Ferritin
Created by Colin Smith
Ferritin is an essential peptide for humans and many other organisms due to its pivotal role in iron metabolism. Perhaps the most important role of iron is in transporting oxygen through the body when complexed in a heme group. Iron is the site at which oxygen binds, thus without it, no oxygen would be delivered to cells. However, free iron can react with peroxides, forming free radicals which can damage DNA, proteins, and other cellular components (1). Iron is also useful because it is can act as an electron donor and acceptor. In this manner, iron is used during redox reactions and in electron transport; two very important processes in oxidative phosphorylation, the primary reaction providing humans, and other aerobic respirators, with energy (1). Iron also has many smaller roles in the body such as: maintaining the immune system, neurotransmitter function in the brain, and connective tissue production (2). Ferritin is the molecule that binds to and stores iron, releasing it in a controlled manner. Effectively, ferritin keeps an essential mineral in a non-toxic state until it can be used, and helps protect against complications of the low iron disease, iron-deficiency anemia, and ironoverload, hemochromatosis.
In a healthy individual, there are 800-4500 iron atoms per ferritin; depending on function and age of the tissue it is present in (3). This globular protein is composed of 24 subunits of two different types. There is a 19 kDa light chain (L-chain) and a 21 kDa heavy chain (H-chain) that compose the subunits of the roughly 474 kDa complete polypeptide. These H and L chains are homopolymers (3). The composition of ferritin can vary depending on the purpose of the specific ferritin protein. A peptide composed mostly of L-chains, used mainly in core formation, has more of a storage role (4). Whereas, when ferritin has a greater H-chain component, the protein is more active in iron metabolism – converting (oxidizing) unstoreable Fe2+ ions to sequesterable Fe3+ions (4). L rich storage ferritins are common in the spleen and liver, while H rich oxidation ferritins are often found in the heart and brain (2). This makes physiological sense because the liver and spleen are large storage areas,but the heart and brain require lots of oxygen and thus lots of physiologically active and useful iron for oxygen transportation. H and L subunits share 55% sequence similarity, with the biggest differences manifest on the outer surface, inner cavity, and hydrophobic channel residues (hydrophilic channels are identical in sequence) (5). The isoelectric point is around 5.4-5.5 for human liver ferritin, the most common place to find the peptide.
Because reduced iron is a toxic molecule to many organisms, yet oxidized iron is necessary, many different species have their own type of ferritin. There is a highly conserved 3-D structure to ferritin. Most oraganisms have ferritin composed of 24 subunits in 4-3-2 symmetry, providing a hollow shell for iron storage (1). Subunits are composed of anti-parallel helical bundles. Four helicies are bundled with a fifth, shorter helix attached at an angle 60° to the larger bundle. Helical pairs are connected by long loop running the length of the helicies, not by short turns at the base of helix pairs. These anti-parallel helix pairs assemble into 12 faces of a ferritin dodecamer (3). Represented by PDB ID 3GVY, the protein bacterioferritin of R. sphaeroides, has a similar structure to human ferritin, 1FHA, because it has a very similar function. It is a globular iron sequestering moleculecomposed of 24 subunits, so it serves a very similar role to human ferritin (6). Represented by PDB ID 2VXX, the DpsA protein of T. elongatus, has an amino acid sequence with similar properties in the same position as human ferritin. This allows for analogous secondary structures which work like those in ferritin. DpsA protein allows for iron ion oxidation, which in turn facilitates the absorption of the essential mineral. Human mitochondrial ferritin (PDB ID: 1R03) has over 50% secondary sequence similarity to regular humanferritin (PDB ID: 1FHA) and over 80% sequence similarity to the human H chain in ferritin. It has two sites for metal ion oxidation, one of which is always filled while the other has variable activity. The protein has similar biochemical properties to H chain ferritin, except it has reduced ferroxidase activity (41% of H chain).
L-chains function as the incorporating subunits in ferritin due to their hydrophobicity. Amino acids with carboxylic groups, specifically Glu-57 and Glu-60, in the bundled helical cavities are the pores used to incorporate ferrous iron (Fe2+) into the ferritin shell (7). Residues 53-59 of the L subunit face inside the protein shell. Glu-53 and Glu-56 are conserved between L and H subunits, but the L-chain also has Glu-57 which is free to rotate. When Glu-57 rotates to unite with Glu-53 and Glu-56, an iron binding site is formed which brings Fe2+into the protein cage (7). There are two iron binding sites for the ferritin molecule. While composition of these sites differs, the stabilizing and bonding is quite similar. In the first site, iron is bound to a His, two Glu, and a water molecule, while in the second site iron is bound to three Glu residues(8). At both sites there are hydrogen bonds that act to stabilize the bound iron, helping to incorporate it into the protein shell. Ferrous iron must be oxidized to ferric iron in order to be stored in the ferritin molecule. This method of oxidation is not completely understood, but it is thought to be either one, 2 electron transfer, or a series of two, single electrontransfers (9). The region of protein responsible for Fe2+ sequestering is not yet known, and the environment around Fe2+ is similarly undefined. A plausible explanation is that O(II) stabilizes in the protein coat by trapping iron in ferrous-ferric oxyhydroxide state. Another possibility is that oxygen encapsulates iron within a layer of hydrous ferric oxide (11). The position of residue side chains has an effect on the rate of iron oxidation. Side chain position is very flexible and can be influenced by buffer or salt concentration, and mutations, thus iron storage rates can be altered greatly.
