F. Wayne Outten and Benjamin S. Twining, University of South Carolina, Columbia, South Carolina
Transition metals are a key component of biological systems. Because of their special properties, they are incorporated into proteins functioning in dioxygen transport, electron transfer, redox transformations, and regulatory control. The metals used in biological systems have been selected throughout evolution based on their availability in the environment and their kinetic lability, resulting in preferential use of first-row transition metals in biology. These essential metals must be obtained from the environment and concentrated within the cell for use in biochemical pathways. Once appropriated, metals must be directed to metalloenzymes or metal storage proteins within the cell. In addition, organisms must be able to distinguish between essential and toxic metals and must have mechanisms for minimizing the toxicity of both essential and toxic metals that are present in excess. Metal homeostasis is broadly defined as the metal uptake, trafficking, efflux, and sensing pathways that allow organisms to maintain an appropriate (often narrow) intracellular concentration range of essential transition metals. This review will introduce several unifying concepts of metal homeostasis with brief illustrative examples for each concept.
Transition metals are key nutrients for nearly all organisms because of their role in critical biochemical pathways such as respiration, photosynthesis, and nitrogen fixation. However, metals cannot be synthesized like other biomolecules and must be obtained from the environment. Once acquired by organisms, metals must be routed to the correct intracellular destination while preventing deleterious side reactions or nonspecific chelation by other cellular components. Metals that are difficult to obtain may be stored for future use by the organism. As intracellular metal concentrations increase, organisms must have the ability to remove (efflux), sequester, or detoxify the excess metal. Finally, organisms must have some ability to distinguish between essential and nonessential metals, despite their similarities, in order to prevent poisoning by nonessential metals. The genetic and biochemical pathways that are used by organisms to acquire, traffic, store, and detoxify metals are collectively known as metal homeostasis systems. Maintenance of intracellular transition metal concentrations within an optimal range has posed a major challenge for biological systems throughout evolution. The study of metal homeostasis has been a key part of the field of bioinorganic chemistry since its inception, providing numerous insights into how transition metals are integrated into biological systems. Note: Although zinc is not considered a transition metal based on the IUPAC definition, zinc has often been included with the transition metals as distinct from the alkali and alkaline earth metals. For the purposes of this review, we will consider zinc as part of the transition metal group with the caveat that it does not strictly meet the IUPAC definition.
Concepts in Metal Homeostasis
There are many functions for transition metals within biological systems (Table 1). The choice of metal used for each function is determined by the chemical characteristics of that metal, including its size as well as its thermodynamic stability and kinetic lability when complexed with biological ligands. Biological systems have mostly incorporated the first-row transition metals. The divalent forms of these metals are particularly common in biology because they have higher ligand exchange rates than their M3+ counterparts. Kinetically labile transition metals are required to allow assembly and disassembly of metal centers and for rapid binding and release of substrates in metal-catalyzed reactions. The relative abundance and availability of the transition metals in the environment has also dictated their use in biology. An excellent discussion of these issues in the context of metal selection for metalloenzyme use is presented in the technical article “Chemistry of Metalloenzymes” by R.J.P. Williams. To ensure an ample supply of transition metals for incorporation into biomolecules, organisms have been selected to contain metal homeostasis systems.
Table 1. Environmental and intracellular metal concentrations and select biological functions for essential metals
Examples of select biological roles
1.4-6.9 x 10-7
1.7 x 10-6
2.4 x 10-6-1.0 x 10-4
1.6-5.0 x 10-4
1.0 x 10-5
1.0 x 10-4
0.7-4.3 x 10-5
1.0-1.9 x 10-4
3.0-4.8 x 10-6
1.0 x 10-5
Dioxygen transport, electron transfer, nitrogen fixation
Alkyl group transfer
Dioxygen transport, electron transfer
Structural stabilization, hydrolase
Nitrogen fixation, oxo transfer
Carbon dioxide reduction/fixation
1Range shown is for both ocean and freshwater aquatic systems.
2Donat JR, Bruland, KW. Trace elements in the oceans. In Trace Elements in Natural Waters. Salbu B, Steinnes E, eds. 1995. CRC Press, Boca Raton, FL, pp. 247-281.
2Borg H. Trace elements in lakes. In Trace Elements in Natural Waters. Salbu B, Steinnes E, eds. 1995. CRC Press, Boca Raton, FL, pp. 177-201.
