Structural basis and mechanism for metallochaperone-assisted assembly of the CuA center in cytochrome oxidase.

The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu1.5+•Cu1.5+ redox center (CuA) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB•CuA center in vitro from apo-CoxB and the holo-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu2+•CoxB complex was shown to be rapidly formed as the first intermediate in the pathway. Moreover, our structural data revealed that PcuC has two copper-binding sites, one each for Cu1+ and Cu2+, and that only PcuC•Cu1+•Cu2+ can release CoxB•Cu2+ from the ScoI•Cu2+•CoxB complex. The CoxB•CuA center was then formed quantitatively by transfer of Cu1+ from a second equivalent of PcuC•Cu1+•Cu2+ to CoxB•Cu2+. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for CuA center formation and the order of their delivery to CoxB.

Copper resistance and its regulation in the sulfate-reducing bacterium Desulfosporosinus sp. OT.

Desulfosporosinus sp. OT is a Gram-positive, acidophilic sulfate-reducing firmicute isolated from copper tailings sediment in the Norilsk mining-smelting area in Siberia and represents the first Desulfosporosinus species whose genome has been sequenced. Desulfosporosinus sp. OT is exceptionally copper resistant, which made it of interest to study the resistance mechanism. It possesses a copUAZ operon which is shown here to be involved in copper resistance. The copU gene encodes a CsoR-type homotetrameric repressor. By electrophoretic mobility shift assay, it was shown that CopU binds to the operator/promoter region of the copUAZ operon in the absence of copper and is released from the DNA by Cu+ or Ag+, implying that CopU regulates the operon in a copper/silver-dependent manner. DOT_CopA is a P1B-type ATPase related to other characterized, bacterial copper ATPases. When expressed in a copper-sensitive Escherichia coli ΔcopA mutant, it restores copper resistance to WT levels. His-tagged DOT_CopA was expressed from a plasmid in E. coli and purified by Ni-NTA affinity chromatography. The purified enzyme was most active in the presence of Cu(I) and bacterial phospholipids. These findings indicate that the copUAZ operon confers copper resistance to Desulfosporosinus sp. OT, but do not per se explain the basis of the high copper resistance of this strain.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403.

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.

How periplasmic thioredoxin TlpA reduces bacterial copper chaperone ScoI and cytochrome oxidase subunit II (CoxB) prior to metallation.

Two critical cysteine residues in the copper-A site (Cu(A)) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu(2+) transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E'0 = -231 mV) as compared with that of TlpA (E'0 = -256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10(4) M(-1) s(-1) that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu(A)-CoxB complex may be bypassed in vivo by high environmental Cu(2+) concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403.

Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.

Thioredoxin-like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI.

TlpA and ScoI of Bradyrhizobium japonicum are membrane-anchored thioredoxin-like proteins oriented towards the periplasm. TlpA is a protein-disulfide reductase. ScoI is a copper chaperone for cytochrome oxidase biogenesis. TlpA with its negative redox potential (E(o') -256 mV) was shown here to reduce oxidized ScoI, for which we determined a less negative E(o') (-160 mV). The fast forward reaction (rate constant 9.4×10(4) M(-1) s(-1)) was typical for physiologically relevant disulfide exchange reactions. A transient TlpA-ScoI heterodisulfide formed between Cys107 of TlpA's active site (C(107)XXC(110)) and Cys78 of ScoI's copper-binding site (C(74)XXXC(78)). We conclude that TlpA recycles ScoI to the dithiol form prior to metallation.

Lactococcus lactis HemW (HemN) is a haem-binding protein with a putative role in haem trafficking.

Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 µM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.

Genome sequence of Desulfovibrio sp. A2, a highly copper resistant, sulfate-reducing bacterium isolated from effluents of a zinc smelter at the Urals.

Desulfovibrio sp. A2 is an anaerobic gram-negative sulfate-reducing bacterium with remarkable tolerance to copper. It was isolated from wastewater effluents of a zinc smelter at the Urals. Here, we report the 4.2-Mb draft genome sequence of Desulfovibrio sp. A2 and identify potential copper resistance mechanisms.

