Human H Ferritin: Caging the iron beast
Created by Daniel Lindberg
The redox chemistry of iron and oxygen presents a puzzling catch-22 to the majority of extant organisms. Each of these elements is essential to the survival of the preponderance of life forms. Dioxygen acts as the final electron acceptor in the electron transport chain of aerobic organisms, permitting the production of life-sustaining energy in the form of ATP, and also serves as a substrate for the synthesis of countless organic molecules. Similarly, iron is an indispensible component of the complexes that carry out electron transfers during the process of oxidative phosphorylation and is the core of the biologically imperative heme group. Yet paradoxically, both iron and oxygen are also potent cellular poisons. The single electron reduction of dioxygen by ferrous iron (Fe2+) produces the reactive superoxide radical O*2-, which can accept another electron and two hydrogens to create hydrogen peroxide, a potentially harmful oxidizing agent (1). Further, H2O2 can subsequently react with additional Fe2+ in the well-characterized Fenton reaction to generate the extremely reactive and damaging hydroxide radical OH* (1). Thus, each cell faces the unique challenge of limiting the concentration of free Fe2+ to merely 10^-8 M while simultaneously overcoming the negligible solubility (~10^-18 M) of ferric Fe3+ so as to harness it for cellular physiology (2). In humans and most other species, this complex cellular requirement is met by the coordinated actions of the ferritin protein and chiefly involves the ferroxidase activity executed by the ferritin H-chain subunit (PDB id = 3AJO) (3).
Human ferritin is a 24-subunit multimer with a molecular weight of approximately 475,000 Da (4). It is comprised of both 19917.6 Da L chains and 21,226 Da H chains whose relative contribution to the 24-mer varies in a stereotypical tissue-specific manner. Organs such as the liver and spleen, which are responsible for the long-term storage of copious ferric Fe3+, express ferritin multimers rich in L subunits. In fact, splenic ferritin consists of 90% L subunits and just 10% H subunits. Conversely, tissues exhibiting rapid metabolism and high ferroxidase activity construct ferritin predominantly from H-chain monomers (1). Brain regions such as the hippocampus are exceptionally active and build ferritin with up to 95% H chain composition, while the human heart uses ferritin made from 90% H subunits (5). This tissue-specific identity is vital to proper iron and oxygen homeostasis. The H chain-dominated forms of ferritin in active tissues permit the rapid ferroxidation of iron for replacement of effete electron carriers in the myriad mitochondria of their energy-consuming cells and simultaneously consume excess O2 and H2O2 produced during rapid cellular metabolism (6). Equally important, the L subunit rich forms of ferritin in iron storage organs allow for the accumulation of iron for later use in blood cell formation and immunological function (7). Thus, the tissue-specific expression of unique ferritin multimers ensures both cellular and organismal health and contributes to the proper utilization and regulation of iron and oxygen species.
To form a functional ferritin protein, a total of 24 individually-translated H and L subunits must coalesce into a single multimeric complex. Though the details of this process have been obscured by the relative instability of the monomeric constituents and the difficulty of isolating multimeric intermediates, numerous cross-linking, dissociation, and mutational studies have revealed important insights into the process of oligomerization. Interestingly, the sequence and structure of human ferritin H chain has proven instrumental in the proper formation of a functional ferritin tetracosamer (8).
Human ferritin H chain is a polypeptide of 182 amino acids and an isoelectric point of 5.31. Nearly three quarters of its residues reside within five helical regions maintained by main-chain hydrogen bonds. These alpha-helices are sequentially labeled A, B, C, D, and E according to their position relative to the N-terminal in the polypeptide primary structure and respectively correspond to residues 13 to 42, 48 to 77, 95 to 125, 126 to 159, and 163 to 174 (4).
