Cu homeostasis in bacteria: The ins and outs

Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells

Oxydases terminales chez la bactérie pourpre Rubrivivax gelatinosus (rôle et mise en place dans la membrane)

La bactérie pourpre Rubrivivax gelatinosus se développe en utilisant la photosynthèse et la respiration. Dans le cadre des études menées sur la régulation des gènes photosynthétiques par l oxygène, nous nous sommes intéressés à l étude des oxydases terminales, enzymes catalysant la réduction de l oxygène en eau dans la respiration. Dans ce travail, nous avons étudié le rôle physiologique de ces complexes et la mise en place de leurs cofacteurs. Nous avons identifié deux oxydases terminales, cbb3 et bd, fonctionnelles dans la souche sauvage. Un suppresseur exprimant l oxydase caa3 a été également sélectionné. Notre travail a permis de montrer que ces oxydases terminales jouent un rôle important dans l initiation de la photosynthèse. Ainsi, l activité de ces complexes a pour conséquence la réduction de l oxygène nécessaire à l expression des gènes photosynthétiques. Nous avons également analysé les caractéristiques spectroscopiques de l oxydase cbb3 par RPE et par spectroscopie UV/visible. L hème c de la sous-unité CcoO de cette oxydase présente deux position du 6ème ligand axial, Met143 et His130. Ce changement de ligand s accompagne d un changement de potentiel redox, probablement important pour l activité de l enzyme. L identification des oxydases de type hème-cuivre, cbb3 dans la souche sauvage et caa3 dans un suppresseur, a permis d initier une étude sur leur biogenèse et particulièrement la mise en place du cuivre. Cette étude a permis d identifier un transporteur putatif CtpA et des chaperonnes qui permettraient la mise en place de ce métal dans ces oxydases mais aussi dans la N2O réductase, enzyme de la chaîne de dénitrification.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The Escherichia coli MFS-type transporter genes yhjE, ydiM, and yfcJ are required to produce an active bo3 quinol oxidase

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family (e.g., CcoA) are required for Cu import and heme-CuB center biogenesis of the cbb3-type cytochrome c oxidases (cbb3-Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo3-type quinol oxidases (bo3-Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo3-Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E. coli bo3-Qox. When respiratory growth is dependent on bo3-Qox, because of deletion of the bd-type Qox enzymes, three candidate genes, yhjE, ydiM, and yfcJ, were found to be critical for E. coli growth. Radioactive metal uptake assays showed that ΔydiM has a slower 64Cu uptake, whereas ΔyhjE accumulates reduced 55Fe in the cell, while no similar uptake defect is associated with ΔycfJ. Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases

Uncovering the Transmembrane Metal Binding Site of the Novel Bacterial Major Facilitator Superfamily-Type Copper Importer CcoA

Uptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox). Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive 64Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter

A Robust Genetic System for Producing Heterodimeric Native and Mutant Cytochrome <i>bc</i>₁

The ubihydroquinone:cytochrome <i>c</i> oxidoreductase, or cytochrome <i>bc</i>₁, is central to the production of ATP by oxidative phosphorylation and photophosphorylation in many organisms. Its three-dimensional structure depicts it as a homodimer with each monomer composed of the Fe–S protein, cytochrome <i>b</i>, and cytochrome <i>c</i>₁ subunits. Recent genetic approaches successfully produced heterodimeric variants of this enzyme, providing insights into its mechanism of function. However, these experimental setups are inherently prone to genetic rearrangements as they carry repeated copies of cytochrome <i>bc</i>₁ structural genes. Duplications present on a single replicon (one-plasmid system) or a double replicon (two-plasmid system) could yield heterogeneous populations via homologous recombination or other genetic events at different frequencies, especially under selective growth conditions. In this work, we assessed the origins and frequencies of genetic variations encountered in these systems and describe an improved variant of the two-plasmid system. We found that use of a recombination-deficient background (<i>recA</i>) minimizes spontaneous formation of co-integrant plasmids and renders the homologous recombination within the cytochrome <i>b</i> gene copies inconsequential. On the basis of the data, we conclude that both the newly improved RecA-deficient and the previously used RecA-proficient two-plasmid systems reliably produce native and mutant heterodimeric cytochrome <i>bc</i>₁ variants. The two-plasmid system developed here might contribute to the study of “mitochondrial heteroplasmy”-like heterogeneous states in model bacteria (e.g., <i>Rhodobacter</i> species) suitable for bioenergetics studies. In the following paper (DOI 10.1021/bi400561e), we describe the use of the two-plasmid system to produce and characterize, in membranes and in purified states, an active heterodimeric cytochrome <i>bc</i>₁ variant with unusual intermonomer electron transfer properties