In addition to iron storage, ferritin transforms ferric iron, Fe3+, into useable ferrous iron, Fe2+. The residues that compile this binding site and polar, and thus charged, so they interact favoably with the iron ion. The structure of ferritin is such that it is a shell with channels allowing movement across the cage, and channels providing reductive powers. The eight channels through the protein whichare lined with hydrophilic amino acids are at 3-fold axes – the meeting point of three subunits. Six channels are lined with hydrophobic residues and are at 4-fold axes – the meeting point of foursubunits (10). Non-charged 4-fold channels are thought to be the site of electron transfers, effectively reducing ferric iron into ferrous iron (5). However, the method by which this reaction occurs is not precisely known. One possible way of reducingferric to ferrous iron is through pores. Pores in the ferritin shell are thought to regulate contact betweenhydrated ferric iron inside the cage and reductants outside the cage, thereby reducing the Fe3+ to transportable Fe2+. The specific residues that act as gates for the pores are Arg-72, Asp-122, Leu-110, and Leu-134 (12).
In the process of conversion from Fe3+ to Fe2+, iron adopts a crystalline structure similar to ferrihydrite, [FeO(OH)]8[FeO(H2PO4)]. The dimensions of each ferrihydrite crystal lattice subunit are approximately 25x25x50 Angstroms (2). These subunits are tightly packed except at the 3-fold channels, so as to allow for iron complex movement out of the protein shell. 90% of Fe3+ is bound to six O(II) ions, while 10% of ferric iron is bound to five O(II) ions and 1 phosphate group. The phosphate group attaches iron to residues on the inside of the ferritin shell, thus stabilizing the stored mineral-protein structure. Normally there is a ratio of about 8 iron:1 phosphate in ferritin, but variations can occur depending on environment. Most of the phosphate in ferritin is on the surface since that location is most conducive for chemical dissolution (1). Because iron is in a crystal lattice in its stored state, it is insoluble. The lattice must be dissolved in order to release useable ferrous iron from ferritin. While ferrihydrite is a good model for visualizing the general properties of the ferritin core, it is a very broad term that encompasses a variety of materials. A major setback of using ferrihydrite as a model for the ferritin core is that there is no phosphate in ferrihydrite (3). Although useful as a basic model for the iron core, these drawbacks serve as warnings that ferrihydrite is not a suitable substitute molecule.
Six water molecules surround Fe2+, creating a soluble cage around iron so thatit can be released into the body. Then iron must exit the protein through the polar 3-fold channels. The polarity of the hydrophilic moieties, mostly Asp and Glu, allows for favorable interaction between ferrous iron and H2O due to electrostatic forces and attraction of opposite charges. On the contrary, since 4-fold channels are lined with hydrophobic amino acids like Leu, there are no forces of attraction acting on ferrous iron, thus no iron movement through 4-fold channels (2). The iron water complex is too large to pass through the 3-fold channels, thus it must lose a few H2O molecules in order for iron to exit storage. Some of the interactions between ferrous iron and water are cleaved, but they are replaced by other polar interactions between iron and residues inside the3-fold channel, thus facilitating iron exit movement (1). Once outside the protein shell, ferrous iron is rehydrated to Fe(H2O)62+, a state that can be picked up, transferred, and used in other areas of the body.
The bacterium Strptomyces pilosus can produce Deferoxamine (DFO), a powerful ferric iron chelator. This product is used to remove excess iron from patients in order to prevent hemochromatosis and other iron overload disorders. DFO induces ferritin degradation via two methods. In thefirst method, DFO provokes ferritin to enter a lysosome where it is degraded and recycled. The second method involves a depletion of iron from the ferritin prior to the protein being sent to a proteasome for degradation and recycling (13).
For your consideration, Image 1 is a Ramachandran plot showing possible dihedral angles of the complete 24mer of ferritin (Image 1). Images 2-4 are plots of closed cavity area vs. volume for L-chain, H-chain, and a complete 24mer of ferritin, respectively (Image 2, Image 3, Image 4).