2Hart BT, Hines T. Trace elements in rivers. In Trace Elements in Natural Waters. Salbu B, Steinnes E, eds. 1995. CRC Press, Boca Raton, FL, pp. 203-221.
3Ho T-Y, Quigg A, Finkel ZV, Milligan AJ, Wyman K, Falkowski PG, Morel FMM. The elemental composition of some marine phytoplankton. J. Phycol. 2003; 39:1145-1159.
3Twining BS, Baines SB, Fisher NS. Elemental stoichiometries of individual phytoplankton cells collected from the Southern Ocean Iron Experiment (SOFeX). Limnol. Oceanogr. 2004; 49:2115-2128.
3Outten CE, O’Halloran TV. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science. 2001; 292:2488-2492.
The metal quota
A key concept in metal homeostasis is the “metal quota.” This quota is simply the amount of a given metal required for normal cellular function under a specific growth condition. In theory the metal quota can vary considerably. For example, metal requirements may change in response to growth state (quiescence or active growth), oxygen availability, carbon source, and a variety of other environmental factors. Similarly, different organisms or different cell types within an organism may have different metal requirements (1). For example, photosynthetic cyanobacteria have been reported to have higher Mn and Fe quotas than other nonphotosynthetic prokaryotes (2). In this case, the higher metal quotas stem directly from increased amounts of specific metalloproteins (Photosystem I and Photosystem II). At the opposite extreme, the obligate parasite that causes Lyme disease,
Borrelia burgdorferi, seems to have no requirement for Fe and lacks many metalloproteins found in other microbes (3). These unusual adaptations may allow B. burgdorferi to survive in the iron-limited environment within the host. In general, however, direct measurement of cellular metal concentrations has shown remarkably small differences between widely divergent organisms, often less than an order of magnitude (Table 1). Thus there does seem to be a well-conserved optimum range of intracellular metal concentrations for most essential metals. The quota of a specific metal is largely dictated by the concentration of cellular proteins that require the metal for their function and by the importance of the metalloproteins (i.e., does the metal loprotein play a critical role or can another protein functionally substitute?). However, metal bioavailability and metal toxicity also strongly influence the metal quota and the nature of each metal-specific homeostasis system.
A critical factor in metal homeostasis is the bioavailability of the specific metal. The “bioavailability” of a metal can be distinct from the overall abundance of the metal in the environment. For example, iron is the fourth-most abundant element in the Earth’s crust. However, in the current-day oxygen-rich atmosphere of Earth, iron is largely present in the ferric (Fe3+) form. In aqueous, aerobic environments at neutral or basic pH, ferric iron forms nearly insoluble iron hydroxides. Because of the insolubility of Fe3+, iron is one of the least bioavailable of the essential transition metals. The concentration of iron in seawater (3 x 10-5 ppm) is nine orders of magnitude lower than the crustal concentration (5 x 104ppm). In contrast, zinc (Zn2+) is present at only 70 ppm in the crust but is found at 1 x 10-3 ppm in seawater (4, 5). The increased solubility of zinc means that it is actually more bioavailable than iron, despite its overall lower abundance. As discussed, iron homeostasis requires strategies for mobilizing iron in the environment, whereas homeostasis of metals such as zinc largely begins with cellular uptake.
The bioavailability of a given metal is influenced by its chemical speciation in the ambient environment. Although some metals occur predominantly in their “free” or “aquo” form (that is, the inner coordination sphere of the metal ion is occupied solely by water molecules), most bioactive metals occur as complexes in the natural environment. Hydroxide, carbonate, and chloride anions can all bind transition metals to a significant extent. For example, Fe3+ forms hydroxide complexes [e.g., Fe(OH)2+ or Fe(OH)3] in natural waters at pH > 7 and Cu tends to form carbonate complexes (CuCO3) under similar conditions. Metals may also bind to organic molecules, which may be expressly produced by resident biota to influence metal availability (such as siderophores for iron and methanobactin for copper) or result from the degradation of cells. It has been shown that most iron, copper, cobalt, and zinc ions in the marine environment are bound to unidentified organic molecules (6-9). Metals in terrestrial, freshwater, and near-shore environments are often bound to humic and fulvic acids produced via decomposition of terrestrial organic matter (10). As a general rule, only the free metal ion can react with uptake or transport proteins (11). However, the ligand exchange kinetics of most inorganic complexes are fast enough that these forms are often considered bioavailable as well. Organic complexes are often not immediately available (except in some cases such as lipophilic complexes); however, some cells have evolved specific biochemical mechanisms for obtaining required metals from organic ligands. For example, iron-siderophore complexes may be directly transported into the cell, and iron bound to nonspecific ligands may be obtained with ferric chelate reductases that reduce Fe3+ to Fe2+ for subsequent transport across the cell membrane (12).
