Probing the Coordination Environment of the Human Copper Chaperone HAH1: Characterization of Hg^II‐Bridged Homodimeric Species in Solution

Abstract

While metal ion homeostasis in cells is often mediated through metallochaperones, there are opportunities for toxic metals to be sequestered through the existing transport apparatus. Proper trafficking of CuI in human cells is partially achieved through complexation by HAH1, the human metallochaperone responsible for copper delivery to the Wilson and Menkes ATPase located in the trans-Golgi apparatus. In addition to binding copper, HAH1 strongly complexes HgII, with the x-ray structure of this complex previously described. We felt it was important to clarify the solution behavior of these systems and, therefore, have probed the binding of HgII to HAH1 over the pH range 7.5 to 9.4 using 199Hg NMR, 199mPAC and UV-visible spectroscopies. We have also examined the metal-dependent protein association over this pH range using analytical gel-filtration. We conclude that at pH 7.5, HgII is bound to a monomeric HAH1 as a two coordinate, linear complex (HgS2), like the HgII-Atx1 X-ray structure (PDB ID: 1CC8). At pH 9.4, HgII promotes HAH1 association, leading to formation of HgS3 and HgS4 complexes, which are in exchange on the μs-ns time scale. Thus, we have characterized structures that may represent central intermediates in the process of metal ion transfer, as well as their exchange kinetics.

Formation of Hg(II) tetrathiolate complexes with cysteine at neutral pH

“…For trigonal planar HgS 3 coordination in Hg( S -cysteinate) 3 the experimental δ( 199 Hg) = −185 ppm was reported. 12, 26 Also, in combination with PAC ( 199m Hg perturbed angular correlation spectroscopy), a 199 Hg NMR signal at δ Hg = −348 ppm obtained for Hg(II) bound to a dimeric HAH1 metallochaperone (pH = 8.5 – 9.4) has been assigned to a mixture of distorted (T-shaped) HgS 3 and HgS 4 structures in equilibrium. 12 …”

“…However, considering the neutral pH of the solution F, it seems more likely that the [Hg( S,N Cys)( S -HCys)] − or dissolved Hg( S -HCys) 2 (aq) dithiolate complexes are present. Using the estimated relative amounts of the di-, tri- and tetrathiolates in solution F (Table 3), and the 199 Hg NMR chemical shifts of the trigonal planar HgS 3 (δ Hg = −185 ppm), 12, 26 and tetrahedral HgS 4 (δ Hg = −340 ppm), 10 a chemical shift between about δ Hg −1140 and −1200 ppm would be expected for the dithiolate species in solution F (see Appendix I in Supporting Materials ), which is within the δ Hg range (−800 to −1200 ppm) for mercury(II) dithiolate complexes. 2 …”

“…12 A relevant question is then whether four-coordinated Hg( S -cysteinate) 4 species could form in solution at physiological pH (~7.4). A recent report on Hg(II) biosorption by E. Coli shows that Hg(II) is bound to 2 – 4 thiol groups at the membrane of non-metabolizing cells and also within metabolizing cells in the absence of externally added organic ligands.…”

Mass spectrometry of B. subtilis CopZ: Cu(i)-binding and interactions with bacillithiol

“…13 One of the best-studied and most prevalent copper trafficking pathways involves a copper chaperone and a P-type ATPase transporter, 14 which function in transmembrane Cu(I)-transport. In humans, the chaperone Hah1 (also called Atox1) delivers Cu(I) to two P-type ATPase transporters, the Menkes and Wilson proteins, 15 while in yeast the chaperone Atx1 delivers Cu(I) to the transporter Ccc2. 16 In the Gram-positive bacterium Bacillus subtilis, as in many prokaryotes, the copper efflux system comprises a copper chaperone (CopZ) and a P-type ATPase (CopA).…”

CERN-MEDICIS (Medical Isotopes Collected from ISOLDE): A New Facility

“…In early 2006, the project was taken over by the biophysics and bioinorganic chemistry groups from the University of Copenhagen, Denmark, led by Lars Hemmingsen. The major focus of the research was continuously kept on investigating the metal ion behavior at the molecular level and under physiological conditions and on the heavy-metal ion toxicity of Hg(II), Pb(II) and Cd(II) metal ions [34][35][36]. In addition, the year 2012 was particularly successful for the biophysics collaboration, because a new apparatus allowing for β-NMR experiments on liquid samples was finally established at ISOLDE and tried in the world's first experiments in an ionic liquid.…”