X-ray Absorption Spectroscopy of the Copper Chaperone HAH1 Reveals a Linear Two-coordinate Cu(I) Center Capable of Adduct Formation with Exogenous Thiols and Phosphines

Abstract

The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the transGolgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase. Although a substantial amount of structural data exist for HAH1 and its yeast and bacterial homologues, details of the copper coordination remain unclear and suggest the presence of two protein-derived cysteine ligands and a third exogenous thiol ligand. Here we report the preparation and reconstitution of HAH1 with Cu(I) using a protocol that minimizes the use of thiol reagents believed to be the source of the third ligand. We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear twocoordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu-S interactions at 2.16 Å, a distance typical of digonal Cu(I) coordination. Binding of exogenous ligands (GSH, dithiothreitol, and tris-(2-carboxyethyl)-phosphine) to the Cu(I) was investigated. When GSH or dithiothreitol was added to the chaperone during the reconstitution procedure, the resulting Cu(I)-HAH1 remained two-coordinate, whereas the addition of the phosphine during reconstitution elicited a threecoordinate species. When the exogenous ligands were titrated into the Cu(I)-HAH1, all formed three-coordinate adducts but with differing affinities. Thus, GSH and dithiothreitol showed weaker binding, with estimated K D values in the range 10 -25 mM, whereas tris-(2-carboxyethyl)-phosphine showed stronger affinity, with a K D value of <5 mM. The implications of these findings for mechanisms of copper transport are discussed.

Yeast Cox17 Solution Structure and Copper(I) Binding

“…For example, DTT can coordinate Cu(I) (37). According to EXAFS studies, DTT contributes a third ligand to the Cu(I)-loaded, two-coordinate Atox1 protein (38). Similarly, DTT included in the purification of Cox17 loaded with copper in vivo (19,21) could provide additional ligands and facilitate the formation of polycopper clusters.…”

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

“…Isothermal Titration Calorimetry Measurements-Starting stock solution protein concentrations were measured using extinction coefficients at 280 nm, determined by amino acid hydrolysis (WD12, 25 ). Protein was concentrated or diluted to 50 M in 3-ml amounts, and 10 mM EDTA was added.…”

“…Although there are no structures of polypeptides comprising all six repeats from WND or MNK, their metal-binding properties have been studied in some detail. Both the WND and MNK N termini bind 5-6 copper ions (22), and x-ray absorption spectroscopic studies indicate that the copper is present as Cu(I), ligated by the sulfurs from two cysteines (23)(24)(25).…”

Unification of the Copper(I) Binding Affinities of the Metallo-chaperones Atx1, Atox1, and Related Proteins

“…In addition, the intrinsic instability of free Cu ϩ in aqueous solution and the tendency to aerial oxidation of cysteine ligands impose special conditions on these experiments. These aspects are complicated further by reports that thiol ligands such as endogenous glutathione (GSH) may expand the Cu I coordination sphere in these proteins or lead to polymeric forms (14,17,18).In an attempt to resolve these fundamental issues for this iconic set of proteins, this study surveys the literature values for the Cu I affinities of the four probe ligands bicinchoninate (Bca), bathocuproine disulfonate (Bcs), dithiothreitol (Dtt), and glutathione (GSH) (Scheme 1). By direct experimental comparison, their relative affinities are unified with the single standard Bcs whose absolute formation constant is also documented.…”