In environments where metal bioavailability is limited by biological or geochemical factors, normal cellular growth and functioning may be impaired. For example, nearly 40% of global ocean waters are characterized by iron concentrations that are low enough to limit the growth of resident phytoplankton (13). In these systems, species have evolved mechanisms to reduce their minimum iron quotas, such as substituting proteins with copper or nonmetallic cofactors for iron metalloproteins (14, 15). Still, this limitation impacts biological productivity in the ocean and the processing of atmospheric CO2. Iron availability can also impact terrestrial organisms, as more than 30% of world soils are considered iron-deficient (16). Plants often experience chlorosis—decreased levels of chlorophyll—under such conditions. Up to two billion people, mostly in the developing world, are chronically iron-deficient because of the limited availability of iron in diets with a high plant component (16).
Despite some major challenges from limited bioavailability, transition metals can also be toxic if present in excess of cellular requirements. Even metals difficult to obtain, such as iron, can cause cellular damage if intracellular concentrations rise too high. Redox-active metals like iron and copper can cycle between different oxidation states under physiologic conditions, which is a characteristic that makes them useful in key electron transfer reactions. However, uncontrolled redox cycling caused by excess metal can generate reactive oxygen species, such as the hydroxyl radical, via the Fenton reaction. Metals that are not redox-active can still mediate considerable toxicity if present in excess. In these cases, toxicity often occurs because the metal binds to biologically inappropriate ligands within the cell, including metalloenzyme active sites intended for other metals. Nonspecific binding of metals to incorrect sites can lead to loss of protein function and cell damage. The Irving-Williams series predicts that available ligands will preferentially bind certain metals over other metals based partially on the ionic radii of the metal (Ca2+ < Mg2+ < Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+). For example, since the Irving-Williams series predicts that any given ligand will have a binding preference for divalent copper over ferrous iron, accumulation of excess copper might lead to copper binding at sites intended for iron. Metal toxicity requires that most metal homeostasis systems have mechanisms for limiting unwanted side-reactions between metals and cellular components. Examples of such mechanisms are the metallochaperones and metal storage proteins (see below).
The bioavailability of the metal in question influences the strategy used to prevent toxicity. Metals that are easily obtained from the environment can simply be removed from the cell by efflux transporters when levels rise too high. In contrast, metals that are difficult to obtain from the environment are often sequestered into storage proteins to preserve the metal for future use. Note: We focus on homeostasis of essential transition metals in this review. However, nonessential toxic metals also have a major impact on biological systems. They pose a dilemma for metal homeostasis because they are chemically similar to some essential transition metals except that they are often inert and will not perform the desired biochemical function. In some cases metal homeostasis systems cannot completely distinguish essential from nonessential metals. One major example for human health is lead. Lead is not normally used in any metalloprotein in biology but can enter cells and cause toxicity. The toxicity is thought to be mediated by Pb(II) binding to sites intended for chemically similar metals such as Zn(II). Silver in the form of Ag(I) is an excellent mimic for Cu(I) but is not redox-active. So silver binding to copper-specific sites also leads to nonfunctional metalloproteins. The study of cellular resistance to toxic metals is a fascinating and fully developed field. Readers are directed to the Further Reading section for more information on this topic.
