Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain heart infusion; BHI) under microaerobic conditions reveals upregulation of 13 genes including adcR and adcCBA, encoding a high affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and a ≈2-fold increase in cell-associated Zn(II). Apo-and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K DNA-Zn = 2.4 ×10 8 M −1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with step-wise Zn(II) affinities K Zn1 and K Zn2 of ≥10 9 M −1 at pH 6.0 and ≥10 12 M −1 at pH 8.0. X-ray absorption spectroscopy (XAS) of the high affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild-type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild-type apo AdcR, but fail to conformationally switch upon Zn(II) binding. These studies clearly identify His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.

Atm1p is an ABC transporter localized in the mitochondrial inner membrane; it functions to export an unknown species into the cytosol and is involved in cellular iron metabolism. Depletion or deletion of Atm1p causes Fe accumulation in mitochondria and a defect in cytosolic Fe/S cluster assembly, but reportedly not a defect in mitochondrial Fe/S cluster assembly. In this study the nature of the accumulated Fe was examined using Mössbauer spectroscopy, EPR, electronic absorption spectroscopy, X-ray absorption spectroscopy, and electron microscopy. The Fe that accumulated in aerobically grown cells was in the form of Fe(III) phosphate nanoparticles similar to that which accumulates in yeast frataxin Yfh1p-deleted or yeast ferredoxin Yah1p-depleted cells. Relative to WT mitochondria, Fe/S cluster and heme levels in Atm1p-depleted mitochondria from aerobic cells were significantly diminished. Atm1p-depletion also caused a build-up of nonheme Fe(II) ions in the mitochondria and an increase in oxidative damage. Atm1p-depleted mitochondria isolated from anaerobically grown cells exhibited WT levels of Fe/S clusters and hemes, and they did not hyper-accumulate Fe. Atm1p-depleted cells lacked Leu1p activity, regardless of whether they were grown aerobically or anaerobically. These results indicate that Atm1p does not participate in mitochondrial Fe/S cluster assembly, and that the species exported by Atm1p is required for cytosolic Fe/S cluster assembly. The Fe/S cluster defect and the Fe-accumulation phenotype, resulting from the depletion of Atm1p in aerobic cells (but not in anaerobic cells), may be secondary effects that are observed only when cells are exposed to oxygen during growth. Reactive oxygen species generated under these conditions might degrade iron-sulfur clusters and lower heme levels in the organelle.

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