SCO1 Human

What is the structural organization of human SCO1?

Human SCO1 (HSco1) is a 301-residue polypeptide anchored to the inner mitochondrial membrane through a single transmembrane helix. The functional part consists of a single soluble domain located in the C-terminal region, while the N-terminus contains a mitochondrial-targeting sequence followed by the transmembrane helix . The protein adopts a thioredoxin-like fold containing four α-helices and seven β-strands organized in two β-sheets .

The metal-binding site of HSco1 is constituted by two cysteine residues present in a conserved CPXXCP motif and a fully conserved histidine residue. This arrangement allows HSco1 to coordinate metals such as Cu(I), Cu(II), and other metal ions like Ni(II) . The structure transitions from an open, conformationally mobile state when metal-free to a closed, rigid conformation upon metal binding, as demonstrated by electrospray ionization mass spectrometry and NMR data .

How does SCO1 function in copper metabolism and CcO assembly?

SCO1 functions primarily as a copper chaperone involved in the biogenesis of the CuA site in cytochrome c oxidase subunit 2 (Cox2). The protein binds copper in a 1:1 stoichiometry using the two cysteine residues and one histidine residue as ligands . This coordination is similar to that found in other copper chaperones, such as ATX1 from Synecocystis .

Research indicates SCO1 may have a dual role:

• As a copper chaperone that transfers copper to the CuA site of Cox2

• As a thioredoxin-like protein involved in redox processes that may reduce the cysteines in the CuA site prior to copper insertion

The mechanism likely involves SCO1 interacting with oxidized Cox2, reducing the cysteine residues of the CuA site, and then transferring copper to the reduced site. After copper transfer, oxidized SCO1 must be reduced again before the next metal transfer cycle, possibly by another protein such as SCO2 or cytochrome c .

What happens when SCO1 function is compromised?

Mutations in the SCO1 gene lead to severe tissue-specific cytochrome c oxidase assembly impairment accompanied by marked copper deficiency . Clinically, SCO1 mutations have been associated with fatal infantile encephalomyopathy and hepatopathy .

In a specific case study, a G132S mutation in SCO1 was associated with early-onset hypertrophic cardiomyopathy, encephalopathy, hypotonia, and hepatopathy . This mutation compromised protein stability, likely by preventing oligomerization, leading to impaired CcO assembly, accumulation of Cox2-containing subcomplexes, and severe copper deficiency in skeletal muscle .

Biochemical analysis of patient muscle mitochondria showed that the CcO holoenzyme content was reduced to approximately 10-20% of control values, with enzyme activity reduced to 8% of the mean reference value when normalized to citrate synthase activity .

How do the structures of apo, Cu(I), and Ni(II) forms of human SCO1 differ, and what insights do they provide about function?

The solution structures of apo, Cu(I), and Ni(II) human SCO1 reveal significant conformational changes upon metal binding. In the metal-free (apo) state, HSco1 adopts an open and conformationally mobile structure. Upon binding Cu(I) or Ni(II), the protein transitions to a closed and rigid conformation .

In Cu(I)HSco1, the metal coordination involves two cysteine residues from the CPXXCP motif and one histidine residue. This trigonal coordination is typical for Cu(I) binding sites . For Ni(II)HSco1 in solution, the coordination sphere is completed by an additional ligand, possibly an aspartate residue, providing the square planar geometry preferred by Ni(II) .

Interestingly, the crystal structure of Ni(II)HSco1 showed a completely different metal binding mode compared to the solution structure. In the crystal, nickel was bound to the oxidized (disulfide-bonded) form of the two cysteines in the CPXXCP motif . This unusual binding mode, where a metal coordinates to an oxidized S-S bond, is rare in the Protein Data Bank .

These structural differences suggest that HSco1 can adopt various conformations throughout its functional cycle, including the potential for metal binding to the oxidized protein at certain stages. This structural plasticity may be crucial for its dual role in copper transfer and redox functions .

What methodological approaches can be used to study SCO1's physical interaction with the CcO complex?

Researchers have successfully employed several complementary techniques to investigate SCO1's physical interaction with the CcO complex:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE): This technique allows separation of protein complexes in their native state. Using BN-PAGE followed by immunoblotting, researchers identified that Sco1 complexes specifically comigrate with the CcO holoenzyme in human muscle mitochondria solubilized with 1% dodecyl maltoside .