The ''labile metal pool''
It is currently routine to quantify the total metal content of cultured cells or organisms using sensitive analytical techniques. For example, inductively coupled plasma mass spectrometry (ICP-MS) enables simultaneous detection of most transition metals in bulk cell culture samples at part per trillion concentrations, and high-resolution magnetic sector instruments are capable of separating all transition metals from common matrix and plasma interferences. These approaches have been used to establish the metal quota under various conditions in various organisms (17, 18). Most of any given metal within the cell will be stably incorporated into metalloenzymes and storage proteins. However, a small but physiologically critical pool of the metal has to be available for incorporation into newly synthesized metalloproteins. This “labile pool” of metal is not “free” in the chemical sense as the metal is likely bound to metallochaperones or other carrier proteins or to small molecules like citrate or glutathione. The labile metal pool is an important aspect of homeostasis because intracellular metal sensors that regulate metal homeostasis likely respond to changes in this subpopulation of metal. Also, the labile metal pool is more likely to undergo spurious side reactions under adverse conditions than metal safely incorporated into metalloenzymes or storage proteins. Thus it is critical to define the concentration of the labile metal pool and to understand how it is maintained as part of overall metal homeostasis. As is evident in the “Chemical Tools and Techniques” section, it is not trivial to establish a value for the labile metal pool.
Cellular Mechanisms to Maintain Metal Homeostasis
Figure 1 shows a generic scheme for a typical metal homeostasis system. We now will consider each component in detail.
Figure 1. General overview of metal homeostasis for a transition metal (Mn+). Not all components shown above are used for all transition metals. For simplicity, metal trafficking to subcellular organelles is not shown but is a key element of metal homeostasis in eukaryotic organisms. Dashed arrows indicate a possible role for metallochaperones in metal delivery to metal storage and metalloregulatory proteins.
Acquisition of metals from the environment
Some metals may need to be mobilized from the environment to make them bioavailable. Iron in particular must be rendered more soluble to be accessible for uptake. Microorganisms and some plants have evolved with secreted ligands known as siderophores (or phytosiderophores). These ligands bind Fe3+ with extraordinary affinity. For example, a complex of the siderophore enterobactin with ferric iron has a formal stability constant of 1049 (19). Once siderophores compete with other environmental ligands for iron, the ferric iron-siderophore complex then binds to specific transport proteins at the microbial cell surface and is taken into the cell. Most microorganisms can synthesize or use multiple siderophores as a source of iron. A similar strategy is used to move iron through the blood of multicellular organisms like mammals. Transferrin is an iron-binding plasma protein that preferentially binds ferric iron. Circulating Fe3+-transferrin complex is recognized by cell-surface receptors for uptake via receptor-mediated endocytosis (20). Because of tight regulation of iron transport throughout the body, pathogenic microbes are faced with the same problem of limited iron availability as their microbial counterparts in other environments. In response to this pressure, many microbial pathogens contain transporters and enzymes that allow them to obtain iron from host sources, such as heme and transferrin (21). Some plants use an alternative strategy for iron acquisition from the environment. Plant cells in the roots (where most iron is obtained) excrete protons in order to acidify the soil. Lowering the pH in the microenvironment around the roots increases the solubility of iron and allows it to be acquired by transporters (16). A similar pH-dependent process allows mammals to render dietary iron more soluble during digestion. In addition, some organisms contain ferric reductases that reduce ferric iron to the more soluble ferrous form. Reduction of ferric iron can occur at the cell surface or within the cytoplasm to release iron from ferric chelates like siderophores (22).
Concentrating metals within cells
Once the metal has been removed from the environment, it must be transported into the cell for use. In most cases, environmental metal levels are significantly lower than the cellular metal quota (Table 1). This dichotomy requires organisms to concentrate metals inside cells via energy-dependent processes. Transport of metals across the lipid bilayer by transport proteins is often linked to ATP hydrolysis or to the proton motive force (PMF) in order to provide the energy for concentrating metals. In some cases, the soluble metal is directly transported into the cell. In other cases (such as iron), metals complexed with acquisition molecules are transported. Once inside the cell, these complexes are disrupted to release the metal. In addition to transmembrane transporters, uptake of some metals requires other accessory proteins. For example, ferrous iron must be oxidized by a multi-copper oxidase enzyme at the cell surface in order to be transported by some eukaryotic transporters (23).