The long helices (A-D) constitute a characteristic four-helix bundle of paired parallel and anti-parallel helices, which when viewed down the bundle axis displays a typical left-handed twist (9). Alternatively, the shorter E helix is disposed at an angle of approximately 60 degrees to the axis of the central helical bundle and is directed towards the interior of the assembled ferritin molecule (2). It makes several hydrophobic contacts with non-polar side-chains present at the start of the B helix and the end of the D, and is linked by hydrogen bonds to the N-terminal ends of the B and C helices. Though all four long helices are moderately bent, a break in intrahelical hydrogen bonding at His-136 of helix D generates a pronounced kink in the helix, thus disrupting the standard alpha-helical phi and psi angles for the relevant histidine and the following tyrosine residue, and allowing the D helix to project to the external surface of the assembled ferritin tetracosamer (4). Additionally, this D helix kink occurs at a position where three subunits come together near the 3-fold axis in the assembled ferritin multimer, thus allowing a channel to form without disrupting the packing of the helices in the remainder of the four-helix bundle (9). As described later, this 3-fold pore is instrumental in shuttling ferrous iron to the H chain ferroxidase center and thus plays a vital role in ferritin antioxidant function (10).
Though the majority of ferritin H chain consists of alpha-helical regions, short non-helical right-handed turns connect helices A and B and helices D and E, and a long non-helical loop stretches the length of the helical bundle to connect the B and C helices. Similarly, the N and C termini assume non-helical conformations (9). The short turn existing between the D and E helices is in fact comprised of three overlapping four-residue turns consisting of amino acids 159 to 162 (GAPE), 160 through 163 (APES), and 161 to 164 (PESG), respectively. Intriguingly, both the AB loop and the N-terminus also consist of overlapping turns, though in these cases only two turns of types I and IV are present. At the N-terminus, residues 6 to 9 form a type I turn, while residues 6 through 12 contribute to the type IV turn. Similarly, residues 43 to 46 of the AB turn form a type I turn, while Asp-44, Asp-45, Val-46, and Ala-47 bend to create a type IV turn (4).
The structure of the trans-molecular loop connecting alpha-helices B and C seems specially adapted to grant stability to the ferritin H chain subunit. For example, the loop twists such that Pro-88 makes van der Waals contacts with Tyr-32, Val-33, and Ser-36, ensuring that the long and flexible loop remains closely associated with the A helix. Additionally, this twist positions Trp-93 within a hydrophobic pocket and allows it to hydrogen bond to Ser-36 of helix A, thus granting additional stability to the BC loop. A puzzling intricacy of this loop connecting helices B and C is that Glu-94, positioned immediately before the first residue of the C helix, assumes disallowed Ramachandran angles with residues 93 and 95 and adopts a classical gamma turn (4). Though there is no accepted explanation for the presence of this conformation, it is possible that a tight gamma turn terminating in glutamate is necessary to allow the C helix to return to the hydrophobic core of the bundled helices in order to bury its own hydrophobic residues. Furthermore, the presence of glutamate may be necessary for yet undiscovered hydrogen bonds between dimerizing subunits of the growing ferritin cage. However, whatever the purpose of this Glu-terminated gamma turn, it is certain that the BC loop in which it resides is instrumental to the formation of the complete ferritin tetracosamer, and is thus vital to the life-sustaining functions of the assembled ferritin protein.
Though the mechanism of multimeric ferritin assembly remains relatively obscure, the isolation and examination of dimerized subunits has provided clues to the method of construction of this complex proteinaceous puzzle. The twisted conformation assumed by the long loop connecting helices B and C in ferritin H chain enables the formation of hydrogen bonds between the main-chain amino and carboxyl groups of identical Ile-85 residues in dimerized subunits (4). This association promotes the formation short anti-parallel pleated sheets between monomers, which combined with the van der Waals interaction between isoleucine side-chains at residues 80 and 85 and leucine side-chains at residue 82 contributes substantial stability to the dimer interface (4,8). Additionally, these hydrophobic residues of the BC loop interdigitate with similar hydrophobic residues located on the A helix of their symmetry-related dimer partner (4). Thus, the unique composition and conformation of the BC loop allows for the formation of stable H ferritin dimers. Furthermore, the isolation of these dimers combined with the examination of additional inter-subunit interactions within the assembled ferritin tetracosamer has led to the development of an accepted model for ferritin self-assembly.
This model chiefly involves the association of eight assembled dimers with eight newly-translated monomers to form eight transient trimers, which subsequently dimerize to generate four hexamers. These hexamers again dimerize to form two dodecamers. Finally, the dimerization of these dodecamers creates a complete and fully functional ferritin cage (8).
