The P174L Mutation in Human Sco1 Severely Compromises Cox17-dependent Metallation but Does Not Impair Copper Binding

Abstract

Sco1 is a metallochaperone that is required for copper delivery to the Cu A site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1⌬ yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu A site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.

Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase

“…The essential requirement of two Sco proteins in humans is therefore accounted for by these different, specific functions. Notably, both human Sco proteins are active in their copper-bound forms, accounting for the observation that mutation of any of the copper ligands severely affects the oxidase activity and cellular respiration (40,41). The most frequent pathological mutations (P174L in Sco1 and E140K in Sco2) are located close to the CX 3 CX n H motif, and thus expected to alter either the copper-binding or the redox capabilities of these proteins.…”

Mapping the Functional Interaction of Sco1 and Cox2 in Cytochrome Oxidase Biogenesis

“…The molecular defect in the P174L mutant Sco1 is an impaired ability to be copper metallated by Cox17 (7,20). The defect is attributed to an attenuated interaction with Cox17 (20), in addition to a modest structural defect that attenuates the Cu(I) binding affinity (7). Defective Cox17-mediated copper metallation of Sco1, and subsequent failure of Cu A site maturation, is the basis for the inefficient assembly of the CcO complex in SCO1 patient fibroblasts.…”

“…The P174L substitution is adjacent to the second Cys in the Cu(I)-binding CX 3 C motif in Sco1, yet the mutant protein retains the ability to bind Cu(I) or Cu(II) when expressed in bacteria. The molecular defect in the P174L mutant Sco1 is an impaired ability to be copper metallated by Cox17 (7,20). The defect is attributed to an attenuated interaction with Cox17 (20), in addition to a modest structural defect that attenuates the Cu(I) binding affinity (7).…”

Building the CuA site of cytochrome c oxidase: A complicated, redox-dependent process driven by a surprisingly large complement of accessory proteins

“…One copper atom is bound per monomer, and mutations that impair Cu(I) or Cu(II) binding result in a non-functional SCO protein, arguing that this property is crucial to their roles in COX assembly (71). The importance of copper binding to SCO1 function is emphasized by two parallel studies that both found the pathogenic P174L substitution in SCO1 severely compromises COX17-dependent copper transfer (72,73). Whether mutations in SCO2 also impair copper transfer from COX17 remains unclear.…”

“…Mutations in residues within this loop 8 motif do not affect physical interactions with Cox17 or copper-binding, arguing that Sco1 uses distinct interfaces to interact with Cox17 and Cox2 (76). Pulse-chase labeling of mitochondrial translation products in human cells established that impaired SCO1 function did not affect COX2 synthesis but resulted in accelerated turnover of the newly synthesized protein (73). In contrast, COX2 synthesis is greatly reduced in SCO2 patient cells, yet the residual protein that is made is much more stable than in control cells (31).…”