Intracellular metal trafficking
Once concentrated within the cell, specific metals must be routed to the proper metalloprotein. This problem is not trivial since many biological ligands are capable of binding essential metals. Nonspecific interaction of metals with inappropriate ligands could prevent the metal from reaching the necessary target metalloprotein. Spurious side reaction of metals with other molecules in the cell may also generate free radicals. For instance, the Fenton reaction between Fe2+ with H2O2, which is a normal byproduct of aerobic respiration, can generate highly dangerous hydroxyl radicals leading to cell damage. The routing of a metal to its correct target protein may occur as a result of the affinity of the metalloprotein for its specific metal. However, this mechanism seems inadequate as the intracellular concentration of a specific metalloprotein may be quite low compared with the concentration of competing, nonspecific ligands such as glutathione, citrate, or nucleic acids. In addition, the presence of multiple transition metals within the cell would make it difficult for a metalloprotein to partition a specific metal in a single step. It has become clear that several metalloproteins require a metallochaperone carrier protein to donate the correct metal to their active sites. For example, the Cu-Zn superoxide dismutase (SOD1) enzyme requires a copper metallochaperone, known as CCS, to donate copper for assembly of its active site (24). The discovery of metallochaperones for copper and nickel is anticipated by the Irving-Williams series. The series predicts that cellular ligands will preferentially bind nickel and copper, if allowed to access them, rather than metals like iron and manganese. Therefore, metallochaperones are required to ensure that nickel and copper are not bound by nonspecific ligands before they reach their correct binding sites. Metallochaperones have not been identified for all essential metals or for all metalloproteins. Some metalloproteins may interact directly with cytoplasmic domains of metal membrane transporters in order to acquire the correct metal before it enters the cytoplasm and can be chelated by other intracellular components. Other metals at the lower end of the Irving-Williams series, such as manganese, may not require dedicated metallochaperones.
In addition to direct trafficking to target metalloproteins, some metals need to enter specialized biosynthetic pathways for metal cofactor assembly. For example, iron in metalloenzymes is usually present as part of heme or Fe-S clusters, so it must be routed into the biosynthetic pathways for these cofactors. In a similar vein, molybdenum is not biologically active unless it is first incorporated with a pterin compound to form molybdenum cofactor (MoCo) (25). Once metal cofactors such as heme, Fe-S clusters, and MoCo are formed, they also require specialized trafficking systems to ensure they are integrated into the correct metalloprotein.
Because of the energy expended for metal acquisition (including extraction from the environment and transport into the cell against a concentration gradient), excess essential metals are often stored by an organism if they are not immediately incorporated into metalloproteins. Metal storage proteins allow the metal to be kept on hand for future use without exposing the cell to the deleterious effects of metal accumulation. The well-studied example of a metal storage protein is ferritin. Ferritin is a multi-subunit protein complex that can incorporate up to 4500 atoms of iron into ferric oxy-hydroxide cores (26). Once sequestered into ferritin, iron is largely inert until it is released by reduction. Another example of a metal storage protein is metallothionein (MT) for copper and zinc, although MT may also have a role in metal trafficking (27).
Metal efflux and detoxification
If metals accumulate to high intracellular concentrations (for example, because of environmental excess), organisms must have homeostasis mechanisms for removing, sequestering, or detoxifying the metals. In the case of toxic metals such as silver, removal of the metal via efflux transporters may be sufficient. Increased expression of metal storage proteins or novel metal sequestration proteins can also protect the cell from excess metal. This strategy seems to predominate for essential metals under most conditions. For example, the model organism Escherichia coli contains multiple ferritin homologues that are differentially regulated to provide excess storage capacity for iron under adverse conditions (28). In eukaryotes and some prokaryotes, the cysteine-rich metallothionein protein family functions to store or buffer excess copper and zinc (27).
Regulation of metal homeostasis
Expression of the systems mentioned must be carefully coordinated within the cell in order to maintain metal homeostasis. Consequently most organisms contain metalloregulatory proteins to regulate metal homeostasis. Metalloregulatory proteins are transcription factors that sense cellular metal levels and either activate or repress transcription of metal homeostasis genes in response to changes in metal levels. Typically the metal in question binds directly to the cognate metalloregulatory protein and acts as an allosteric switch for activating or inhibiting the transcription factor. In addition to regulation at the transcriptional level, some metal homeostasis systems are also regulated post-transcriptionally. For example, bacteria and mammals regulate iron homeostasis at the mRNA level. In bacteria, a small regulatory RNA, RyhB, binds target mRNAs involved in iron homeostasis and metabolism and regulates their stability or their translation. The ryhB gene itself is under the control of a metalloregulatory protein, Fur, that senses cellular iron (29). In mammals, cytoplasmic aconitase, also known as iron regulatory protein (IRP1), regulates the mRNAs of iron transport and storage proteins by directly binding to 5' or 3' untranslated regions within the mRNA and altering translation initiation or mRNA stability. IRP directly senses cellular iron levels by virtue of an Fe-S cluster present in the protein (30). A detailed discussion of these metalloregulatory proteins is presented in the technical article “Metalloregulatory Proteins” by Deborah Zamble.
