Human Ferrochelatase
Created by Rachel Brinkley
Human ferrochelatase is a homodimeric mitochondrial membrane-associated enzyme. Enzymes are proteins that catalyze chemical reactions. This specific enzyme catalyzes the terminal step in heme biosynthesis by inserting ferrous iron into protoporphyrin IX to form protoheme, heme. Due to the many critical roles of heme, the vast majority of organisms require the synthesis of it.
Since ferrocheletase is involved in the heme biosynthetic pathway, decreased levels of this enzyme's activity have been associated with the inherited disorder of erythropoietic protoporphyria (EPP). A common symptom of this disorder is photosensitivity. These decreased levels of ferrochelatase lead to high levels of protoporphyrin, which is a carrier molecule (4). Protoporphyrin together with iron make the body of the heme group found in hemoglobin and myoglobin. Without ferrochelatase the iron is not inserted into the protoporphyrin, leading to the high levels and heme not being formed. Therefore, the activity of ferrochelatase is essential to the body in order to make these oxygen-involved proteins. 5%-20% of patients with EPP develop liver disease because of the accumulation of protoporphyrin (4). The ferrochelatase gene (FECH) is located on chromosome 18q21.3 spanning 45 kb and encompassing 11 exons. More than 50 different mutations have been identified in the FECH gene resulting in EPP, including missense, nonsense and splicing mutations and both large and small insertions and deletions (3).
Ferrochelatase has a molecular weight of 86 kDa and an isoelectric point of 5.3. It is a specific kind of enzyme known as a chelatase. Chelatases catalyze reactions through the insertion of a metal into various tetrapyrroles (5). Ferrochelatase is located in the mitochondrial membrane, where it inserts the ferrous form of iron into protoporphyrin IX to form heme.
It's secondary structure includes alpha helices, beta sheets, random coils, and turns. It is made up of 47.7% alpha helices and 13.5% beta strands. This polypeptide is composed of two identical subunits. These two dimers form a small water channel in the interface. This water channel is partially due to the large amount of hydrogen bonding and hydrophobic interactions. Within each subunit there are two similar domains each with a four-stranded parallel beta sheets flanked by alpha helices in a beta-alpha-beta motif. Each subunit is made up of 359 residues, residues 65-423 (1). The first 64 residues of the sequence represent the mitochondrial targeting sequence that is removed (1). Also each subunit contains one [2Fe-2S] cluster. Along with this cluster, there are three cholate detergent molecules found in the active site pocket.
The active site in each subunit is created by the coming together of a helix-turn-helix from both of the domains in that subunit. Among the two active sites, one in each subunit, the residues are identical and highly conserved. In the active site, binding pocket, three cholate detergents are observed. Theses ligands extend from the core to the molecular surface. The position of the cholate molecules describes a possible protoporphyrin entry pathway and heme exit pathway for the active site (1).
Also in the active site is the [2Fe-2S] cluster, which in mammals is easily altered. If destructed or eliminated, there is a loss of enzyme activity. Studies have shown that this [2Fe-2S] cluster is nitric-oxide sensitive. The NO seems to rapidly and effectively cause the dissasembly of the cluster by reacting directly with it (6). This NO sensitivity is thought to be the physiological regulation of this enzyme (6).
The active site is composed of two hydrophobic lips and may be involved in membrane association (1). The lower lip consists of highly conserved residues that are arranged as hydrophobic ridges. This organization has themembrane associating face of the enzyme and also the access to the active site on the same surface. This enables the poorly soluble protoporphyrin substrate to enter and the heme product to leave through the membrane (1).The inside of the active site is relatively hydrophilic. In the active site there are many conserved residues that form an acidic pathway that goes from the protein's surface to the centrally located H263 residue and is possibly involved in proton extraction (1). Two other residues that are in contact with the substrate, both inside and outside of the active site, are R-114 and R-115 and they are located on the upper lip (8). There are also some residues that have van der Waals interactions with the substrate and may be involved in the closure of the active site, excluding the surrounding water (8).
In heme sythesis, ferrochelatase inserts iron into protoporphyrin IX. In other words, the metallation of the pyrrole nitrogen of protoporphyrin occurs. In order to do so, protoporphyrin is bound in the active site of ferrochelatase and the enzyme engulfs the substrate in a snug pocket so the metallation can occur (2). Ferrochelatase binds protoporphyrin with micromolar affinity by using the conserved histine residue, H-263, which is located centrally in the active site, for ligation with the heme iron (8). The binding of protoporphyrin to ferrochelatase involves a backbone structural movement that causes the active site mouth to close around the protoporphyrin and results in the reorientation that reshapes the pocket (8). The key player in all of this structureal rearrangement seems to be the H-263 residue. Once the protoporphyrin and iron are bound, the substrate is released as heme. Although it is still not clear what would cause the destabilization of the heme molecule for release (8).
Another form of ferrochelatase that is structurally similiar to the human form, but not identical, is the yeast, Saccharomyces cerevisiae, form ( PDB 1L8X). This ferrochelatase has the same main function in the heme biosynthetic pathway. But instead of binding with an iron complex as the human form does, the yeast form binds with a cobalt complex. A second comparison is made between the human form of ferrochelatase with and without protoporphyrin bound. The structure of human ferrochelatase with bound protoporphyrin (PDB ID: 3HCN) has significant differences in the position of several loop regions (residues 90-115, 302-313, and 349-361) when compared with the structure of human ferrochelatase without bound substrate (2). A common mutant that many experiments are performed on is the E343K variant of human ferrochelatase. It differs from the normal strian because it is made up of two copies of the biological dimer, for a total of four monomers (2). It also has four active sites, opposed to the normal strain, which has one dimer and two active sites.
The ferrochelatase enzyme is crucial for the synthesis of heme and in consequence is essential for a majority of organisms. Ferrochelatase is an evolutionarily old protein and has been crystallized from many different kinds of organisms (8). The absense or defect of ferrochelatase has biological consequences and proves how important this enzyme is.