Abstract
Sco1 is implicated in the copper metallation of the Cu(A) site in Cox2 of cytochrome oxidase. The structure of Sco1 in the metallated and apo-conformers revealed structural dynamics primarily in an exposed region designated loop 8. The structural dynamics of loop 8 in Sco1 suggests it may be an interface for interactions with Cox17, the Cu(I) donor and/or Cox2. A series of conserved residues in the sequence motif (217)KKYRVYF(223) on the leading edge of this loop are shown presently to be important for yeast Sco1 function. Cells harboring Y219D, R220D, V221D, and Y222D mutant Sco1 proteins failed to restore respiratory growth or cytochrome oxidase activity in sco1Delta cells. The mutant proteins are stably expressed and are competent to bind Cu(I) and Cu(II) normally. Specific Cu(I) transfer from Cox17 to the mutant apo-Sco1 proteins proceeds normally. In contrast, using two in vivo assays that permit monitoring of the transient Sco1-Cox2 interaction, the mutant Sco1 molecules appear compromised in a function with Cox2. The mutants failed to suppress the respiratory defect of cox17-1 cells unlike wild-type SCO1. In addition, the mutants failed to suppress the hydrogen peroxide sensitivity of sco1Delta cells. These studies implicate different surfaces on Sco1 for interaction or function with Cox17 and Cox2.
Highlights
Sco1-Cox2 Interaction Interface activity in SCO1 patient fibroblasts is partially rescued by overexpression of the P174L mutant protein, but the cellular copper deficiency is only rescued by overexpression of SCO2
We showed previously that neither Gln233 nor Asp234 was important for Sco1 function, but Asp238 was functionally important and influenced Cu(II) binding to Sco1 (16)
Supplemental copper salts cannot confer growth of sco1⌬ cells (4), but partial growth was restored in sco1⌬ cells harboring Y219D, R220D, and V221D, but not Y222D mutant alleles when plated on rich medium with glycerol/lactate as carbon sources
Summary
An additional important aspect of human and yeast Sco function is the ability to bind a Cu(II) ion in a type II-like site with a higher coordination number than the trigonal Cu(I) site (16). It is unclear whether Sco transfers both Cu(I) and Cu(II) ions to build the mixed valent, binuclear CuA site in Cox. SCO1 patient fibroblasts exhibit CcO deficiency along with a severe cellular copper deficiency (19). Defective Cox17-mediated copper metallation of Sco, and subsequent failure of CuA site maturation, is the basis for the inefficient assembly of the CcO complex in SCO1 patient fibroblasts
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