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The interaction between 5-hydroxy-1,4-naphthoquinone and cysteine was found to give a clearly resolved voltammetric signature that could be used as an electrochemical assay for the thiol. A structure-function study assessed the nature of the resulting voltammetric profile and the highly selective response to cysteine. The translation of the system as the basis of an assay for the determination of cystine was investigated using mercaptopropanol as the in situ reducing agent. The lack of response to the latter and high recovery performance of the label opens up a new direction for the one pot, mercury free determination of cystine.

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through its ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized DUF1775 domain-containing YcnI. DUF1775 domains are found across bacterial phylogeny and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance (EPR) and inductively-coupled plasma-mass spectrometry (ICP-MS) find that it can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper binding site featuring a unique mono-histidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.

No abstract

Most living organisms encode an ensemble of protein transporters and chaperones to regulate copper levels. The ycn operon of the bacterium Bacillus subtilisis regulated in response to copper, suggesting that the three proteins it encodes may engage in direct interactions with copper ions to facilitate transport and homeostasis of this transition metal. Here, we use a combination of structural, biochemical, biophysical, and bioinorganic approaches to determine the structures of individual proteins and their interactions with copper and other binding partners. The YcnI protein encoded by the operon contains a domain of unknown function (DUF1775) at its N‐terminus. We determined the structure of this domain and characterized its interactions with copper ions, finding that it coordinates the metal using a unique “mono‐histidine brace” site that is highly conserved across other members of the DUF1775 family. Our data also suggest a potential role for this domain to serve as a chaperone to facilitate transfer of metal ions. Furthermore, we also identify potential copper binding sites within the transcriptional regulator encoded within the same operon. Together, these studies reveal new insights into the structure and copper‐binding properties of proteins encoded by the ycnoperon, providing additional perspectives on how they may work together to regulate copper homeostasis. As homologs of these proteins are shared by many other bacterial species, it is likely that analogous mechanisms exist for copper uptake and homeostasis beyond B. subtilis.