Apoferritin oxidation

We now consider how the biomineralization chamber which is constituted by the interior of the apoferritin protein shell influences the growth of the core. Once sufficient core has been developed (>100 Fe atoms), Fe(II) oxidation and hydrolysis can proceed (Yang et ah, 1998) on the mineral surface of the growing core (equation 8) ... 

Iron oxidized by ferritin must be at or near the outer surface of the apoferritin molecule, since iron appears to be exchanged between ferritin molecules, as shown by Mossbauer spectroscopy (Bauminger et ah, 1991a,b) and by the observation that iron oxidized by ferritin can be taken up directly by apotransferrin (Bakker and Boyer, 1986 Jin and Crichton, 1987). 

It is unfortunately the case that when we incubate apoferritin with a certain number of iron atoms (for example as ferrous ammonium sulfate), the product, after elimination of non-protein-bound iron, does not have a homogeneous distribution of iron molecules which were able to (i) take up iron rapidly through the three fold channels, (ii) quickly transfer it and form a diiron centre on a ferroxidase site, and (iii) to transfer the iron inward to a nucleation site, where (iv) it will begin to catalyse iron oxidation on the surface of the growing crystallite, will accumulate iron much more rapidly, and in much greater amounts than molecules in which steps (i), (ii) and (iii) are slower, for whatever reasons (perhaps most importantly subunit composition, and the disposition of subunits of the two types H and L, one with regard to the other). This polydispersity makes the analysis of the process of iron uptake extremely difficult. 

Figure 3. Comparison of the rate of oxidation of Fe(II) when mixed with apoferritin coats (480 Fe/molecule) in 0.15 M Hepes Na, pH 7.0, using absorbance at 420 nm ( a—s— ), availability to react with -phenanthroline ( o—o—o ), change in the x-ray absorption near edge structure (XANES) ( — — ). All three types of measurements were made under the same experimental conditions, including the sample holder. (Data are taken from Ref.

Formation of ferritin involves assemblage of the protein subunits to form the apo-ferritin shell which is then filled with the phosphated ferrihydrite core. The mechanism by which ferritin is filled and the iron core built up, has been investigated intensively in vitro. The experiments usually involved incubating apoferritin (from horse spleen) with Fe salts in the presence of an oxidant such as molecular oxygen. They showed that ferritin could be reconstituted from apoferritin and a source of Fe both the iron and the oxygen enter the protein shell, whereupon oxidation of Fe is catalysed by the interior surface of the protein shell (Macara et al., 1972). 

Iron can be deposited in ferritin by allowing apoferritin to stand with an Fe(II) salt and a suitable oxidant, which may be 02. Physiological transfer of Fe(III) from transferrin to ferritin is thought to require prior reduction to Fe(II). The reoxidation by 02 to Fe(III) for deposition in the ferritin core (Eq. 16-2) is catalyzed by ferrooxidase sites located in the centers of the helical bundles of the H-chains.70-73... 

Mineralization in apoferritin involves a prior oxidation of Fe(II) as it enters channels in the protein shell. The ferroxidase center seems to be composed of Glu (Gin) and His residues situated between four helices (P. M. Harrison, personal communication). There is scope for exploring the design of agents that could block the entry of iron into the core of the protein or hasten its passage out. It is possible that non-redox-active metal ions such as Ga(III), In(III), and Al(III) can act in this way. The nature of the Fe(II) complex in the cytoplasm, which acts as a donor to ferritin, is not clear yet, but perhaps it could be Fe(II) glutathione. 

In vitro a crystalline iron core can be laid down in apoferritin by the addition of an oxidant, such as O2, to an aqueous solution of a ferrous salt and apoferritin (32, 132, 140). The reconstituted core of horse ferritin prepared in the absence of phosphate and with O2 as oxidant is very similar to the native core in terms of its size and Mossbauer properties (85). Electron microscopy, however, reveals that it is less well ordered. Reconstitution in the presence of phosphate leads to smaller cores. Reconstituted A. vinelandii cores in the absence of phosphate were more ordered than were the native cores, and clearly contained ferrihydrite particles and, in some cases, crystal domains (85). Thus the nature of the core is not determined solely by the protein coat the conditions of core formation are also important. This is also indicated by Mossbauer spectroscopy studies of P. aeruginosa cells grown under conditions different than those employed for the large-scale pu-... 

The ease with which the core of ferritin and bacfer can be reconstituted with Fe and an oxidant has led to work with Fe and other metals. Fe, added as a citrate, oxalate, or nitrilotriacetate complex, to horse holoferritin does enter the core, but only a small amount of Fe is taken up (139). No Fe + was taken up by apoferritin. This work emphasizes the requirement for core formation to occur by the oxidation of Fe +, a subject we discuss in the following section. 

The extensive series of studies on in vitro core formation reported by Harrison and co-workers (32, 63, 82, 140) and others (133) has led to the development of a three-step hypothesis for iron uptake. In the first step, iron entry through the channels, Fe + passes from the outside of the protein through the channels in the apoferritin coat to the interior cavity. The second step, nucleation, involves iron binding to groups on the inner surface of the protein in such a way that a small cluster of coupled Fe ions is formed. The final step, formation of the core, involves the extension of a small nucleating cluster by the addition and oxidation of Fe +. This stage is characterized by an initial catalytic phase, during which the small cluster rapidly expands, followed by a reduced rate of expansion once the core has attained a particular size (—1000-1500 iron atoms per molecule). 

The presence on apoferritin of ferroxidase centers is suggested by four observations (1) that tbe initial oxidation step when Fe(II) is added to apoferritin requires a specific oxidant, dioxygen (48Y, (2) that Fe(III) can be produced from Fe(II) in the presence of apoferritin—and intercepted by transferrin under conditions in wbicb hydrolysis is kept... 

Once ferrihydrite particles have formed inside the apoferritin cavity they provide alternative oxidation centers for Fe(II) on the iron core particle surface 15). Evidence for this includes the following observations (1) the stoichiometry of Fe(II) oxidation by dioxygen increases from one to approximately four Fe(II)/02 as a core formation proceeds (55) (2) Fe(II) oxidation can be effected by oxidants other than O2 once a core is present 48) (3) Fe(II) can bind directly to core surfaces, as shown hy Mbsshauer spectroscopy, and the bound Fe(II) can be oxidized (56, 57) (4) added Fe binds preferentially to any existing iron core clusters rather than to the protein shell, again as shown by analysis of Mbssbauer spectra (see Table III and Fig. 3) (57) (5) after addition of an excess of Fe(II) to a small Fe(III) core, ag = 1.87 EPR signal... 

The deposition of Fe into apoferritin using 02 as the oxidant shows that only 3-4% of the oxygen atoms in the core were derived from O2, irrespective of the amount of iron added. Use of H2 0 as solvent confirms that nearly all the oxygen atoms in the core were derived from the solvent. This work gave a stoichiometry of two Fe per 02." It is known that this stoichiometry can vary from 2 1 to 4 1 under different conditions. In the case of the lower stoichiometry, superoxide or peroxide must be produced, although they have not been detected. 

Two proposals have been made for the mechanism of oxidation of apoferritin-bound Fe. In the crystal growth model, the bulk of the Fe is oxidized to Fe(0)(0H) on the surface of the growing crystallite." " It is initiated at catalytic sites on the interior surface of the protein. There is evidence that the mechanism of iron uptake by ferritin changes after the initial uptake. Initial uptake occurs more effectively with O2 as the oxidizing agent, but in the latter stages O2 and KIO3 are equally effective. 

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