The storage of zinc, copper, vanadium, chromium, molybdenum, cobalt, nickel, and manganese

Ions of nonferrous transition metals require a much less complex biological storage system, because the solubilities are much higher (210 - 8 M) than those for Fe 3+ . As a result, the storage of nonferrous transition metals is less obvious, and information is more limited. In addition, investigations are more difficult than for iron, because the amounts in biological systems are so small. Essentially nothing is known yet about the storage of vanadium, chromium, molybdenum, cobalt, nickel, and manganese, with the possible exception of accumulations of vanadium in the blood cells of tunicates.
 

Zinc and copper, which are used in the highest concentrations of any of the non-ferrous transition metals, are specifically bound by the protein metallothionein 35,36 (see Figure 1.10). Like the ferritins, the metallothioneins are a family of proteins, widespread in nature and regulated by the metals they bind. In contrast to ferritin, the amounts of metal stored in metallothioneins are smaller (up to twelve atoms per molecule), the amount of protein in cells is less, and the template (mRNA) is not stored. Because the cellular concentrations of the metallothioneins are relatively low and the amount of metal needed is relatively small, it has been difficult to study the biological fate of copper and zinc in living organisms, and to discover the natural role of metallothioneins. However, the regulation of metallothionein synthesis by metals, hormones, and growth factors attests to the biological importance of the proteins. The unusual metal environments of metallothioneins have attracted the attention of bioinorganic chemists.
 

Metallothioneins, especially in higher animals, are small proteins 35,36 rich in cysteine (20 per molecule) and devoid of the aromatic amino acids phenylalanine and tyrosine. The cysteine residues are distributed throughout the peptide chain. However, in the native form of the protein (Figure 1.10), the peptide chains fold to produce two clusters of -SH, which bind either three or four atoms of zinc, cadmium, cobalt, mercury, lead, or nickel. Copper binding is distinct from zinc, with 12 sites per molecule.
 

In summary, iron is stored in iron cores of a complicated protein. Ferritin, composed of a hollow protein coat, iron-protein interface, and an inorganic core, overcomes the problems of redox and hydrolysis by directing the formation of the quasi-stable mineral hydrous ferric oxide inside the protein coat. The outer surface of the protein is generally hydrophilic, making the complex highly soluble; equivalent concentrations of iron are :::::0.25 M. By contrast to iron, storage of zinc, copper, chromium, manganese, vanadium, and molybdenum is relatively simple, because solubility is high and abundance is lower. Little is known

about the molecules that store these metals, with the possible exception of metallothionein, which binds small clusters of zinc or copper.

Chemical properties of zinc, copper, vanadium, chromium, molybdenum, and cobalt

The chemical properties of the other essential transition elements simplify their transport properties. For zinc there is only the +2 oxidation state, and the hydrolysis of this ion is not a limiting feature of its solubility or transport. Zinc is an essential element for both animals and plants. 8,9,20,21 In general, metal ion uptake into the roots of plants is an extremely complex phenomenon. A crosssectional diagram of a root is shown in Figure 1.6. It is said that both diffusion

and mass flow of the soil solution are of significance in the movement of metal ions to roots. Chelation and surface adsorption, which -are pH dependent, also affect the availability of nutrient metal ions. Acid soil conditions in general retard uptake of essential divalent metal ions but increase the availability (sometimes with toxic results) of manganese, iron, and aluminum, all of which are normally of very limited availability because of hydrolysis of the trivalent ions. Vanadium is often taken up as vanadate, in a pathway parallel to phosphate. However, its oxidation state within organisms seems to be highly variable. Unusually high concentrations of vanadium occur in certain ascidians (the specific transport behavior of which will be dealt with later). The workers who first characterized the vanadium-containing compound of the tunicate, Ascidia nigra, coined the name tunichrome.  The characterization of the compound as a dicatecholate has been reported. 

 

Quite a different chemical environment is found in the vanadium-containing material isolated from the mushroom Amanita muscaria. Bayer and Kneifel, who named and first described amavadine,24 also suggested the structure shown in Figure 1. 7. 25 Recently the preparation, proof of ligand structure, and (by implication) proof of the complex structure shown in Figure 1.7 have been established.  Although the exact role of the vanadium complex in the mushroom

 

 

remains unclear, the fact that it is a vanadyl complex is now certain, although it may take a different oxidation state in vivo.
 

The role of chromium in biology remains even more mysterious. In human beings the isolation of "glucose tolerance factor" and the discovery that it contains chromium goes back some time. This has been well reviewed by Mertz, who has played a major role in discovering what is known about this elusive and apparently quite labile compound.27 It is well established that chromium is taken up as chromic ion, predominantly via foodstuffs, such as unrefined sugar, which presumably contain complexes of chromium, perhaps involving sugar hydroxyl groups. Although generally little chromium is taken up when it is administered as inorganic salts, such as chromic chloride, glucose tolerance in many adults and elderly people has been reported to be improved after supplementation with 150-250 mg of chromium per day in the form of chromic chloride. Similar results have been found in malnourished children in some studies in Third World countries. Studies using radioactively labeled chromium have shown that, although inorganic salts of chromium are relatively unavailable to mammals,

brewer's yeast can convert the chromium into a usable form; so l:irewer's yeast is today the principal source in the isolation of glucose tolerance factor and has been used as a diet supplement.
 

Although chromium is essential in milligram amounts for human beings as the trivalent ion, as chromate it is quite toxic and a recognized carcinogen. 30 The uptake-reduction model for chromate carcinogenicity as suggested by Connett and Wetterhahn is shown in Figure 1.8. Chromate is mutagenic in bacterial and mammalian cell systems, and it has been hypothesized that the difference between chromium in the +6 and +3 oxidation states is explained by the' 'uptake- reduction" model. Chromium(III), like the ferric ion discussed above, is readily hydrolyzed at neutral pH and extremely insoluble. Unlike Fe 3+ , it undergoes extremely slow ligand exchange. For both reasons, transport of chromium( III) into cells can be expected to be extremely slow unless it is present as specific complexes; for example, chromium(III) transport into bacterial cells has been reported to be rapid when iron is replaced by chromium in the siderophore iron-uptake mediators. However, chromate readily crosses cell membranes and enters cells, much as sulfate does. Because of its high oxidizing power, chromate can undergo reduction inside organelles to give chromium(m), which binds to small molecules, protein, and DNA, damaging these cellular components.

In marked contrast to its congener, molybdenum is very different from chromium in both its role in biology and its transport behavior, again because of fundamental differences in oxidation and coordination chemistry properties. In contrast to chromium, the higher oxidation states of molybdenum dominate its chemistry, and molybdate is a relatively poor oxidant. Molybdenum is an essential element in many enzymes, including xanthine oxidase, aldehyde reductase, and nitrate reductase. 19 The range of oxidation states and coordination geometries of molybdenum makes its bioinorganic chemistry particularly interesting and challenging.
 

The chemistry of iron storage and transport is dominated by high concentrations, redox chemistry (and production of toxic-acting oxygen species), hydrolysis (pKa is about 3, far below physiological pH), and insolubility. High-affinity chelators or proteins are required for transport of iron and high-capacity sequestering protein for storage. By comparison to iron, storage and transport of the other metals are simple. Zinc, copper, vanadium, chromium, manganese, and molybdenum appear to be transported as simple salts or loosely bound protein complexes. In vanadium or molybdenum, the stable anion, vanadate or molybdate, appears to dominate transport. Little is known about biological storage of any metal except iron, which is stored in ferritin. However, zinc and copper are bound to metallothionein in a fonn that may participate in storage.

Biological Significance of Iron, Zinc, Copper, Molybdenum, Cobalt, Chromium, Vanadium, and Nickel

Living organisms store and transport transition metals both to provide appropriate concentrations of them for use in metalloproteins or cofactors and to protect themselves against the toxic effects of metal excesses; metalloproteins and metal cofactors are found in plants, animals, and microorganisms. The normal concentration range for each metal in biological systems is narrow, with both deficiencies and excesses causing pathological changes. In multicellular organisms, composed of a variety of specialized cell types, the storage of transition metals and the synthesis of the transporter molecules are not carried out by all types of cells, but rather by specific cells that specialize in these tasks. The form of the metals is always ionic, but the oxidation state can vary, depending on biological needs. Transition metals for which biological storage and transport are significant are, in order of decreasing abundance in living organisms: iron, zinc, copper, molybdenum, cobalt, chromium, vanadium, and nickel. Although zinc is not strictly a transition metal, it shares many bioinorganic properties with transition metals and is considered with them in this chapter. Knowledge of iron storage and transport is more complete than for any other metal in the group. The transition metals and zinc are among the least abundant metal ions in the sea water from which contemporary organisms are thought to have evolved (Table 1.1).1-5 For many of the metals, the concentration in human blood plasma 

greatly exceeds that in sea water. Such data indicate the importance of mechanisms for accumulation, storage, and transport of transition metals and zinc in living organisms.
 

The metals are generally found either bound directly to proteins or in cofactors such as porphyrins or cobalamins, or in clusters that are in tum bound by the protein; the ligands are usually 0, N, S, or C. Proteins with which transition metals and zinc are most commonly associated catalyze the intramolecular or intermolecular rearrangement of electrons. Although the redox properties of the metals are important in many of the reactions, in others the metal appears to contribute to the structure of the active state, e.g., zinc in the Cu-Zn dismutases and some of the iron in the photosynthetic reaction center. Sometimes equivalent reactions are catalyzed by proteins with different metal centers; the metal binding sites and proteins have evolved separately for each type of metal center.

 

Iron is the most common transition metal in biology. 6,7 Its use has created a dependence that has survived the appearance of dioxygen in the atmosphere ca. 2.5 billion years ago, and the concomitant conversion of ferrous ion to ferric ion and insoluble rust (Figure 1.1 See color plate section, page C-1.). All plants, animals, and bacteria use iron, except for a lactobacillus that appears to maintain high concentrations of manganese instead of iron. The processes and reactions in which iron participates are crucial to the survival of terrestrial organisms, and include ribonucleotide reduction (DNA synthesis), energy production (respiration), energy conversion (photosynthesis), nitrogen reduction, oxygen transport (respiration, muscle contraction), and oxygenation (e.g., steroid synthesis, solubilization and detoxification of aromatic compounds). Among the transition metals used in living organisms, iron is the most abundant in the environment. Whether this fact alone explains the biological predominance of iron or whether specific features of iron chemistry contribute is not clear.

Many of the other transition metals participate in reactions equivalent to those involving iron, and can sometimes substitute for iron, albeit less effectively, in natural Fe-proteins. Additional biological reactions are unique to nonferrous transition metals.
 

Zinc is relatively abundant in biological materials. 8 ,9 The major location of zinc in the body is metallothionein, which also binds copper, chromium, mercury, and other metals. Among the other well-characterized zinc proteins are the Cu-Zn superoxide dismutases (other forms have Fe or Mn), carbonic anhydrase (an abundant protein in red blood cells responsible for maintaining the pH of the blood), alcohol dehydrogenase, and a variety of hydrolases involved in the metabolism of sugars, proteins, and nucleic acids. Zinc is a common element in nucleic-acid polymerases and transcription factors, where its role is considered to be structural rather than catalytic. Interestingly, zinc enhances the stereoselectivity of the polymerization of nucleotides under reaction conditions designed to simulate the environment for prebiotic reactions. Recently a group of nucleic-acid binding proteins, with a repeated sequence containing the amino acids cysteine and histidine, were shown to bind as many as eleven zinc atoms necessary for protein function (transcribing DNA to RNA). 10 Zinc plays a structural role, forming the peptide into multiple domains or "zinc fingers" by means of coordination to cysteine and histidine (Figure 1.2A See color plate section, page C-l.). A survey of the sequences of many nucleic-acid binding proteins shows that many of them have the common motif required to form zinc fingers. Other zinc-finger proteins called steroid receptors bind both steroids such as progesterone and the progesterone gene DNA (Chapter 8). Much of the zinc in animals and plants has no known function, but it may be maintaining the structures of proteins that activate and deactivate genes. 11
 

Copper and iron proteins participate in many of the same biological reactions:

1. reversible binding of dioxygen, e.g., hemocyanin (Cu), hemerythrin (Fe), and hemoglobin (Fe);
2. activation of dioxygen, e.g., dopamine hydroxylase (Cu) (important in the synthesis of the hormone epinephrine), tyrosinases (Cu), and catechol dioxygenases (Fe);
3. electron transfer, e.g., plastocyanins (Cu), ferredoxins, and c-type cytochromes (Fe);
4. dismutation of superoxide by Cu or Fe as the redox-active metal (superoxide dismutases).

The two metal ions also function in concert in proteins such as cytochrome oxidase, which catalyzes the transfer of four electrons to dioxygen to form water during respiration. Whether any types of biological reactions are unique to copper proteins is not clear. However, use of stored iron is reduced by copper deficiency, which suggests that iron metabolism may depend on copper proteins, such as the serum protein ceruloplasmin, which can function as a ferroxidase, and the cellular protein ascorbic acid oxidase, which also is a ferrireductase.

Cobalt is found in vitamin B12 , its only apparent biological site. 12 The vitamin is a cyano complex, but a methyl or methylene group replaces CN in native enzymes. Vitamin-B 12 deficiency causes the severe disease of perniCious anemia in humans, which indicates the critical role of cobalt. The most common type of reaction in which cobalamin enzymes participate results in the reciprocal exchange of hydrogen atoms if they are on adjacent carbon atoms, yet not with hydrogen in solvent water:

(An important exception is the ribonucleotide reductase from some bacteria and lower plants, which converts ribonucleotides to the DNA precursors, deoxyribonucleotides, a reaction in which a sugar -OH is replaced by -H. Note that ribonucleotide reductases catalyzing the same reaction in higher organisms and viruses are proteins with an oxo-bridged dimeric iron center.) The cobalt in vitamin B12 is coordinated to five N atoms, four contributed by a tetrapyrrole (corrin); the sixth ligand is C, provided either by C5 of deoxyadenosine in enzymes such as methylmalonyl-CoA mutase (fatty acid metabolism) or by a methyl group in the enzyme that synthesizes the amino acid methionine in bacteria.
 

Nickel is a component of a hydrolase (urease), of hydrogenase, of CO dehydrogenase, and of S-methyl CoM reductase, which catalyzes the terminal step in methane production by methanogenic bacteria. All the Ni-proteins known to date are from plants or bacteria. 13,14 However, about 50 years elapsed between the crystallization of jack-bean urease in 1925 and the identification of the nickel component in the plant protein. Thus it is premature to exclude the possibility of Ni-proteins in animals. Despite the small number of characterized Ni-proteins, it is clear that many different environments exist, from apparently direct coordination to protein ligands (urease) to the tetrapyrrole F430 in methylreductase and the multiple metal sites of Ni and Fe-S in a hydrogenase from the bacterium Desulfovibrio gigas. Specific environments for nickel are also indicated for nucleic acids (or nucleic acid-binding proteins), since nickel activates the gene for hydrogenase. 

 

Manganese plays a critical role in oxygen evolution catalyzed by the proteins of the photosynthetic reaction center. The superoxide dismutase of bacteria and mitochondria, as well as pyruvate carboxylase in mammals, are also manganese proteins. 16,17 How the multiple manganese atoms of the photosynthetic reaction center participate in the removal of four electrons and protons from water is the subject of intense investigation by spectroscopists, synthetic inorganic chemists, and molecular biologists. 

Vanadium and chromium have several features in common, from a bioinorganic viewpoint. 18a First, both metals are present in only small amounts in most organisms. Second, the biological roles of each remain largely unknown. 18 Finally, each has served as a probe to characterize the sites of other metals, such as iron and zinc. Vanadium is required for normal health, and could act in vivo either as a metal cation or as a phosphate analogue, depending on the oxidation state, V(lV) or V(V), respectively. Vanadium in a sea squirt (tunicate), a primitive vertebrate (Figure 1.2B), is concentrated in blood cells, apparently as the major cellular transition metal, but whether it participates in the transport of dioxygen (as iron and copper do) is not known. In proteins, vanadium is a cofactor in an algal bromoperoxidase and in certain prokaryotic nitrogenases. Chromium imbalance affects sugar metabolism and has been associated with the glucose tolerance factor in animals. But little is known about the structure of the factor or of any other specific chromium complexes from plants, animals, or bacteria.

Molybdenum proteins catalyze the reduction of nitrogen and nitrate, as well as the oxidation of aldehydes, purines, and sulfite. 19 Few Mo-proteins are known compared to those involving other transition metals. Nitrogenases, which also contain iron, have been the focus of intense investigations by bioinorganic chemists
and biologists; the iron is found in a cluster with molybdenum (the iron-molybdenum cofactor, or FeMoCo) and in an iron-sulfur center (Chapter 7). Interestingly, certain bacteria (Azotobacter) have alternative nitrogenases, which are produced when molybdenum is deficient and which contain vanadium and iron or only iron. All other known Mo-proteins are also Fe-proteins with iron centers, such as tetrapyrroles (heme and chlorins), Fe-sulfur clusters, and, apparently, non-heme/non-sulfur iron. Some Mo-proteins contain additional cofactors such as the Havins, e.g., in xanthine oxidase and aldehyde oxidase. The number of redox centers in some Mo-proteins exceeds the number of electrons transferred; reasons for this are unknown currently.