• Co-immunoprecipitation: To directly investigate the physical association of Sco1 with CcO, researchers immunoprecipitated CcO from human muscle mitochondria and HEK-293 cell mitochondria solubilized with dodecyl maltoside. The immunoprecipitates were then analyzed using denaturing immunoblots to detect the presence of Sco1 . This approach demonstrated that a fraction of Sco1 physically associated with the CcO complex in human muscle mitochondria .

• Two-dimensional BN-SDS-PAGE: This technique combines native separation of complexes with subsequent denaturing separation of individual proteins. Using two-dimensional BN-SDS-PAGE immunoblot analysis with antibodies against various CcO subunits (Cox1, Cox2, Cox4, and Cox5a), researchers can visualize CcO subcomplexes and determine their composition .

When implementing these methods, it's important to:

• Use appropriate detergents (typically dodecyl maltoside) for solubilization that maintain native protein interactions

• Include controls to account for nonspecific binding in immunoprecipitation experiments

• Verify equal loading using antibodies against unrelated respiratory complexes (e.g., complex III)

How can researchers distinguish between SCO1's roles as a copper chaperone versus a thioredoxin-like redox protein?

The dual nature of SCO1 as both a copper chaperone and a thioredoxin-like redox protein presents a methodological challenge for researchers. Several approaches can help distinguish between these functions:

• Site-directed mutagenesis: Introducing specific mutations in the conserved CPXXCP motif or the histidine residue can selectively impair copper binding while potentially preserving redox activity. Analyzing the functional consequences of these mutations can provide insights into the relative importance of each role .

• Structural analysis of different conformational states: Solution structures of apo, Cu(I), and Ni(II) forms of HSco1 provide clues about the protein's functional states. The unusual finding of Ni(II) bound to oxidized cysteines in the crystal structure suggests that SCO1 may interact with metals even in its oxidized form, supporting a functional cycle involving both metal transfer and redox activities .

• Functional complementation studies: Testing whether SCO1 can be functionally replaced by either pure copper chaperones or pure thioredoxins can help determine which function is essential in different contexts.

• Analysis of CcO assembly intermediates: In SCO1-deficient samples, researchers can detect the accumulation of specific Cox2-containing subcomplexes, suggesting a role for SCO1 in the incorporation of Cox2 into the CcO complex . The pattern of these assembly intermediates can provide clues about the primary defect.

A proposed model suggests that SCO1 may function sequentially as both a redox protein and a copper chaperone: first reducing the cysteine residues in the CuA site of Cox2 and then transferring copper to the reduced site . This integrated model explains how the thioredoxin fold of SCO1 has evolved to accommodate both functions.

What is the significance of SCO1 oligomerization for its function and stability?

Research suggests that SCO1 oligomerization, particularly dimerization, plays a crucial role in its stability and function. Several lines of evidence support this:

• Native protein complexes: Blue Native PAGE analysis of human muscle mitochondria reveals that Sco1 exists in multiple high-molecular-weight complexes, including a ~70-kDa complex that likely represents a homodimer .

• Effect of mutations: The G132S mutation in SCO1, which is associated with severe disease, appears to compromise the protein's ability to oligomerize. This mutation occurs within a region required for dimerization of Sco1 . BN immunoblot analysis failed to detect the mutant Sco1 in its ~70-kDa homodimeric form, suggesting that the mutation might abrogate the ability of Sco1 to oligomerize .

• Stability implications: The inability to form oligomers appears to reduce the stability of the monomeric form, as evidenced by substantially attenuated steady-state levels of G132S mutant Sco1 in patient muscle mitochondria .

• Structural considerations: The amino acid substitution G132S occurs within a juxtamembrane region separating the N-terminal transmembrane helix from the globular domain. This region has been shown to be required for dimerization of Sco1 . The substitution of a highly compact, neutral glycine with a hydrophilic serine likely alters the character of this region significantly .

