SummaryIn bacteria, most Cu + -ATPases confer tolerance to Cu by driving cytoplasmic metal efflux. However, many bacterial genomes contain several genes coding for these enzymes suggesting alternative roles. Pseudomonas aeruginosa has two structurally similar Cu + -ATPases, CopA1 and CopA2. Both proteins are essential for virulence. Expressed in response to high Cu, CopA1 maintains the cellular Cu quota and provides tolerance to this metal. CopA2 belongs to a subgroup of ATPases that are expressed in association with cytochrome oxidase subunits. Mutation of copA2 has no effect on Cu toxicity nor intracellular Cu levels; but it leads to higher H2O2 sensitivity and reduced cytochrome oxidase activity. Mutation of both genes does not exacerbate the phenotypes produced by single-gene mutations. CopA1 does not complement the copA2 mutant strain and vice versa, even when promoter regions are exchanged. CopA1 but not CopA2 complements an Escherichia coli strain lacking the endogenous CopA. Nevertheless, transport assays show that both enzymes catalyse cytoplasmic Cu + efflux into the periplasm, albeit CopA2 at a significantly lower rate. We hypothesize that their distinct cellular functions could be based on the intrinsic differences in transport kinetic or the likely requirement of periplasmic partner Cu-chaperone proteins specific for each Cu + -ATPase.

Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu+ transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures.

Cu + -ATPases play a key role in bacterial Cu + homeostasis by participating in Cu + detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P 1B-1 type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu + with high affinity in a trigonal planar geometry. The cytoplasmic Cu + chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu + is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu + -ATPases drive cytoplasmic Cu + efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu + -efflux pumps responsible for Cu + tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments.

P1B-type ATPases are polytopic membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic transition metals. This article reviews recent progress in our understanding of the structure and function of these proteins in bacteria. These are members of the P-type superfamily of transport ATPases. Cu+-ATPases are the most frequently observed and best-characterized members of this group of transporters. However, bacterial genomes show diverse arrays of P1B-type ATPases with a range of substrates (Cu+, Zn2+, Co2+). Furthermore, because of the structural similarities among transitions metals, these proteins can also transport non-physiological substrates (Cu2+, Cd2+, Pb2+, Au+, Ag+). P1B-type ATPases have six or eight transmembrane segments (TM) with metal coordinating amino acids in three core TMs flanking the cytoplasmic domain responsible for ATP binding and hydrolysis. In addition, regulatory cytoplasmic metal binding domains are present in most P1B-type ATPases. Central to the transport mechanism is the binding of the uncomplexed metal to these proteins when cytoplasmic substrates are bound to chaperone and chelating molecules. Metal binding to regulatory sites is through a reversible metal exchange among chaperones and cytoplasmic metal binding domains. In contrast, the chaperone-mediated metal delivery to transport sites appears as a largely irreversible event. P1B-ATPases have two overarching physiological functions: to maintain cytoplasmic metal levels and to provide metals for the periplasmic assembly of metalloproteins. Recent studies have shown that both roles are critical for bacterial virulence, since P1B-ATPases appear key to overcome high phagosomal metal levels and are required for the assembly of periplasmic and secreted metalloproteins that are essential for survival in extreme oxidant environments.

Transition metals are essential components of important biomolecules, and their homeostasis is central to many life processes. Transmembrane transporters are key elements controlling the distribution of metals in various compartments. However, due to their chemical properties, transition elements require transporters with different structural-functional characteristics from those of alkali and alkali earth ions. Emerging structural information and functional studies have revealed distinctive features of metal transport. Among these are the relevance of multifaceted events involving metal transfer among participating proteins, the importance of coordination geometry at transmembrane transport sites, and the presence of the largely irreversible steps associated with vectorial transport. Here, we discuss how these characteristics shape novel transition metal ion transport models.Micronutrient transition metals (manganese, iron, cobalt, nickel, copper, zinc, molybdenum, and tungsten) serve catalytic and structural functions as prosthetic groups in metalloproteins. In these roles, they are required for a number of diverse physiological processes, ranging from gene transcription to respiration (1). However, despite their essential roles and ubiquitous presence, metals can cause deleterious effects by catalyzing the production of free radicals or by simply impairing metalloenzyme functions by substituting for the optimal metal cofactors. Consequently, organisms strive to maintain a tightly controlled homeostasis of these elements through the coordinated action of transmembrane transporters; chaperone, sequestering, and storage molecules; and metal-responsive transcriptional regulators (2-4). These components distribute the ions to appropriate targets and maintain adequate metal quotas, keeping the cellular compartments essentially free of unsequestered metals (3,5,6).This minireview focuses on the structural and functional aspects of transmembrane transporters that participate in the homeostasis of transition metals. The current understanding of ion transmembrane transport is rooted in 6 decades of research characterizing alkali (H ϩ , Na ϩ , and K ϩ ) and alkali earth (Mg 2ϩ and Ca 2ϩ ) channels, carriers, and pumps. These ions are free (hydrated) and abundant in biological systems. Therefore, their transport mechanisms are shaped by electrochemical gradients and governed by their reversible interaction with transmembrane transport sites constituted by polar amino acid side chains (7). Consideration of the physicochemical differences between alkali/alkali earth and transition metal ions quickly reveals that the existing models describing ion transmembrane translocation cannot explain the mechanism of transport of transition metals. In this context, emerging paradigms for the transport of uncomplexed metal ions are discussed here. Transporters of metal complexes (siderophore-metal, heme, etc.) will not be considered because their selectivity and mechanism might not be determined by the bound metal, but rather by the co...