The newly assembled ferritin multimer is a hollow cage that can be related by two, three, and fourfold symmetry with an outer diameter of approximately 120 Å and an inner cavity around 80 Å wide (10). It is an empty prison capable of containing up to 4500 Fe3+ atoms, thus protecting the cell from the damaging effects of iron oxidation and radical production (2). During this process, the H subunit of ferritin acts as the warden of a proteinaceous prison, directing the entry, ferroxidation, and release iron from the crystalline core (1). Additionally, through the ferroxidase activity of the ferritin H chain, Fe2+ participates in the cellular service of removing excess oxygen from the cytoplasm (6). This simultaneously shields the cell from the deleterious effects of incomplete O2 reduction and transforms the insidious Fe2+ into a more manageable ferrous form that can easily be released to perform various cellular and physiological services (3).
The eight 4 Å entrances to the ferritin core are evenly spaced about the outer protein cage and are located at points of three-fold symmetry (1). These appropriately named threefold channels direct positively-charged Fe2+ to the active site of the ferroxidase center through the strategic placement of positively and negatively charged amino acid residues on the H subunit (2). Acidic amino acids line the opening to the pore, with Asp-131 and Glu-134 significantly contributing to the iron recruitment process. Conversely, patches of positive potential surround the entrance. This arrangement leads to the formation of electrostatic fields pointing into the pore, thereby directing ferrous cations to the numerous entrances of the ferritin cage (10).
Up to three Fe2+ ions may occupy the threefold channel (1). Until recently the method of translocation of these ions from the pore entrance to the ferroxidase center was unknown. It now appears that multiple residues participate in this process. After being recruited to the pore opening by His-118, His-128, and Cys-130, ferrous Fe2+ is passed to the slightly less superficial Asp-131 and Glu-134, which subsequently deliver the ions to the Glu-140 transit site via Thr-135, His-136, and Tyr-137. This highly flexible glutamate residue is located near the middle of the long D helix, and by altering its configuration, may pass ions deeper into the helical bundle to the dinuclear A site of the well-characterized ferroxidase center (10).
The ferroxidase center is the second of two active sites present on the assembled ferritin tetracosamer (the first being the hydrophilic channel located around the threefold symmetry axes) (6). Here, dinuclear A and B binding sites confer catalytic activity to the H subunit (3). The dinuclear A site is the initial acceptor of ferrous iron, and consists of a central Glu-27 hydrogen bound to both Glu-62 and His-65. These noncovalent interactions are critical to the function of the catalytic site and allow for the stable ionic interaction with Fe2+ (1). Similarly, the dinuclear B site forms ionic interactions with ferric iron and is comprised of four amino acids widely separated in the primary structure of the H chain polypeptide (4). Furthermore, it possesses several interhelical hydrogen bonds, as Tyr-34 of the A helix and Gln-141 of the D helix are noncovalently associated with Glu-107 of the C helix. Glu-62 of the B helix, which acts at both the A and B sites, does not participate in these hydrogen bonds, yet is equally important in conferring catalytic activity to the dinuclear B site (1). Thus, the tertiary structure of human ferritin H chain can easily be seen as critical to the catalytic actions performed at the dinuclear A and B sites. The rhomboid pairs of parallel and antiparallel alpha-helices form a relatively condensed bundle of helices, which, by contributing amino acids to the ferroxidase center enable the protein to catalyze its life-sustaining antioxidant and iron-storage functions (2).
