BIOLOGICAL SYSTEMS OF METAL STORAGE, TRANSPORT, AND MINERALIZATION

1. The storage of iron
 

Three properties of iron can account for its extensive use in terrestrial biological reactions:
 

(a) facile redox reactions of iron ions;
 

(b) an extensive repertoire of redox potentials available by ligand substitution or modification (Table 1.4);
 

(c) abundance and availability (Table 1.1) under conditions apparently extant when terrestrial life began (see Section LB.).
 

Ferrous ion appears to have been the environmentally stable form during prebiotic times. The combination of the reactivity of ferrous ion and the relatively large amounts of iron used by cells may have necessitated the storage of ferrous ion; recent results suggest that ferrous ion may be stabilized inside ferritin long enough to be used in some types of cells. As primitive organisms began to proliferate, the successful photosynthetic cells, which trapped solar energy by reducing CO2 to make carbohydrates (CH20)n and produce O2 , exhausted from the environment the reductants from H2 or H2S or NH3 . The ability of primitive organisms to switch to the use of H20 as a reductant, with the concomitant production of dioxygen, probably produced the worst case of environmental pollution in terrestrial history. As a result, the composition of theatmosphere, the course of biological evolution, and the oxidation state of environmental iron all changed profoundly. Paleogeologists and meteorologists estimate that there was a lag of about 200-300 million years between the first dioxygen production and the appearance of significant dioxygen concentrations in the atmosphere, because the dioxygen produced at first was consumed by the oxidation of ferrous ions in the oceans. The transition in the atmosphere, which occurred about 2.5 billion years ago, caused the bioavailability of iron to plummet and the need for iron storage to increase. Comparison of the solubility of Fe 3+ at physiological conditions (about 10 - 18 M) to the iron content of cells (equivalent to 10 -5 to 10 -8 M) emphasizes the difficulty of acquiring sufficient Iron.
 

Iron is stored mainly in the ferritins, a family * of proteins composed of a protein coat and an iron core of hydrous ferric oxide [Fe203(H20)n] with various amounts of phosphate.6,7 As many as 4,500 iron atoms can be reversibly stored inside the protein coat in a complex that is soluble; iron concentrations equivalent to 0.25 M [about 10 16-fold more concentrated than Fe(III) ions] can be easily achieved in vitro (Figure 1.1). Ferritin is found in animals, plants, and even in bacteria; the role of the stored iron varies, and includes intracellular use for Fe-proteins or mineralization, long-term iron storage for other cells, and detoxification of excess iron. Iron regulates the synthesis of ferritin, with large amounts of ferritin associated with iron excess, small or undetectable amounts associated with iron deficiency. [Interestingly, the template (mRNA) for ferritin synthesis is itself stored in cells and is recruited by intracellular iron or a derivative for efficient translation into protein. 31 Iron does not appear to interact directly with ferritin mRNA nor with a ferritin mRNA-specific regulatory (binding) protein; however, the specific, mRNA regulatory (binding) protein has sequence homology to aconitase, and formation of an iron-sulfate cluster prevents RNA binding.] Because iron itself determines in part the amount of ferritin in an organism, the environmental concentration of iron needs to be considered before one can conclude that an organism or cell does not have ferritin.
 

Ferritin is thought to be the precursor of several forms of iron in living organisms, including hemosiderin, a form of storage iron found mainly in animals. The iron in hemosiderin is in a form very similar to that in ferritin, but the complex with protein is insoluble, and is usually located within an intracellularmembrane (lysosomes). Magnetite (Fe304) is another form of biological iron derived, apparently, from the iron in ferritin. Magnetite plays a role in the behavior of magnetic bacteria, bees, and homing pigeons (see Section II.C). The structure of ferritin is the most complete paradigm for bioinorganic chemistry because of three features: the protein coat, the iron-protein interface, and the iron core. 6,7