Chemical Tools and Techniques
Genetic approaches have been used with considerable success to study metal homeostasis in genetically tractable model organisms like E. coli and S. cerevisiae. These approaches involve deleting genes that encode metal homeostasis proteins (such as metal transporters or metallochaperones) and analyzing the resulting phenotypes. For example, the Atx1 copper metallochaperone was originally identified in yeast as part of a genetic screen to isolate novel antioxidant factors. In addition, gene reporter constructs and DNA microarray analysis have been used to characterize metal responsive gene transcription and have helped to identify new genes that encode metal homeostasis proteins. Global transcriptional analysis has also led to a deeper understanding of how perturbations in metal homeostasis impact other cellular pathways. For example, DNA microarray analysis in E. coli has revealed that extensive remodeling of iron metalloproteins occurs when cells shift from an iron-rich environment to an iron-poor environment. Overall iron metalloprotein content is reduced in order to conserve available iron (28, 29). However, this remodeling has profound effects on cellular metabolism since many metabolic proteins contain iron in the form of Fe-S clusters or heme. Similar processes have also been observed in eukaryotic organisms (23). Thus, genetic techniques are allowing investigators to determine how metal homeostasis is integrated with overall cellular metabolism and physiology.
Biochemical characterization of metal homeostasis components
Purification and in vitro characterization of metal homeostasis proteins has provided a wealth of information about protein function. Specific metal binding sites have been characterized in metal transporters, metallochaperones, and metalloregulatory proteins using physical inorganic techniques such as extended x-ray absorbance fine structure (EXAFS) spectroscopy, Mossbauer spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and electroparamagnetic resonance (EPR) spectroscopy. For example, spectroscopic analysis of the Cu(I)-Atx1 complex revealed the coordination chemistry of the copper binding site and led to a chemical exchange model for step-by-step copper donation from Atx1 to the target metalloprotein CCC2 (31). Three-dimensional crystal structures of metal homeostasis components have also given researchers insight into the mechanisms of homeostasis. Proteins involved in metal transport (32), metal trafficking (33, 34), metal storage (35-37), and metal sensing (38, 39) have been crystallized (the references listed herein are only a small, representative sample from a large body of literature).
Measurement of the ''labile metal pool''
As mentioned, accurate determination of the “labile metal pool” is a key goal of metal homeostasis research. Most recent efforts have focused on measuring the labile metal pool in situ without disrupting the cell. This trend toward nondisruptive approaches stemmed from the realization that metal localization and speciation could be greatly perturbed by diluting cellular reductants, exposing the intracellular milieu to oxygen, disrupting subcellular organelles, and other adverse consequences of cell breakage (40). The general approach to define the labile metal pool relies on chelation of the labile metal pool in situ followed by detection and measurement of the chelator-metal complex. For example, to measure labile iron in E. coli, the ferric iron chelator desferrioxamine can be added to cells resulting in stabilization of all labile iron in the ferric form. The ferric iron can then be measured using EPR spectroscopy (41). Similarly, metal-specific fluorescent probes can be added to cells and the concentration of “available” metal measured by changes in fluorescent signal. Probes such as calcein and zinquin have been used to quantify the labile pools of iron and zinc, respectively (40, 42, 43). However, interpreting the results from these approaches can be difficult. If the metal binding affinity of the chelator or probe is sufficiently higher than the metal binding affinities of cellular metalloproteins, the probe may strip metals from metalloproteins and artificially increase the amount of “labile” metal (for a thorough discussion of troubleshooting the measurement of labile iron, see Reference 40). In addition, the cell membrane permeability, intracellular localization, and toxicity of the probe must be considered when evaluating its use.
