Kinetic analysis of copper transfer from a chaperone to its target protein mediated by complex formation.

Chaperone proteins that traffic copper around the cell minimise its toxicity by maintaining it in a tightly bound form. The transfer of copper from chaperones to target proteins is promoted by complex formation, but the kinetic characteristics of transfer have yet to be demonstrated for any chaperone-target protein pair. Here we report studies of copper transfer between the Atx1-type chaperone CopZ from Bacillus subtilis and the soluble domains of its cognate P-type ATPase transporter, CopAab. Transfer of copper from CopZ to CopAab was found to occur rapidly, with a rate constant at 25 °C of ∼267 s-1, many orders of magnitude higher than that for Cu(i) dissociation from CopZ in the absence of CopAab. The data demonstrate that complex formation between CopZ and CopAab, evidence for which is provided by NMR and electrospray ionisation mass spectrometry, dramatically enhances the rate of Cu(i) dissociation from CopZ.

Redox and chemical activities of the hemes in the sulfur oxidation pathway enzyme SoxAX.

BACKGROUND: SoxAX enzymes initiate microbial oxidation of reduced inorganic sulfur compounds. Their catalytic mechanism is unknown. RESULTS: Cyanide displaces the CysS(-) ligand to the active site heme following reduction by S(2)O(4)(2-) but not Eu(II). CONCLUSION: An active site heme ligand becomes labile on exposure to substrate analogs. SIGNIFICANCE: Elucidation of SoxAX mechanism is necessary to understand a widespread pathway for sulfur compound oxidation. SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E(m)) at pH 7.0 of approximately +210, -340, and -400 mV for the His/Met, His/Cys(-), and active site His/CysS(-)-ligated heme, respectively. Exposing SoxAX to S(2)O(4)(2-), a substrate analog with E(m) ~-450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E(m) ~-1140 mV), allows cyanide to displace the cysteine persulfide (CysS(-)) ligand to the active site heme. This provides the first evidence for the dissociation of CysS(-) that has been proposed as a key event in SoxAX catalysis.

A tetranuclear Cu(I) cluster in the metallochaperone protein CopZ.

Copper trafficking proteins and copper-sensitive regulators are often found to be able to bind multiple Cu(I) ions in the form of Cu(I) clusters. We have determined the high-resolution X-ray crystal structure of an Atx1-like copper chaperone protein from Bacillus subtilis containing a novel tetranuclear Cu(I) cluster. The identities and oxidation states of the cluster ions were established unambiguously by refinement of X-ray energy-dependent anomalous scattering factors. The [Cu(4)(S-Cys)(4)(N-His)(2)] cluster geometry provides new structural insights into not only the binding of multiple cuprous ions by metallochaperones but also protein-associated tetranuclear Cu(I) clusters, including those found in eukaryotic copper-responsive transcription factors.

Distinct characteristics of Ag+ and Cd2+ binding to CopZ from Bacillus subtilis.

The chaperone CopZ together with the P-type ATPase transporter CopA constitute a copper-detoxification system in Bacillus subtilis that is commonly found in bacteria and higher cells. Previous studies of the regulation of the copZA operon showed that expression is significantly upregulated in response to elevated concentrations of environmental silver and cadmium, as well as copper. Here, we have used spectroscopic and bioanalytical methods to investigate in detail the capacity of CopZ to bind these metal ions (as Ag(+) and Cd(2+)). We demonstrate that Ag(+) binding mimics closely that of Cu(+): Ag(+)-mediated dimerisation of the protein occurs, and distinct Ag(+)-bound species are formed at higher Ag(+) loadings. Cd(2+) also binds to CopZ, but exhibits significantly different behaviour. Cd(2+)-mediated dimerisation is only observed at low loadings, such that at 0.5 and one Cd(2+) per CopZ the protein is present mainly in a monomeric form; and multinuclear higher-order forms of Cd(2+)-CopZ are not observed. Competition binding studies reveal that Ag(+) binds with an affinity very similar to that of Cu(+), while Cd(2+) binding is significantly weaker. These data provide support for the proposal that CopZ may be involved in the detoxification of silver and cadmium, in addition to copper.

Structure and Cu(I)-binding properties of the N-terminal soluble domains of Bacillus subtilis CopA.

CopA, a P-type ATPase from Bacillus subtilis, plays a major role in the resistance of the cell to copper by effecting the export of the metal across the cytoplasmic membrane. The N-terminus of the protein features two soluble domains (a and b), that each contain a Cu(I)-binding motif, MTCAAC. We have generated a stable form of the wild-type two-domain protein, CopAab, and determined its solution structure. This was found to be similar to that reported previously for a higher stability S46V variant, with minor differences mostly confined to the Ser(46)-containing beta3-strand of domain a. Chemical-shift analysis demonstrated that the two Cu(I)-binding motifs, located at different ends of the protein molecule, are both able to participate in Cu(I) binding and that Cu(I) is in rapid exchange between protein molecules. Surprisingly, UV-visible and fluorescence spectroscopy indicate very different modes of Cu(I) binding below and above a level of 1 Cu(I) per protein, consistent with a major structural change occurring above 1 Cu(I) per CopAab. Analytical equilibrium centrifugation and gel filtration results show that this is a result of Cu(I)-mediated dimerization of the protein. The resulting species is highly luminescent, indicating the presence of a solvent-shielded Cu(I) cluster.

CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA.

The structure of the hypothetical copper-metallochaperone CopZ from Bacillus subtilis and its predicted partner CopA have been studied but their respective contributions to copper export, -import, -sequestration and -supply are unknown. DeltacopA was hypersensitive to copper and contained more copper atoms cell(-1) than wild-type. Expression from the copA operator-promoter increased in elevated copper (not other metals), consistent with a role in copper export. A bacterial two-hybrid assay revealed in vivo interaction between CopZ and the N-terminal domain of CopA but not that of a related transporter, YvgW, involved in cadmium-resistance. Activity of copper-requiring cytochrome caa(3) oxidase was retained in deltacopZ and deltacopA. DeltacopZ was only slightly copper-hypersensitive but deltacopZ/deltacopA was more sensitive than deltacopA, implying some action of CopZ that is independent of CopA. Significantly, deltacopZ contained fewer copper atoms cell(-1) than wild-type under these conditions. CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.

Copper-mediated dimerization of CopZ, a predicted copper chaperone from Bacillus subtilis.

Understanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer. Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif. We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers. The nature of the copper(I)-CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein. The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)-CopZ occurs mainly as a monomer species. Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein. We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer.