A Histidine Residue and a Tetranuclear Cuprous‐thiolate Cluster Dominate the Copper Loading Landscape of a Copper Storage Protein from Streptomyces lividans

Abstract

The chemical basis for protecting organisms against the toxic effect imposed by excess cuprous ions is to constrain this through high‐affinity binding sites that use cuprous‐thiolate coordination chemistry. In bacteria, a family of cysteine rich four‐helix bundle proteins utilise thiolate chemistry to bind up to 80 cuprous ions. These proteins have been termed copper storage proteins (Csp). The present study investigates cuprous ion loading to the Csp from Streptomyces lividans (SlCsp) using a combination of X‐ray crystallography, site‐directed mutagenesis and stopped‐flow reaction kinetics with either aquatic cuprous ions or a chelating donor. We illustrate that at low cuprous ion concentrations, copper is loaded exclusively into an outer core region of SlCsp via one end of the four‐helix bundle, facilitated by a set of three histidine residues. X‐ray crystallography reveals the existence of polynuclear cuprous‐thiolate clusters culminating in the assembly of a tetranuclear [Cu4(μ2‐S‐Cys)4(Νδ1‐His)] cluster in the outer core. As more cuprous ions are loaded, the cysteine lined inner core of SlCsp fills with cuprous ions but in a fluxional and dynamic manner with no evidence for the assembly of further intermediate polynuclear cuprous‐thiolate clusters as observed in the outer core. Using site‐directed mutagenesis a key role for His107 in the efficient loading of cuprous ions from a donor is established. A model of copper loading to SlCsp is proposed and discussed.

Mercaptothiacalixarenes Steer 24 Copper(I) Centers to form a Hollow‐Sphere Structure Featuring Cu2S2 Motifs with Exceptionally Short Cu⋅⋅⋅Cu Distances

“…In nature the latter expresses itself in a number of copper proteins, which show cysteine/methionine ligation, such as the family of metallothioneins, the blue copper centers or Cu A centers . Furthermore, a family of bacterial proteins use a Cys‐rich four‐helix bundle to store large quantities of Cu I . We have therefore explored the binding of copper(I) at LH 4 , which—having found suitable conditions and reagents—led us reproducibly and in good yields to a unique cluster featuring a hollow‐sphere structure composed of 24 Cu I ions connected by six L 4− ligands.…”

Copper Storage Protein From Streptomyces Lividans

“…57 For 5-equivalents, anomalous electron density associated with bound cuprous ions was only observed in the outer core (Figure 7(a)), revealing two types of multinuclear copper clusters. 57 Outer core is broken as two Cys thiolates display monodentate Cu I coordination (Figure 7(b)). In both clusters, only one cuprous ion occupies a binding site observed in the fully Cu I -loaded protein (Cu17).…”

“…The in vitro mechanism of cuprous ion loading to SlCsp3 has been investigated using X-ray crystallography, stopped-flow kinetics, and site-directed mutagenesis. 57 Visualization of Cu I loading to SlCsp3 through X-ray crystallography X-ray crystal structures of SlCsp3 have been determined following the addition of substoichiometric equivalents (5and 10-equivalents) of cuprous ions. 57 For 5-equivalents, anomalous electron density associated with bound cuprous ions was only observed in the outer core (Figure 7(a)), revealing two types of multinuclear copper clusters.…”

“…57 Visualization of Cu I loading to SlCsp3 through X-ray crystallography X-ray crystal structures of SlCsp3 have been determined following the addition of substoichiometric equivalents (5and 10-equivalents) of cuprous ions. 57 For 5-equivalents, anomalous electron density associated with bound cuprous ions was only observed in the outer core (Figure 7(a)), revealing two types of multinuclear copper clusters. 57 Outer core is broken as two Cys thiolates display monodentate Cu I coordination (Figure 7(b)).…”

Mercaptothiacalixarenes Steer 24 Copper(I) Centers to form a Hollow‐Sphere Structure Featuring Cu2S2 Motifs with Exceptionally Short Cu⋅⋅⋅Cu Distances

“…[8] Furthermore, a family of bacterial proteins use a Cys-rich four-helix bundle to store large quantities of Cu I . [9] We have therefore explored the binding of copper(I) at LH 4 , whichhaving found suitable conditions and reagents-led us reproducibly and in good yields to a unique cluster featuring a hollow-sphere structure composed of 24 Cu I ions connected by six L 4À ligands.…”