To avoid these sorts of problems, some investigators have relied on the metal binding affinities of metallochaperones or metalloregulatory proteins themselves to estimate intracellular labile metal concentration (44). For example, two zinc metalloregulatory proteins, Zur and ZntR, control expression of zinc homeostasis genes in E. coli by directly binding Zn to sense cellular zinc levels. Zn-Zur represses zinc uptake systems when cellular zinc levels are adequate, whereas Zn-ZntR activates zinc efflux systems when zinc levels rise too high. Thus, the two regulators are thought to sense zinc at the lower and upper concentration limits of the labile zinc pool in E. coli . Measuring zinc-dependent DNA binding by Zur and ZntR allowed the investigators to establish the threshold zinc concentrations for activation of each regulator. These values were then reported as the upper and lower concentration limits of the labile zinc pool in E. coli (18). This approach assumes that both metalloregulatory proteins are in equilibrium with the labile zinc pool. It is not clear if this assumption applies in vivo since regulation of homeostasis proteins at all levels (from gene transcription to protein degradation) necessarily requires a lag between metal sensing and the desired change in the activity of the target homeostasis components, such as transporters and storage proteins. This lag may prevent a true equilibrium from forming under typical conditions where metal availability is not constant. Intermediate proteins may also act as carriers to load Zn into Zur or ZntR in vivo, which further complicates the labile zinc measurement since the additional protein-protein interactions required in vivo could significantly alter the zinc concentrations at which the regulators respond.
Measurements of labile iron range from about 0.9 to 12.3 x 10-6 M in the cytoplasm of a range of organisms from bacteria to mammals (40, 41). In contrast, some approaches have estimated cytoplasmic pools of labile zinc and copper to be 10-15 and 10-18 M, respectively (18, 45), whereas other studies have measured labile zinc at around 5 x 10-12 M (43). Although the absolute measurements vary somewhat, these studies clearly demonstrate that labile pools of copper and zinc are tightly controlled in vivo to avoid accumulation of these metals. The identity of the biological ligands that maintain such small labile pools of zinc and copper remains a topic of some controversy. As we can see, measurement of the labile metal pool is strongly influenced by the technique used and final determination of labile metal pools will likely require a convergence of approaches.
In vivo localization of cellular metals
The specific localization of transition metals within subcellular compartments has been characterized by a variety of methods. Subcellular organelles, such as mitochondria, purified by traditional centrifugation methods have been directly analyzed to identify subcellular populations of metals like copper (46, 47). Use of fluorescent probes coupled with microscopy allows investigators to visualize the subcellular location of metals, especially in eukaryotic cells because of their larger size. Studies of metal homeostasis in vivo have also benefited greatly from recent advances in analytical instrumentation. Subcellular mapping and quantification of transition metals within cells is possible using electron, proton, and X-ray microbeam techniques. Electron microprobes are capable of a spatial resolution of several nanometers (48) but require that cells be sectioned before analysis. Although widely available, electron microprobes also lack sensitivity to transition metals. Much higher sensitivity is possible when X rays are used to excite characteristic fluorescence, and impressive advances have been made with synchrotron X-ray fluorescence (SXRF) microprobes (49) (Fig. 2). Through the use of Fresnel zone plate optics at third-generation synchrotron facilities, spatial resolutions approaching 50 nm are now possible, and detection limits on the order of 10-18 mol per cell are routinely achieved for the transition metals (50). SXRF has been used to determine subcellular localization of transition metals in cardiomyocytes (51), human leukemia cells (52), and plant seeds (53). SXRF can also be combined with immunofluorescence probes (54) or metal-specific fluorescent sensors. For example, Yang et al. (55) visualized the localization of intracellular “labile” copper in the mitochondria and Golgi apparatus of mouse fibroblast cells using both fluorescent probes and X-ray analysis.