The Escherichia coli MFS-type transporter genes yhjE, ydiM, and yfcJ are required to produce an active bo3 quinol oxidase.

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family (e.g., CcoA) are required for Cu import and heme-CuB center biogenesis of the cbb3-type cytochrome c oxidases (cbb3-Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo3-type quinol oxidases (bo3-Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo3-Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E. coli bo3-Qox. When respiratory growth is dependent on bo3-Qox, because of deletion of the bd-type Qox enzymes, three candidate genes, yhjE, ydiM, and yfcJ, were found to be critical for E. coli growth. Radioactive metal uptake assays showed that ΔydiM has a slower 64Cu uptake, whereas ΔyhjE accumulates reduced 55Fe in the cell, while no similar uptake defect is associated with ΔycfJ. Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases

Recent advances in cytochrome bc1: Inter monomer electronic communication?

AbstractThe ubihydroquinone: cytochrome c oxidoreductase, or cytochrome bc1, is a central component of photosynthetic and respiratory energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy production (ATP). The three-dimensional structures of cytochrome bc1 indicate that its two monomers are intertwined to form a symmetrical homodimer. This unusual architecture raises the issue of whether the monomers operate independently, or function cooperatively during the catalytic cycle of the enzyme. In this review, recent progresses achieved in our understanding of the mechanism of function of dimeric cytochrome bc1 are presented. New genetic approaches producing heterodimeric enzymes, and emerging insights related to the inter monomer electron transfer between the heme b cofactors of cytochrome bc1 are described

Intermonomer Electron Transfer between the <i>b</i> Hemes of Heterodimeric Cytochrome <i>bc</i>₁

The ubihydroquinone:cytochrome <i>c</i> oxidoreductase, or cytochrome <i>bc</i>₁, is a central component of respiratory and photosynthetic energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy (ATP) production. The three-dimensional structures of cytochrome <i>bc</i>₁ show a homodimeric organization of its three catalytic subunits. The unusual architecture revived the issue of whether the monomers operate independently or function cooperatively during the catalytic cycle of the enzyme. In recent years, different genetic approaches allowed the successful production of heterodimeric cytochrome <i>bc</i>₁ variants and evidenced the occurrence of intermonomer electron transfer between the monomers of this enzyme. Here we used a version of the “two-plasmid” genetic system, also described in the preceding paper (DOI: 10.1021/bi400560p), to study a new heterodimeric mutant variant of cytochrome <i>bc</i>₁. The strain producing this heterodimeric variant sustained photosynthetic growth of <i>Rhodobacter capsulatus</i> and yielded an active heterodimer. Interestingly, kinetic data showed equilibration of electrons among the four <i>b</i> heme cofactors of the heterodimer, via “reverse” intermonomer electron transfer between the <i>b</i>_L hemes. Both inactive homodimeric and active heterodimeric cytochrome <i>bc</i>₁ variants were purified to homogeneity from the same cells, and purified samples were subjected to mass spectrometry analyses. The data unequivocally supported the idea that the cytochrome <i>b</i> subunits carried the expected mutations and their associated epitope tags. Implications of these findings on our interpretation of light-activated transient cytochrome <i>b</i> and <i>c</i> redox kinetics and the mechanism of function of a dimeric cytochrome <i>bc</i>₁ are discussed with respect to the previously proposed heterodimeric Q cycle model

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron\u2011sulfur protein (ISP) with its Fe2S2 cluster, cyt c1 and cyt b, forming two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the electron transfer pathways originating from QH2 oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc1 and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Qo site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc1 activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc1. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH2 oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe2S2 cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH2 oxidation at the Qo site of cyt bc1