Genome sequence of Desulfosporosinus sp. OT, an acidophilic sulfate-reducing bacterium from copper mining waste in Norilsk, Northern Siberia.

We have sequenced the genome of Desulfosporosinus sp. OT, a Gram-positive, acidophilic sulfate-reducing Firmicute isolated from copper tailing sediment in the Norilsk mining-smelting area in Northern Siberia, Russia. This represents the first sequenced genome of a Desulfosporosinus species. The genome has a size of 5.7 Mb and encodes 6,222 putative proteins.

Killing of bacteria by copper surfaces involves dissolved copper.

Bacteria are rapidly killed on copper surfaces. However, the mechanism of this process remains unclear. Using Enterococcus hirae, the effect of inactivation of copper homeostatic genes and of medium compositions on survival and copper dissolution was tested. The results support a role for dissolved copper ions in killing.

Response of gram-positive bacteria to copper stress.

The Gram-positive bacteria Enterococcus hirae, Lactococcus lactis, and Bacillus subtilis have received wide attention in the study of copper homeostasis. Consequently, copper extrusion by ATPases, gene regulation by copper, and intracellular copper chaperoning are understood in some detail. This has provided profound insight into basic principles of how organisms handle copper. It also emerged that many bacterial species may not require copper for life, making copper homeostatic systems pure defense mechanisms. Structural work on copper homeostatic proteins has given insight into copper coordination and bonding and has started to give molecular insight into copper handling in biological systems. Finally, recent biochemical work has shed new light on the mechanism of copper toxicity, which may not primarily be mediated by reactive oxygen radicals.

Copper-induced oxidative stress in rainbow trout gill cells.

Copper is known to pose a serious threat to aquatic organisms. However, the mechanisms of its toxicity still remain unclear. Cu is known to exert its toxicity partly due to the formation of reactive oxygen species (ROS). The purpose of this work was therefore to link the exposure to copper at pH 6 and 7 to cellular formation of ROS and effects like cell viability and genotoxicity using the rainbow trout gill cell line RTgill-W1. To relate effects to bioavailable copper, free Cu(2+) concentrations in the medium were calculated using the programm ChemEQL 3.0. 2',7'-Dichlorodihydrofluorescein-diacetate (H(2)DCF-DA) was used as cell-permeant indicator of ROS formation. Cell viability was assessed using the fluorogenic probe 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). DNA strand breaks were assessed using the comet assay, and lipid peroxidation was investigated using the thiobarbituric acid-reactive substances assay (TBARS). Copper treatment resulted in a dose-dependent elevation in cytotoxicity and formation of cellular ROS. Cell viability was significantly reduced at total copper (Cu(T)) concentrations of 5 microM (corresponding to a free Cu(2+) of 0.11 microM at pH 7) and higher, resulting in an EC(50) of Cu(T)=29.2 microM (Cu(2+)=0.63 microM, pH 7). Neither an impairment concerning the viability of control cells due to growth at pH 6 was observed nor significant differences for cytotoxicity in cells exposed to the same nominal Cu(T) concentrations at pH 6 compared to pH 7. Cellular ROS concentrations increased significantly and decreased with loss of cell viability. After normalizing ROS formation to cell viability, ROS induction up to 25-35-fold compared to the control was detected, but mainly for rather high concentrations (Cu(T) > or = 100 microM; Cu(2+) > or = 2.2 microM, pH 7). ROS formation rates were slightly higher when cells were exposed to Cu at pH 6 compared to pH 7, correlating with the higher free Cu(2+) concentrations. A significant induction of DNA strand breaks was noted at Cu(T) of 1 and 2.5 microM with greater effects at pH 6 due to higher free Cu(2+) concentrations than at pH 7. No effects on lipid peroxidation were observed. These results lead to the hypothesis that copper-induced loss in viability and genotoxicity in trout gill cells are partially triggered by the generation of ROS and related to the free Cu(2+).