Heme serves as a prosthetic group for enzymes and proteins involved in a broad range of important cellular processes such as oxygen transport and storage, photosynthesis, redox, signaling and drug metabolism. Iron-protoporphyrin IX, or heme b, is the parent molecule whose function depends on the associated polypeptide, and its modifications yield other forms of heme. Four pyrroles linked by a methine bridge form the planar tetrapyrrole unit of heme; each pyrrole coordinates the iron atom via a n
itrogen atom. The fifth and sixth coordinating positions, above and below the plane of the ring, can be filled by the amino acids of ligand proteins or by small molecules. Eight positions carry side chain modifications, primarily methyl (C2, 7, 12 and 18), but also vinyl (C3 and 8) and propionyl (C13 and 17). Substitutions to these carbons with other side chains give rise to several other, and important, heme types. Heme b, and heme c and a, are found in many organisms and are involved in processes such as electron transfer, oxygen transport and photosynthesis; heme d, m and o are species-specific and involved in specialized functions. The biosynthesis of heme is a conserved pathway that consists of single pyrrole synthesis, assembly of pyrroles into the tetrapyrrole ring, side chain modification and iron insertion into the ring. Details of the individual steps are provided below.
The formation of 5-aminolevulinic acid (ALA), the precursor providing the only source of heme's carbon and nitrogen atoms, is the first and rate-limiting step of heme biosynthesis. In what is known as the Shemin pathway, ALA synthase (Alas), residing on the matrix side of the inner mitochondrial membrane (IMM), carries out the condensation of glycine and the succinyl-CoA intermediate of the citric acid cycle. A second pathway, known as C5 and present in plants, archea and some bacteria, produces ALA in a two-step reaction. There are two Alas isoforms in mammals: Alas1 which is ubiquitously expressed, and Alas2 which is erythrocyte specific. Both enzymes are homodimers that require pyridoxal 5'-phosphate (PLP) for the reaction. As glycine and succinyl-CoA are condensed, CO2 and coenzyme A are released as byproducts. ALA exits the mitochondria and serves as a substrate for the next four enzyme-driven conversions. Details about its export are not well established; the Slc25a38 member of the Slc25 family of IMM transporters is thought to be involved. In this scenario, the transporter would facilitate import of glycine in exchange for ALA export. Recent studies indicate that the yeast and human proteins are the mitochondrial glycine transporters and are necessary for heme biosynthesis.
In the cytosol, ALA is used as the building block for the synthesis of uroporphyrinogen III in three consecutive steps as follows. In the first step, two ALAs are combined by delta-aminolevulinate dehydratase Alad (also known as porphobilinogen synthase, PBGS) to generate porphobilinogen (PBG). Alad is a homooctamer and each dimer harbors one catalytic site. Each site binds one ALA at two distinct sites and one zinc atom. Of the total of eight Zn atoms, four confer structural stability and the other four participate in catalytic activity. Four PBG molecules are subject to polymerization by hydroxymethylbilane synthase Hmbs (or porphobilinogen deaminase, known as PBGD). The enzyme uses a covalently attached dipyrromethane cofactor to prime the polymerization; the cofactor is made of two linked PBG molecules. The six PBG molecules form a linear hexapyrrole which is then cleaved to yield 1-hydroxymethylbilane. The product, an unstable tetrapyrrole, is the substrate for uroporphyrinogen synthase, Uros. Uros functions as a monomer which carries out ring inversion and closure of the tetrapyrrole to form uroporphyrinogen III. Spontaneous closure to urophorphyrinogen I can take place, but the molecule cannot be converted to heme. Uroporphyrinogen decarboxylase, Urod, subsequently decarboxylates all four acetate groups of uroporphyrinogen III to form the methyl groups of coproporphyrinogen III.
The final steps of heme biosynthesis are carried out by enzymes associated with the IMM. Coproporphyrinogen III transport into the mitochondrion is believed to be facilitated by translocator protein (TSPO), also known as the peripheral-type benzodiazepine receptor (Pbr), located on the outer mitochondrial membrane (OMM). The arrangement of enzymes is indicative of a possible multiprotein complex mediating substrate channeling. Coproporphyrinogen oxidase Cpox, located in the mitochondrial intermembrane space, promotes the oxidative decarboxylation of propionyl side chains on two of the coproporphyrinogen III pyrrole rings to form protoporphyrinogen IX. Protoporphyrinogen IX, in turn, is oxidized to protoporphyrin IX by protoporphyrinogen IX oxidase, Ppox. Ppox is a homodimer located in the IMM and uses FAD as a cofactor. Finally, ferrochelatase (Fech) inserts ferrous iron into protoporphyrin IX to form heme b. The enzyme is a dimer with several regions in each monomer and a [2Fe-2S] catalytic cluster. A detailed mechanistic understanding of metallation is still lacking. Structural and molecular dynamic simulation studies point to structural changes in the enzyme and possible distortions of the protoporphyrin, movement of Fe2+ from the exterior of the enzyme to the site of metallation, key residues possibly involved in proton abstraction from the substrate during catalysis, re-arrangements of hydrogen bonds, and movement of heme out of the active site and heme release. To see a crystal structure of the human enzyme, click here
The biosynthesis of heme is regulated at many levels. Alad, Hmbs and Uros are transcriptionally regulated. Alas2, which has an 'iron response element' in the 5'-UTR of its mRNA, is post-transcriptionally regulated. Mutations in several genes of heme biosynthesis are associated with a number of diseases, most notably porphyrias, of which there are nine major types. To see the ontology report for annotations, GViewer and download, click here...(less)