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

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

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.

The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a MnIII2-tyrosyl radical (Y•) or a FeIII2-Y• cofactor in the NrdF subunit. Whereas FeIII2-Y• can self-assemble from FeII2-NrdF and O2, activation of MnII2-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O2. The crystal structures reported here of E. coli MnII2-NrdF and FeII2-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of MnII2-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF MnII2 active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of MnIII2-Y• cofactor assembly.

N-nitroso-containing small molecules, such as the bacterial natural product streptozotocin, are prominent carcinogens 1,2 and important cancer chemotherapeutics 3,4 . Despite this functional group's significant impact on human health, dedicated enzymes involved in N-nitroso assembly have not been identified. Here, we describe a metalloenzyme from streptozotocin biosynthesis (SznF) that catalyzes an oxidative rearrangement of the guanidine group of N ω -methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF uncovered two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. The discovery of this biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-heme iron-dependent enzymes to include N-N bond formation. We find biosynthetic gene clusters encoding SznF homologs are widely distributed among bacteria, including environmental organisms, plant symbionts, and human pathogens, suggesting an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites. Streptozotocin (SZN) (Zanosar®) is an N-nitrosourea natural product and approved cancer chemotherapeutic (Fig. 1a) 3,5 . SZN is also used to induce type I diabetes in animal models due to its toxicity towards pancreatic beta cells (> 28,500 PubMed references) 6 . Like other N-nitrosoureas, SZN exerts its activity in vivo by generating electrophilic DNA alkylating Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://

Copper trafficking proteins, including the chaperone Atox1 and the P 1B -type ATPase ATP7B, have been implicated in cellular resistance to the anticancer drug cisplatin. We have determined two crystal structures of cisplatin-Atox1 adducts that reveal platinum coordination by the conserved CXXC copper-binding motif. Direct interaction of cisplatin with this functionally relevant site has significant implications for understanding the molecular basis for resistance mediated by copper transport pathways.Cisplatin (cis-diamminedichloroplatinum(II)) is a highly effective anticancer therapeutic in wide clinical use. The use of cisplatin and related drugs is limited by intrinsic and acquired cellular resistance, however. 1 Although many different systems are implicated in cisplatin trafficking within the cell, 1 mounting evidence suggests a linkage between cisplatin resistance and the human copper homeostatic proteins Atox1 and ATP7A or ATP7B (Menkes and Wilson disease proteins). 2 The copper chaperone Atox1 binds Cu(I) with a conserved CXXC motif and delivers it to the N-terminal metal binding domains (MBDs) of ATP7B and ATP7A, which are Cu(I) specific P 1B -type ATPases. 3 Each human Cu(I) ATPase has six MBDs, which also bind Cu(I) with CXXC motifs and resemble Atox1 in overall structure. 4 6 As a model for cisplatin interaction with these domains, we have investigated cisplatin binding to the human copper chaperone Atox1. The crystal structures reported here establish that cisplatin binds specifically to the Cu(I) binding site in Atox1.The structure of a stoichiometric cisplatin-Atox1 adduct (Pt-Atox1) was determined to 1.6 Å resolution (Table S1, PDB accession code 3IWL). Inductively-coupled plasma atomic emission spectroscopy (ICP-AES) analysis shows 1.0 ± 0.03 Pt ions per Atox1 (Supporting Information). There is one monomer in the asymmetric unit, in contrast to previous metalbridged dimeric structures solved in the presence of Cu(I), Cd(II), and Hg(II). 7 The overall fold is similar to that of Atox1 in the dimeric structures ( Figure S1) with an average root mean square deviation (rmsd) of 0.391 Å for 65 Cα coordinates. The Pt(II) ion is coordinated by Cys12 and Cys15 from the CXXC motif with Cys(S)-Pt distances of 2.30 and 2.35 Å, respectively ( Figure 1B and Figure S1). These distances are similar to those reported for cisplatin bound to metallothionein. 8 Broad spectral features indicative of Pt(II)-S ligand-tometal charge transfer (LMCT) transitions are consistent with cysteine ligation ( Figure S2). 9 The orientations of the coordinating cysteines are quite similar to those observed in the other metal-loaded Atox1 structures, with Cys15 occupying the identical position, and the sulfur and amide nitrogen atoms of Cys12 deviating slightly ( Figure 1B). The geometry is square planar with the two cysteine ligands oriented trans to one another. The remaining ligands are provided by the backbone amide nitrogen of Cys12 at 2.27 Å and an exogenous donor best modeled as a TCEP molecule with a TCEP(P)-Pt ...

The radical SAM (RS) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys 355) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys 355 is S-methylated and located proximal to the SAM methyl group, suggesting that SAM involved in the initial methyl transfer binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site.