu­(II) over Cu­(I) defines a redox-driven affinity switch for Bs SCO, a hypothesis distinctly different from the entatic-state model that explains the facile electron transfer functionality observed for cupredoxin proteins such as azurin .…”

“…38 The binding of Cu(II) leads to loss of approximately 70% of the fluorescence intensity from reduced apo-BsSCO (Figure 2a and Table 1), and we propose that much of this quenching is due to energy transfer mediated by the spectral overlap of tryptophan emission with the copper absorbance spectrum centered at 352 nm (Figure 2b). Titrations of apo-BsSCO with copper reveal such a tight interaction that binding affinity is difficult to assess quantitatively 39 but is estimated to be in the picomolar range by competition experiments 21,40 and by the effect of copper binding on thermal denaturation. 20 Circular dichroism in the far-UV region (i.e., UV-CD) is useful for assessing the secondary structural elements that compose a protein's overall structure and for characterizing changes that might occur upon ligand binding, 41 upon protein− protein interactions, 42 or during unfolding.…”

“…The binding of Cu­(II) leads to loss of approximately 70% of the fluorescence intensity from reduced apo- Bs SCO (Figure a and Table ), and we propose that much of this quenching is due to energy transfer mediated by the spectral overlap of tryptophan emission with the copper absorbance spectrum centered at 352 nm (Figure b). Titrations of apo- Bs SCO with copper reveal such a tight interaction that binding affinity is difficult to assess quantitatively but is estimated to be in the picomolar range by competition experiments , and by the effect of copper binding on thermal denaturation …”

“…10,19 Building on the copper transfer model of SCO functionality, we have observed for BsSCO and for SCO1 from yeast a strong preferential affinity for Cu(II) over Cu(I). 20,21 For BsSCO, we have reported a Cu(II) binding constant of 3.5 pM and a Cu(I) binding constant of 10 μM. This extreme preference for Cu(II) over Cu(I) defines a redoxdriven affinity switch for BsSCO, a hypothesis distinctly different from the entatic-state model that explains the facile electron transfer functionality observed for cupredoxin proteins such as azurin.…”

Lipoproteins in Gram-Positive Bacteria: Abundance, Function, Fitness

“…SCO homologs are found in many bacteria. They have a high affinity for Cu 2+ and are required for the proper synthesis of cytochrome c oxidase (Xu et al, 2015). Moreover, in S. aureus QoxA is an Lpp.…”