Introduction 4760 1.1. Background 4760 1.2. Scope 4761 2. Overview of Copper Trafficking Pathways 4761 2.1. Eukaryotic Systems 4761 2.2. Prokaryotic Systems 4762 3. Ctr Transporters 4763 3.1. Human Ctr1 4764 3.2. Yeast Ctr1 4764 4. Atx1-like Chaperones 4764 4.1. Yeast Atx1 4764 4.1.1. Hg(II)-Atx1 Crystal Structure 4764 4.1.2. Apo-Atx1 (Oxidized) Crystal Structure 4765 4.1.3. Cu(I)-Atx1 NMR Structure 4765 4.1.4. Apo-Atx1 (Reduced) NMR Structure 4765 4.1.5. Cu(I)-Atx1 Spectroscopy 4765 4.2. Human Atox1 4765 4.2.1. Hg(II)-Atox1 Crystal Structure 4765 4.2.2. Cd(II)-Atox1 Crystal Structure 4765 4.2.3. Cu(I)-Atox1 Crystal Structure 4766 4.2.4. Apo and Cu(I)-Atox1 NMR Structures 4766 4.2.5. Atox1 Spectroscopy 4766 4.3. Bacterial Atx1 Homologues 4766 4.3.1. E. hirae Apo and Cu(I)-CopZ NMR Structures 4766 4.3.2. B. subtilis Apo and Cu(I)-CopZ NMR 5.3.2. A. fulgidus CopA ATPBD (N and P Domains) Crystal Structure 4771 5.3.3. A. fulgidus CopA A Domain Crystal Structure 4772 5.3.4. A. fulgidus CopA Cryoelectron Microscopy Structure 4772 6. Complexes between Atx1-like Chaperones and Target MBDs 4772
A 2.4-Å-resolution x-ray crystal structure of the carrier-protein independent halogenase, WelO5, in complex with its welwitindolinone precursor substrate, 12-epi-fischerindole U, reveals that the C13 chlorination target is proximal to the anticipated site of the oxo group in a presumptive cis-halo-oxo-iron(IV) (haloferryl) intermediate. Prior study of related halogenases forecasts substrate hydroxylation in this active-site configuration, but x-ray crystallographic verification of C13 halogenation in single crystals mandates that ligand dynamics must reposition the oxygen ligand to enable the observed outcome. Ser189Ala WelO5 effects a mixture of halogenation and hydroxylation products, showing that an outer sphere hydrogen bonding group orchestrates ligand movements to achieve a configuration that promotes halogen transfer.
MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.
Base excision repair (BER) enzymes maintain the integrity of the genome, and in humans, BER mutations are associated with cancer. Given the remarkable sensitivity of DNA-mediated charge transport (CT) to mismatched and damaged base pairs, we have proposed that DNA repair glycosylases (EndoIII and MutY) containing a redox-active [4Fe4S] cluster could use DNA CT in signaling one another to search cooperatively for damage in the genome. Here, we examine this model, where we estimate that electron transfers over a few hundred base pairs are sufficient for rapid interrogation of the full genome. Using atomic force microscopy, we found a redistribution of repair proteins onto DNA strands containing a single base mismatch, consistent with our model for CT scanning. We also demonstrated in Escherichia coli a cooperativity between EndoIII and MutY that is predicted by the CT scanning model. This relationship does not require the enzymatic activity of the glycosylase. Y82A EndoIII, a mutation that renders the protein deficient in DNA-mediated CT, however, inhibits cooperativity between MutY and EndoIII. These results illustrate how repair proteins might efficiently locate DNA lesions and point to a biological role for DNA-mediated CT within the cell.DNA charge transport ͉ DNA damage ͉ iron-sulfur proteins ͉ oxidative stress
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