Protein Coat Twenty-four peptide chains (with about 175 amino acids each), folded into ellipsoids, pack to form the protein coat, * which is a hollow sphere about 100 A in diameter; the organic surface is about 10 A thick (Figure 1.9). Channels which occur in the protein coat at the trimer interfaces may be involved in the movement of iron in and out of the protein. 62,63,65 Since the protein coat is stable with or without iron, the center of the hollow sphere may be filled with solvent, with Fe203' H20, or, more commonly, with both small aggregates of iron and solvent. Very similar amino-acid sequences are found in ferritin from animals and plants. Sorting out which amino acids are needed to form the shape of the protein coat and the ligands for iron core formation requires the continued dedication of bioinorganic chemists; identification of tyrosine as an Fe(III)-ligand adds a new perspective. 64
 

Iron-Protein Interface Formation of the iron core appears to be initiated at an Fe-protein interface where Fe(II)-O-Fe(Ill) dimers and small clusters of Fe(Ill) atoms have been detected attached to the protein and bridged to each other by oxo/hydroxo bridges. Evidence for multiple nucleation sites has been obtained from electron microscopy of individual ferritin molecules (multiple core crystallites were observed) and by measuring the stoichiometry of binding of metal ions, which compete with binding of monoatomic iron, e.g., VO(IV) and Th(m) (about eight sites per molecule). EXAFS (Extended X-ray Absorption Fine Structure) and Mossbauer spectroscopies suggest coordination of Fe to the protein by carboxyl groups from glutamic (Glu) and aspartic (Asp) acids. Although groups of Glu or Asp are conserved in all animal and plant ferritins, the ones that bind iron are not known. Tyrosine is an Fe(III)-ligand conserved in rapid mineralizing ferritins identified by Uv-vis and resonance Raman spectroscopy.

Iron Core Only a small fraction of the iron atoms in ferritin bind directly to the protein. The core contains the bulk of the iron in a polynuclear aggregate with properties similar to ferrihydrite, a mineral found in nature and formed experimentally by heating neutral aqueous solutions of Fe(III)(N03h. X-ray diffraction data from ferritin cores are best fit by a model with hexagonal closepacked layers of oxygen that are interrupted by irregularly incomplete layers of octahedrally coordinated Fe(III) atoms. The octahedral coordination is confirmed by Mossbauer spectroscopy and by EXAFS, which also shows that the average Fe(In) atom is surrounded by six oxygen atoms at a distance of 1.95 A and six iron atoms at distances of 3.0 to 3.3 A.
 

Until recently, all ferritin cores were thought to be microcrystalline and to be the same. However, x-ray absorption spectroscopy, Mossbauer spectroscopy, and high-resolution electron microscopy of ferritin from different sources have revealed variations in the degree of structural and magnetic ordering and/or the level of hydration. Structural differences in the iron core have been associated with variations in the anions present, e.g., phosphate 29 or sulfate, and with the electrochemical properties of iron. Anion concentrations in tum could reflect both the solvent composition and the properties of the protein coat. To understand iron storage, we need to define in more detail the relationship of the ferritin protein coat and the environment to the redox properties of iron in the ferritin core.
 

Experimental studies of ferritin formation show that Fe(n) and dioxygen are needed, at least in the early stages of core formation. Oxidation to Fe(nI) and hydrolysis produce one electron and an average of 2.5 protons for iron atoms incorporated into the ferritin iron core. Thus, formation of a full iron core of 4,500 iron atoms would produce a total of 4,500 electrons and 11,250 protons. After core formation by such a mechanism inside the protein coat, the pH would drop to 0.4 if all the protons were retained. It is known that protons are released and electrons are transferred to dioxygen. However, the relative rates of proton release, oxo-bridge formation, and electron transfer have not been studied in detail. Moreover, recent data indicate migration of iron atoms during the early stages of core formation and the possible persistence of Fe 2+ for periods of time up to 24 hours. When large numbers of Fe(n) atoms are added, the protein coat appears to stabilize the encapsulated Fe(n).34a,b Formation of the iron core of ferritin has analogies to surface corrosion, in which electrochemical gradients are known to occur. Whether such gradients occur during ferritin formation and how different protein coats might influence proton release or alter the structure of the core are subjects only beginning to be examined.