Figure 2. An example of the use of SXRF in combination with genetics to study metal localization and metal homeostasis. Col-O is the wild-type control Arabidopsis plant, whereas the vit1-1 mutant lacks a vacuolar iron uptake transporter needed for proper iron homeostasis in Arabidopsis seeds. X-ray fluorescence microtomography of seeds shows clear differences in Fe localization within the seeds of the mutant compared with the wild-type plant, whereas Zn and Mn localization was not altered. (A) Light micrograph cross-section of a mature Arabidopsis seed; bar: 62 pm. (B and C) Total X-ray absorption tomographic slices of Col-0 and vit1-1 seeds; bar: 100 pm. (D) X-ray fluorescence tomographic slices of Fe Ka (blue), Mn Ka (green), and Zn Ka (red) fluorescence lines collected from Col-0 and vit1-1 with metal abundances indicated in mg kg-1 (smaller images), and composite images of Fe, Mn, and Zn abundance of Col-0 and vit1-1 (larger images). (E) Three-dimensional rendering of total X-ray absorption of a wild-type Arabidopsis seed. (F) In silico-sectioned (y-axis, upper 50% removed) rendering of total X-ray absorption shown in (E). (G and H) Three-dimensional rendering of Fe Ka X-ray fluorescence in Col-0 and vit1-1, respectively, with both seeds identically oriented. From Reference 53, reprinted with permission from AAAS.
Future Research Directions
The metallome and metallome homeostasis
Initial study of metal homeostasis tended to focus on a single metal. However, proper functioning of most organisms requires careful balancing of multiple essential transition metals. The sum of all essential metals used by an organism for cellular function is known as the “metallome.” The metallome consists of the total metal content of a cell but also includes all specific metal-biomolecule complexes that are present in a given cell. Characterizing this global entity is one of the most exciting future research directions in the field of metal homeostasis. Measuring the metal quota for each essential metal is still a key requirement for defining the metallome. Recently it has become possible to simultaneously measure all essential transition metals in an organism. For example, ICP-MS has been used to measure metal quotas in a diverse range of prokaryotic and eukaryotic organisms under controlled growth conditions. If the number of cells in the sample and the cell volume are known, ICP-MS measurements can be converted into total cellular concentrations to facilitate the comparison of metal quotas in organisms with a wide range of cell sizes. The power of approaches like ICP-MS is that they allow investigators to simultaneously measure cellular concentrations of essential transition metals in different organisms, under different environmental conditions, and in different genetic backgrounds to more fully define the metal quota. Another dimension of the metallome concept is the specific location of transition metals within the cell (as opposed to their total concentrations in the entire cell). In this context, location can refer to the protein or biomolecule component where the metal is bound as well as the subcellular localization of the metal in various organelles. One approach to define metal localization will be to combine metal analysis techniques with proteomics in order to identify all metalloproteins, including their specific transition metal content, within an organism (56, 57). Global analysis of the metalloprotein portion of the metallome will also lead to the identification of previously unknown metalloproteins. Identifying new metalloproteins, whether they are novel metalloenzymes or new homeostasis components, will point to new roles for metals in biology. Other mass spectrometry techniques should also be applied to study of the metallome in order to characterize metals colocalized with nonprotein cellular components such as lipids, metabolites, and nucleic acids.
Some of the most intriguing differences between the metallomes of different cell types occur in subcellular organelles or vesicles. Eukaryotic cells in particular have carefully compartmentalized essential transition metals for specific biological purposes. The mitochondria and the chloroplast both contain high levels of metalloproteins relative to the cytoplasm and may have distinct metal quotas. Mammalian cerebrocortical neurons possess zinc-filled vesicles with labile zinc pools that approach millimolar concentrations (58) and other eukaryotic cells, including yeast, possess vesicular “zincosomes” for zinc storage (59). Strategies that combine well-defined fluorescent probes with microscopy or SXRF are needed to clarify the subcellular localization and concentration of the essential transition metals. The goal of these studies is to establish baseline concentrations and locations for all transition metals within the cell. Once this baseline is established, a host of different experiments can be performed to test how the metallome is perturbed by adverse environmental conditions and in disease states. Another future direction for metal homeostasis research is to understand how the individual metal homeostasis systems overlap and interact so that all required essential metals are obtained and routed to the correct locations. These interactions we refer to as metallome homeostasis. That such interactions exist is clear. For example, copper homeostasis is intertwined with iron acquisition because a multi-copper oxidase enzyme is needed for iron uptake at the cell surface (23). There are also numerous reports of transporters, such as the NRAMP family, that have the ability to transport multiple metals (60). Clearly metallome homeostasis is complex and metal homeostasis of individual metals does not occur in isolation. Future characterization of the metallome and metallome homeostasis will help complete our understanding of metal homeostasis and likely open exciting new avenues of bioinorganic research.
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Metal Transport Through Membranes
Chemistry of Metallochaperones
Chemistry of Metalloenzymes