The details of the chemical reaction occurring at the feroxidase center are complex and incompletely understood. It likely involves several intermediate steps, and is convoluted by the fact that once formed, the ferric mineral core can catalyze the deposition of additional iron onto its crystalline surface in a manner independent of the H chain ferroxidase site. The best current model of the ferroxidase reaction begins with the formation of a mu-1,2peroxodi-iron(III) intermediate created from the reaction of two ferric Fe2+ ions, four molecules of water, and a single molecule of O2. This intermediate remains bound to the feroxidase center and is extremely unstable, rapidly decaying (within 150 ms) into one or more mu-oxo(hydroxo)-bridged di-iron intermediates, which form clusters and ultimately lead to the formation of the mineral core itself. Over the course of this reaction, H2O2 is released in stoichiometric amounts. Were this not consumed within the ferritin cage, this could lead to significant oxidative damage and cellular pathology. However, harmful peroxide can react with two ferrous ions held at the feroxidase center and two more molecules of water to generate more ferric mineral to deposit at the crystalline core and four relative innocuous protons. (1)
Thus, the catalytic activity of ferritin H chain performs vital antioxidant functions for cells containing oxygen and iron. Additionally, by converting ferrous Fe2+ to Fe3+, the H subunit of the ferritin complex permits the later use of iron for both cellular and organismal physiology. The necessity of functional H chain ferritin is demonstrated by its conservation across species and its existence across millions of years of evolution. In fact, comparison of human H chain ferritin and a bacterial ferritin isolated from the gram-negative bacteria Campylobacter jejuni reveals sequence similarity and structural identity, as demonstrated by the E value of 1x10^-7 obtained from use of BLAST and Z score of 22.7 acquired from a search of the Dali database. Though its sequence similarity with human ferritin L chain is significantly better (E value = 3x10^-70), the tertiary similarities between the functional L and H chain are only slightly improved (Z score = 26.7) compared to the structural identity observed between human H chain ferritin and ferritin from C. jejuni. This demonstrates the conserved structure-function relationship in the ferritin family and exemplifies the vital function of this three-dimensional structure in performing the life-sustaining functions of the ferritin H chain protein.
As an example of the exceptional interspecies conservation of ferritin H chain secondary and tertiary structures, evolutionary structural biologists often cite the structure of horse L ferritin. Though a complete discussion of the structural similarities and differences between human H and equine L ferritins is rendered moot by the observation that all alpha-carbons superimpose to within 0.5 Å rms deviation, some interesting subtle differences warrant mentioning. First, as previously explained, Glu-94 of human H ferritin takes up disallowed Ramachandran angles with residues 93 to 95, adopting a classical gamma turn, whereas Gly-94 in equine L ferritin adopts generously allowed torsional angles. Additionally, while the DE turn in human H ferritin adopts three overlapping turns of types VIa1, IV, and VIII, extending from residues 159 to 162, 160 to 163, and 161 to 164, respectively, an immediate turn at Ser-161 occurs in horse L ferritin. This is because the DE turn is essentially absent, as helix D terminates with Ser161 and the E helix begins with Gln-162. Finally, and perhaps most significantly, there are differences in the feroxidase sites of human H and equine L ferritin. Though the dinuclear B site residues Glu-62, Glu-107, and Gln-141 are conserved in all species thus far examined, Glu-27 of the dinuclear A site is replaced by a tyrosine which forms hydrogen bonds with a water molecule that, in turn, binds to Gln-141. Additionally, Glu-62, which in human H ferritin participates in both dinuclear sites, is replaced by a lysine, whose positive charge forms an ion pair with the negative charge of Glu-107 (4). Thus, while human H and horse L ferritins may show 87% sequence identity, their secondary and tertiary structures demonstrate greater functional conservation than their high level of sequence similarity would suggest (9).
The incredible conservation of ferritin H chain structure through evolutionary history and the devastating or lethal consequences of its mutation or deletion exemplify its importance in the maintenance of life. In humans, mutations in ferritin H chain have been associated with iron dysregulation in the central nervous system, resulting in substantial oxidative damage and neuronal loss. In fact, there is mounting evidence that defects in neuronal or astocytic iron homeostasis contributes to several of the most prevalent neurodegenerative diseases, including both Alzheimer's and Parkinson's Disease. It is, thus, a testament to the paramount importance of the ferritin H chain protein that even slight alterations in its structure or function can produce devastating pathologies; more significant alterations are almost certainly lethal (5).
Through the detailed analysis of the structure, function, and conservation of ferritin H chain, one observation becomes almost inescapable. Ferritin H chain is compulsory for life on earth. It enables organisms to survive in a paradoxically nutritive and poisonous environment of oxygen and iron. Its extraordinary evolutionary conservation suggests an ancient origin. In fact, it is more than likely that its ancestral precursor emerged over two billion years ago when levels of atmospheric oxygen rose high enough to support aerobic life (3). Though the cellular and physiological roles of ferritin H chain have diversified, its basic function remains the same, and the fact remains that ferritin H chain makes life with iron and oxygen possible.