title: Lumenal loop M672-P707 of the Menkes protein (ATP7A) transfers copper to peptidylglycine monooxygenase.
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 (15)N-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.
authors.affiliation: Institute of Environmental Health, Oregon Health & Sciences University, Beaverton, Oregon 97006, USA.
authors.forename: Adenike
authors.initials: A
authors.lastname: Otoikhian
authors.forename: Amanda N
authors.initials: AN
authors.lastname: Barry
authors.forename: Mary
authors.initials: M
authors.lastname: Mayfield
authors.forename: Mark
authors.initials: M
authors.lastname: Nilges
authors.forename: Yiping
authors.initials: Y
authors.lastname: Huang
authors.forename: Svetlana
authors.initials: S
authors.lastname: Lutsenko
authors.forename: Ninian J
authors.initials: NJ
authors.lastname: Blackburn
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics; D003300:Copper / Q000737:chemistry; D000073840:Copper-Transporting ATPases; D006801:Humans; D051379:Mice; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016415:Sequence Alignment; D056928:X-Ray Absorption Spectroscopy
chemical_list: D027682:Cation Transport Proteins; D009097:Multienzyme Complexes; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases