CHAPTER 1: Overview

This book has the objective of describing the binding, transport and storage, mainly of metal ions, and many of their functions in organisms. It does not cover the link between these functions and enzyme action. The widest possible range of metal ions and metalloids are described. The functions include intracellular and extracellular activities but the book does not look at the transport into prokaryote cells through siderophores and similar molecules. Great stress is placed on healthy rather than diseased conditions.

Looking first at binding by all kinds of biological ligands we must remember that in an organism selection between the metal ions and their ligands is essential. The selection is constrained differently inside different compartments by the limitations imposed by transport of metal ions through membranes. The specific transport by inward or outward pumps may give either high concentrations of a given element in a vesicle (for example, in the message vesicles of zinc and calcium) or low concentrations. We must also note that binding in any compartment can be either weak or strong depending on the ligand. For example adenosine triphosphate, ATP, binds weakly to all the metal ions in cells. Hence ATP is only bound by an ion of the element magnesium, which is in high free ion concentration, much higher than that of any other ions. ATP and Mg²⁺ are overwhelmingly retained in one compartment, the cytoplasm. There are further factors governing stronger metal ligand selective binding at equilibrium. In addition to controlling metal ion concentration the ligand can also be controlled in its concentration by transport through membranes into vesicles. Clearly the two can then give selectivity in bound states. Examples are the small molecule organic anions in zinc vesicles and the specific proteins in calcium vesicles. An additional control on ligand concentration is synthesis, to which we turn later. As oxidative/reductive potentials are fixed differently in certain compartments so the resultant binding at equilibrium is different and ligands are selected for this purpose. We note that ferrous and cuprous states are usually found inside the cytoplasm of cells whereas in the extracellular fluids and the environment, ferric and cupric states prevail. With those metal ions that are responsible for activity in membranes there must be transport to them and selective binding in the membranes. Membranes then form separate compartments and in them there is little interaction between fixed sites and free ions except at pumps.

Worthy of note are the orders of selection of metal ions with ligands at equilibrium. In the cases of Mg²⁺ and Ca²⁺ the binding selectivity between the two is dependent upon the sizes of the metal ions and the cavity provided by the ligand. For a small cavity magnesium ions bind most strongly but for a cavity that cannot close around the small magnesium its hydration makes binding weak. However, calcium may fit such a constrained space very well when the order of binding strength becomes Ca²⁺>Mg²⁺. The possibility of selection is very different in the cytoplasm where Mg and K ions are accumulated while the other two ions of Na and Ca are rejected from this space. This makes gradients across membranes; see later. While here the Mg/Ca selectivity is related to different ion size fittings, similarly so is that of Na⁺ relative to K⁺. This discrimination based on size is not very powerful in the equilibrium binding of the transition metal ions in the observed Irving–Williams series of their complexes:

The ions here are of similar though declining size but the order arises from the electron affinity of the ions. This order is found in vitro with almost every ligand and it is very probably true in biological compartments to which there is access to all of them. All these ions are present to some extent in the cytoplasm. Fe²⁺ ions are not found in vesicles but Mn²⁺ is.

It is important to observe that the series is broken by change of spin state, to some degree by steric demands of ligands (crystal field effects) and of course by redox state changes of some ions. The spin-state changes are observed for example in iron ortho-phenanthroline complexes in vitro and in porphyrins in vivo. The binding of low-spin ions is then such that Fe²⁺=Cu²⁺. Steric factors influence the relative binding of some pairs of ions. For example Zn²⁺ prefers tetrahedral but Ni²⁺ prefers octahedral coordination. Thus the order of binding can be Ni²⁺>Zn²⁺ or Zn²⁺>Ni²⁺ according to the geometric disposition of liganding groups. A discussion of all the above bindings is given in ref. 1.

We turn from thermodynamic binding in a compartment to the kinetic factors generating selection. Most obviously the production of protein ligands from translated RNA is controlled, started or stopped, often by transcription factors and promoters bound to DNA. These factors and promoters are often activated themselves by the binding of metal ions or the action of small organic molecules. This control leads to limiting concentrations of specific protein ligands. Proteins can also be transferred to selected binding sites and to compartments. Since most proteins are not very stable they are constantly slowly degraded and must be replaced. A flow system in steady state is then a limiting factor on such concentrations. Some ions in proteins are selective transcription factors such as zinc fingers bound to DNA or are hydrolases such as metallophosphatases giving rise to phosphate, both of which control gene expression.