For experimental approaches to study SCO1 oligomerization, researchers can employ:

• Cross-linking studies to capture transient interactions

• Size-exclusion chromatography to separate different oligomeric states

• Multi-angle light scattering to determine absolute molecular weights of complexes

• Analytical ultracentrifugation to assess the distribution of oligomeric species in solution

What techniques are most effective for structural characterization of SCO1 and its metal complexes?

Structural characterization of SCO1 and its metal complexes requires a multifaceted approach combining several complementary techniques:

When studying SCO1, it's crucial to consider the oxidation state of both the protein (particularly the CPXXCP motif) and the metal ions, as these factors significantly influence structure and function. Researchers should employ oxygen-free conditions when working with reduced forms of the protein to prevent unwanted oxidation.

How can researchers assess the impact of SCO1 mutations on CcO assembly and function?

To comprehensively evaluate how SCO1 mutations affect cytochrome c oxidase assembly and function, researchers can implement the following methodological approaches:

• Enzyme activity assays: Measuring CcO activity normalized to citrate synthase (CS) activity provides a quantitative assessment of functional impairment. In SCO1 patient samples, the CcO-to-CS ratio was reduced to 8% of the reference value .

• Blue Native PAGE analysis: This technique allows quantification of assembled CcO holoenzyme levels. In muscle mitochondria from a SCO1 patient, the content of CcO holoenzyme was reduced to approximately 10-20% of control values .

• Two-dimensional BN-SDS-PAGE: This approach enables detection and characterization of CcO assembly intermediates that accumulate due to impaired assembly. SCO1 patient mitochondria showed accumulation of several CcO subcomplexes, including Cox1-containing subcomplexes and unexpected Cox2-containing assemblies .

• Immunoblot analysis: Using antibodies against various CcO subunits (Cox1, Cox2, Cox4, Cox5a), researchers can determine the subunit composition of assembly intermediates. This revealed that a ~110-kDa subcomplex in SCO1 patient mitochondria lacked both Cox4 and Cox5a subunits but contained significant amounts of Cox2 .

• Expression studies in cellular models: Introducing SCO1 mutations into cellular models allows for controlled assessment of their effects on CcO assembly and function. This approach can help establish causality between specific mutations and observed phenotypes.

• Copper content analysis: Since SCO1 mutations lead to copper deficiency, measuring cellular or tissue copper levels provides additional insights into the functional consequences of mutations.

When conducting these analyses, it's important to include appropriate controls and to consider tissue-specific effects, as SCO1 mutations can affect different tissues to varying degrees .

What are the best approaches for studying the interaction between SCO1 and copper in living cells?

Investigating the interaction between SCO1 and copper in living cells presents unique challenges due to the dynamic nature of copper homeostasis and the mitochondrial localization of SCO1. Several methodological approaches can be employed:

• Fluorescent copper sensors: Genetically encoded or synthetic fluorescent sensors can be used to visualize copper distribution and dynamics in living cells. These can be targeted to mitochondria to specifically monitor the copper pool relevant to SCO1 function.

• Radioactive copper tracing: Using 64Cu or 67Cu isotopes allows tracking of copper movement between cellular compartments and proteins. This approach can reveal the kinetics of copper acquisition by SCO1 and its subsequent transfer to Cox2.

• Proximity-based labeling: Techniques like BioID or APEX2, where SCO1 is fused to a promiscuous biotin ligase or peroxidase, can identify proteins that come into close proximity with SCO1 in its native environment, potentially revealing components of the copper delivery pathway.

• Metal-binding mutants: Creating SCO1 variants with mutations in the copper-binding residues (cysteines and histidine) can help dissect the specific role of copper binding in SCO1 function and cellular copper homeostasis.

• Copper chelation and supplementation: Manipulating cellular copper levels through chelators or supplementation, combined with monitoring SCO1-dependent phenotypes, can reveal the sensitivity of SCO1 function to copper availability.

• Mitochondrial isolation and fractionation: This approach allows for biochemical analysis of copper distribution in mitochondrial subcompartments and can help determine how SCO1 influences mitochondrial copper pools.

The unusual finding that SCO1 mutations lead to tissue-specific copper deficiency suggests that SCO1 plays a broader role in cellular copper homeostasis beyond its function in CcO assembly. Understanding this role requires methods that can distinguish between direct effects on copper handling and indirect effects resulting from impaired CcO assembly.