Abstract
Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1–2) × 1017 M−1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.
Highlights
PMMO is housed on specialized intracytoplasmic membranes[23] that are either contiguous with the periplasm or form discrete compartments and pMMO may be a rare example of a bacterial cytoplasmic copper enzyme
Regardless of the lack of a currently identified intracellular requirement for copper, a protein (YcnJ) with homology to both CopD and CopC, the latter a periplasmic copper metallochaperone found in Gram negative bacteria, at its C- and N-termini respectively has been suggested to be involved in copper uptake into the cytosol of B. subtilis[46]
To understand the role of Csp3s in cytosolic copper-handling by Gram negative and Gram positive bacteria we have characterized the proteins from M. trichosporium OB3b (MtCsp3) and B. subtilis (BsCsp3)
Summary
A cytosolic Csp[3] is present in Bacillus subtilis, whose metal ion homeostasis has been studied in detail[33,34,35] This model Gram positive bacterium possesses the copper-dependent transcriptional activator CsoR36–38, which regulates expression of both CopA and CopZ (BsCopZ)[36]. To understand the role of Csp3s in cytosolic copper-handling by Gram negative and Gram positive bacteria we have characterized the proteins from M. trichosporium OB3b (MtCsp3) and B. subtilis (BsCsp[3]). This has included investigating their Cu(I)-binding and release properties in vitro.
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