Apart from these conditions of the kinetics of ligand concentrations there are also kinetic limitations on exchange of metal ions from binding sites. This is particularly true for very strongly bound ions such as those of copper and zinc. These ions are found with different protein partners inside and outside cells. This condition is brought about by transport extending from across membranes to carrier proteins, often called metallochaperones. The first observed example of the second kind of transport is that across the cytoplasm of epithelial cells of calcium by the intestinal calcium binding protein. Subsequently many other metallochaperones have been found. Different chaperones convey a given metal ion to a target site where it may be virtually irreversibly bound. A very good example that has been known for some time is the range of chelatases, protein ligands, which carry different metal ions to different porphyrins in proteins. Six metal ions are involved, ferrous, cobalt, nickel, magnesium and, much more rarely, copper and zinc. Recognition of insertion sites is by selective protein/protein binding. Porphyrin binding of metal ions is not exchangeable and is then irreversible. Irreversible binding cannot be included in the Irving–Williams series, which only applies to reversible equilibrium binding. This does not mean, however, that selection at early steps by chaperones and by pumps is also excluded. In most cases of chelatases binding it is reversible. This may be true of other chaperones and many of the ion pumps where binding, though different on the two different sides of a membrane, is reversible with the contents of the two compartments which are separated by the membrane. It may be that free ion concentration (and binding) is selective in the cytoplasm following the Irving–Williams series, common to all cells.

Our analysis so far has largely concerned the cytoplasm of all cells. A different kind of selection is apparent in quite different compartments including the internal extracellular media of multicellular organisms. This book is not concerned with the binding of elements in the environment even where it is known that molecules, even proteins, are exported to scavenge for a particular ion, for example ferric ion by siderophores. On the other hand scavenging by ligands between cells in multicellular organisms is included in the chapters.

We must now turn to the functions of selective handling of metal ions in organisms. The most obvious is that strong selection by ligand sites allows subsequent strict selection of enzyme action. In no way must acid/base catalysis, mainly a function of zinc and magnesium ions, be confused with redox activity dominated by iron and copper ions. It is very noticeable that many enzyme sites are associated with one metal only, allowing experimental extraction and fractionation of specific metalloenzymes and proteins, such as copper oxidases and zinc fingers. This is not universally true and certain sites may be bound by one cation but be replaceable by another. A recent example are forms of carbonic anhydrase in which zinc can be replaced by cadmium in one organism and perhaps by cobalt in several. This is very different from the finding of different metal ions for a given function in very different organisms, for example there are iron, manganese, nickel and zinc/copper superoxide dismutases even in different cell compartments. The most obvious use of metal ion binding to proteins, RNA or DNA, is to give them structure. The structures can remain mobile between large domains or very locally. The mobility allows the proteins in particular to act as triggers of actions. Valence states or simple spin-state changes can adjust structure to induce uptake cooperativity as of oxygen in haemoglobin.

Returning to vesicles, or similar containers together with the extracellular fluids inside multicellular organisms, their redox and/or acidity is usually different from those of the fluids in the cytoplasm. Certain vesicles for the uptake of iron are acidic and the extracellular fluids and these and other vesicles are controlled. In these compartments iron is ferric not ferrous and copper is cupric not cuprous. There are exceptions as the circulating cupric oxygen carrier in some organisms, haemocyanin, is a cuprous complex but another oxygen carrier is a ferric protein. Of course all metal ions have to be circulated and apart from the free ions of Na, K, Mg and Ca, more strongly bound ions, such as Cu, are usually transferred in complexes or by proteins such as albumins.

Many cells have vesicles or other structures for storage even within the cytoplasm of cells. The well-known examples are the storage of iron in ferritin, of haem iron in haemoglobin and myoglobin in muscle tissue. The haemoglobin iron in cells is reduced iron, needed for oxygen binding. Many organisms store somewhat poisonous metal ions in vesicles rejecting the vesicles later. In this way stores can act in purification. Plants that can survive in nickel-rich soils carry the nickel to vesicles in the leaves and then it is thrown away as the leaf falls. Cadmium is stored in the animal multi-metal ion protein, metallothionein, in the kidney cells, while plants and more primitive organisms contain it in a peptide, glutathione. Other kinds of vesicle storage are the precursors of external biominerals of which the most obvious are calcium carbonates or phosphates, which on export are placed in the outer surface of lower organisms. The most studied example is that of the coccolith. By way of contrast animals store calcium phosphates in bone and plants store silica in small granules, concentrated in large numbers internally. While the silica is very free from other ions bone is not. There are many other biomineral storages such as magnetite (Fe), Sr and Ba in sulfates, and even Zn in the teeth of some organisms. Of course transport is common to movement of vesicles or of ions by chaperones.

Finally binding of ions can give structure to proteins, DNA and RNA. Particularly protein folding often occurs only on binding calcium hence calcium acts as a trigger messenger of action. The structure of ribosomes is dependent on the presence of magnesium and some part of DNA depends on potassium binding.

A way of understanding the vast range of metalloproteins, even excluding enzymes, is to analyse their evolution through their changing DNA sequences with the changing environment through the ages.²  This analysis reveals relatively sudden changes of the numbers of such proteins at the two major stages of organization of cells. The first is the evolution of single-cell eukaryotes from prokaryotes while the second is that of multicellular organisms. Different functions of the metalloproteins can be seen in the three different groups of organisms.

In conclusion the book then covers a very wide range of the various metal ion functions quite apart from in enzyme catalysis. These functions are of equal importance with those of catalysis and like it they have evolved over billions of years.