A lytic polysaccharide monooxygenase-like protein functions in fungal copper import and meningitis.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms.

Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans.

The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro; however, the details of these interactions are poorly understood, and their implications for in vivo antifungal activity have not been established. Here, we show that the availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that co-treatment of Hist-5 with Cu improved the EC from ∼5 to ∼1 μM, whereas co-treatment with a high-affinity Cu-specific chelator abrogated antifungal activity.

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases.

Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2.

The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn(2+)-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe(2+)) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity.

Interactions of Cu(I) with selenium-containing amino acids determined by NMR, XAS, and DFT studies.

Cu(I) coordination by organoselenium compounds was recently reported as a mechanism for their prevention of copper-mediated DNA damage. To establish whether direct Se-Cu coordination may be involved in selenium antioxidant activity, Cu(I) coordination of the selenoamino acids methyl-Se-cysteine (MeSeCys) and selenomethionine (SeMet) was investigated. NMR results in D(2)O indicate that Cu(I) binds to the Se atom of both MeSeCys and SeMet as well as the carboxylic acid oxygen atom(s) or amine nitrogen atoms.

A comparison of methionine, histidine and cysteine in copper(I)-binding peptides reveals differences relevant to copper uptake by organisms in diverse environments.

The N-terminal, extracellular regions of eukaryotic high affinity copper transport (Ctr) proteins vary in composition of the Cu(i) binding amino acids: methionine, histidine, and cysteine. To examine why certain amino acids are exploited over others in Ctrs from different organisms, the relative Cu(i) binding affinity and the dependence of binding on pH were examined for 3 peptides of the sequence MG(2)XG(2)MK, where X is either Met, His, or Cys. Cu(i) affinity was examined using an ascorbic acid oxidation assay, an electrospray ionization mass spectrometry technique, and spectrophotometric titration with a competitive Cu(i) chelator.

A comparison of methionine, histidine and cysteine in copper(I)-binding peptides reveals differences relevant to copper uptake by organisms in diverse environments.

The N-terminal, extracellular regions of eukaryotic high affinity copper transport (Ctr) proteins vary in composition of the Cu(i) binding amino acids: methionine, histidine, and cysteine. To examine why certain amino acids are exploited over others in Ctrs from different organisms, the relative Cu(i) binding affinity and the dependence of binding on pH were examined for 3 peptides of the sequence MG(2)XG(2)MK, where X is either Met, His, or Cys. Cu(i) affinity was examined using an ascorbic acid oxidation assay, an electrospray ionization mass spectrometry technique, and spectrophotometric titration with a competitive Cu(i) chelator.

Histidine 103 in Fra2 is an iron-sulfur cluster ligand in the [2Fe-2S] Fra2-Grx3 complex and is required for in vivo iron signaling in yeast.

The BolA homologue Fra2 and the cytosolic monothiol glutaredoxins Grx3 and Grx4 together play a key role in regulating iron homeostasis in Saccharomyces cerevisiae. Genetic studies indicate that Grx3/4 and Fra2 regulate activity of the iron-responsive transcription factors Aft1 and Aft2 in response to mitochondrial Fe-S cluster biosynthesis. We have previously shown that Fra2 and Grx3/4 form a [2Fe-2S](2+)-bridged heterodimeric complex with iron ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue.

Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I).

Cellular acquisition of copper in eukaryotic organisms is primarily accomplished through high-affinity copper transport proteins (Ctr). The extracellular N-terminal regions of both human and yeast Ctr1 contain multiple methionine residues organized in copper-binding Mets motifs. These motifs comprise combinations of methionine residues arranged in clusters of MXM and MXXM, where X can be one of several amino acids.

The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation.

The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear.

Metal complexes and free radical toxins produced by Pfiesteria piscicida.

Metal-containing organic toxins produced by Pfiesteria piscicida were characterized, for the first time, by corroborating data obtained from five distinct instrumental methods: nuclear magnetic resonance spectroscopy (NMR), inductively coupled plasma mass spectrometry (ICP-MS), liquid chromatography particle beam glow discharge mass spectrometry (LC/PB-G DMS), electron paramagnetic resonance spectroscopy (EPR), and X-ray absorption spectroscopy (XAS). The high toxicity of the metal-containing toxins is due to metal-mediated free radical production. This mode of activity explains the toxicity of Pfiesteria, as well as previously reported difficulty in observing the molecular target, due to the ephemeral nature of radical species.

EXAFS spectroscopic evidence for an Fe=O unit in the Fe(IV) intermediate observed during oxygen activation by taurine:alpha-ketoglutarate dioxygenase.

The Fe(II)- and alpha-ketoglutarate-dependent dioxygenases catalyze hydroxylation reactions of considerable biomedical and environmental significance. Recently, the first oxidized iron intermediate in the reaction of a member of this family, taurine:alpha-ketoglutarate dioxygenase (TauD), was detected and shown to be a high-spin Fe(IV) complex. In this study we have used X-ray absorption spectroscopy to demonstrate the presence of a short (1.

Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli.

The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway.