skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level

Abstract

Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu K{alpha}-edge on the recombinant N-terminal domain of WNDP (N-WNDP). Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) inmore » which the copper concentration was 10-20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections {mu}-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.« less

Authors:
;  [1];  [2]
  1. Dept of Biochemistry and Molecular Biology, Oregon Health and Science University (OHSU), Portland, OR 97239-3098 (United States)
  2. Dept of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University (OHSU), Beaverton, OR 97006-8921 (United States)
Publication Date:
OSTI Identifier:
21054623
Resource Type:
Journal Article
Resource Relation:
Journal Name: AIP Conference Proceedings; Journal Volume: 882; Journal Issue: 1; Conference: XAFS13: 13. international conference on X-ray absorption fine structure, Stanford, CA (United States), 9-14 Jul 2006; Other Information: DOI: 10.1063/1.2644515; (c) 2007 American Institute of Physics; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; ABSORPTION SPECTROSCOPY; COORDINATION NUMBER; COPPER; CYSTEINE; DISEASES; FLUORESCENCE; GENES; LIGANDS; LIVER; LIVER CELLS; METABOLISM; MICE; MOLECULES; PHOSPHINES; PROTEINS; VALENCE; X-RAY FLUORESCENCE ANALYSIS; X-RAY SPECTRA; X-RAY SPECTROSCOPY

Citation Formats

Ralle, Martina, Lutsenko, Svetlana, and Blackburn, Ninian J. Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level. United States: N. p., 2007. Web. doi:10.1063/1.2644515.
Ralle, Martina, Lutsenko, Svetlana, & Blackburn, Ninian J. Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level. United States. doi:10.1063/1.2644515.
Ralle, Martina, Lutsenko, Svetlana, and Blackburn, Ninian J. Fri . "Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level". United States. doi:10.1063/1.2644515.
@article{osti_21054623,
title = {Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level},
author = {Ralle, Martina and Lutsenko, Svetlana and Blackburn, Ninian J.},
abstractNote = {Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu K{alpha}-edge on the recombinant N-terminal domain of WNDP (N-WNDP). Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) in which the copper concentration was 10-20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections {mu}-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.},
doi = {10.1063/1.2644515},
journal = {AIP Conference Proceedings},
number = 1,
volume = 882,
place = {United States},
year = {Fri Feb 02 00:00:00 EST 2007},
month = {Fri Feb 02 00:00:00 EST 2007}
}
  • Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu K{sub {alpha}}-edge on the recombinant N-terminal domain of WNDP (N-WNDP).more » Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) in which the copper concentration was 10--20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections {mu}-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.« less
  • A pair of cyanide-bridged iron-copper molecular assemblies relevant to cyanide-inhibited heme-copper oxidases has been studied by X-ray absorption spectroscopy at both Fe and Cu K-edges. These two complexes, [(py)(OEP)Fe-CN-Cu(Me{sub 6}tren)]{sup 2+} (2) and [(py)(OEP)Fe-CN-Cu(TIM)]{sup 2+} (3), contain a unique four-body Fe-C-N-Cu bridge with an Fe-C-N angle of 179{degree}. They, however, differ significantly in the Cu-N-C angle (174{degree} in 2 and 147{degree} in 3). We have calculated theoretical four-body MS signals for the bridge configuration and other four-body pathways in the structures, and performed least-square fittings of the theoretical signals to the experimental data using the GNXAS programs. A strong long-rangemore » Fe...Cu interaction (4.94 A) has been observed from both the Fe and Cu K-edge data for 2. GNXAS analysis shows that this long-range interaction is attributed to the MS amplitude enhancement form the linear four-body Fe-C-N-Cu configuration. This interaction is not observable from the Fe and Cu K-edge data for 3 where the angle deviates significantly from linearity. An angle-dependence study of MS effects on the Cu-N-C angle shows that there is a large enhancement of MS intensity where the angle approaches linearity, and that this MS effect is negligible when the angle is below nearly 160{degree}. Essentially no Fe-Cu interaction is detectable for a configuration with such an angle. 33 refs., 11 figs., 2 tabs.« less
  • An X-ray absorption spectroscopy study has been carried out at the Fe and Cu K-edges for two bridged molecular assemblies, both of which contain an Fe-X-Cu (X = O{sup 2{minus}}, OH{sup {minus}}) bridge unit, some of whose features are relevant to the binuclear site of cytochrome c oxidase. The two complexes [(OEP)Fe-O-Cu(Me{sub 6}tren)]{sup 1+} and [(OEP)-Fe-(OH)-Cu(Me{sub 5}tren)(OC1O{sub 3})]{sup 1+} have similar structural fragments around the metal centers except that they differ significantly in the bridge structure (the former contains a linear oxo bridge while the latter has a bent hydroxo bridge). The authors report a comparative study of these complexesmore » using multiple-scattering (MS) EXAFS analysis and the program package GNXAS. It is found that there is a dramatic increase in the amplitude of the Fe-X-Cu MS pathway as the bridge unit approaches linearity. Full EXAFS MS analysis enables accurate quantitation of bridge metrical details and geometry for both complexes. The accuracy of the structural determination for the bridge units is evaluated by a statistical analysis methodology in which correlations among fitting parameters are identified and contour plots are used to determine random error. The relevance of this study to the structural definition of binuclear bridged sites in cytochrome c oxidase and other metalloenzymes is considered.« less