Lumenal loop M672-P707 of the menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase

Adenike Otoikhian, Amanda N. Barry, Mary Mayfield, Mark Nilges, Yiping Huang, Svetlana Lutsenko, Ninian Blackburn

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper-translocating ATPase (ATP7A), but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased toward 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met, whereas at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

Original languageEnglish (US)
Pages (from-to)10458-10468
Number of pages11
JournalJournal of the American Chemical Society
Volume134
Issue number25
DOIs
StatePublished - Jun 27 2012

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Copper
Proteins
Adenosine Triphosphatases
peptidylglycine monooxygenase
X-Ray Absorption Spectroscopy
Extended X ray absorption fine structure spectroscopy
X ray absorption near edge structure spectroscopy
Sequence Deletion
Secretory Pathway
Scaffolds (biology)
Chelation
Histidine
Scaffolds
Methionine
Paramagnetic resonance
Enzymes
Resins
Ligands
Pumps
Scattering

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Lumenal loop M672-P707 of the menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase. / Otoikhian, Adenike; Barry, Amanda N.; Mayfield, Mary; Nilges, Mark; Huang, Yiping; Lutsenko, Svetlana; Blackburn, Ninian.

In: Journal of the American Chemical Society, Vol. 134, No. 25, 27.06.2012, p. 10458-10468.

Research output: Contribution to journalArticle

Otoikhian, Adenike ; Barry, Amanda N. ; Mayfield, Mary ; Nilges, Mark ; Huang, Yiping ; Lutsenko, Svetlana ; Blackburn, Ninian. / Lumenal loop M672-P707 of the menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase. In: Journal of the American Chemical Society. 2012 ; Vol. 134, No. 25. pp. 10458-10468.
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AU - Lutsenko, Svetlana

AU - Blackburn, Ninian

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