Direct Spectroscopic Ferrochelatase Assay.

Ferrochelatases (E.C. 4.

Impact of Phosphorylation at Various Sites on the Active Pocket of Human Ferrochelatase: Insights from Molecular Dynamics Simulations.

Ferrochelatase (FECH) is the terminal enzyme in human heme biosynthesis, catalyzing the insertion of ferrous iron into protoporphyrin IX (PPIX) to form protoheme IX (Heme). Phosphorylation increases the activity of FECH, and it has been confirmed that the activity of FECH phosphorylated at T116 increases. However, it remains unclear whether the T116 site and other potential phosphorylation modification sites collaboratively regulate the activity of FECH.

The alternative coproporphyrinogen III oxidase (CgoN) catalyzes the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III.

Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in , employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based identification of a gene called , encoding a putative oxygen-independent counterpart, which we propose to term CgoN, from () .

Exploiting Differences in Heme Biosynthesis between Bacterial Species to Screen for Novel Antimicrobials.

The final three steps of heme biogenesis exhibit notable differences between di- and mono-derm bacteria. The former employs the protoporphyrin-dependent (PPD) pathway, while the latter utilizes the more recently uncovered coproporphyrin-dependent (CPD) pathway. In order to devise a rapid screen for potential inhibitors that differentiate the two pathways, the genes associated with the protoporphyrin pathway in an YFP strain were replaced with those for the CPD pathway from (SA) through a sliding modular gene replacement recombineering strategy to generate the strain -CPD-YFP.

Proteomic Analysis of Ferrochelatase Interactome in Erythroid and Non-Erythroid Cells.

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored.

A primer on heme biosynthesis.

Heme (protoheme IX) is an essential cofactor for a large variety of proteins whose functions vary from one electron reactions to binding gases. While not ubiquitous, heme is found in the great majority of known life forms. Unlike most cofactors that are acquired from dietary sources, the vast majority of organisms that utilize heme possess a complete pathway to synthesize the compound.

New Avenues of Heme Synthesis Regulation.

During erythropoiesis, there is an enormous demand for the synthesis of the essential cofactor of hemoglobin, heme. Heme is synthesized de novo via an eight enzyme-catalyzed pathway within each developing erythroid cell. A large body of data exists to explain the transcriptional regulation of the heme biosynthesis enzymes, but until recently much less was known about alternate forms of regulation that would allow the massive production of heme without depleting cellular metabolites.

Ferrochelatase: Mapping the Intersection of Iron and Porphyrin Metabolism in the Mitochondria.

Porphyrin and iron are ubiquitous and essential for sustaining life in virtually all living organisms. Unlike iron, which exists in many forms, porphyrin macrocycles are mostly functional as metal complexes. The iron-containing porphyrin, heme, serves as a prosthetic group in a wide array of metabolic pathways; including respiratory cytochromes, hemoglobin, cytochrome P450s, catalases, and other hemoproteins.

Ironing out the distribution of [2Fe-2S] motifs in ferrochelatases.

Heme, a near ubiquitous cofactor, is synthesized by most organisms. The essential step of insertion of iron into the porphyrin macrocycle is mediated by the enzyme ferrochelatase. Several ferrochelatases have been characterized, and it has been experimentally shown that a fraction of them contain [2Fe-2S] clusters.

Mitochondrial contact site and cristae organizing system (MICOS) machinery supports heme biosynthesis by enabling optimal performance of ferrochelatase.

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents.

The immunometabolite itaconate inhibits heme synthesis and remodels cellular metabolism in erythroid precursors.

As part of the inflammatory response by macrophages, Irg1 is induced, resulting in millimolar quantities of itaconate being produced. This immunometabolite remodels the macrophage metabolome and acts as an antimicrobial agent when excreted. Itaconate is not synthesized within the erythron but instead may be acquired from central macrophages within the erythroid island.

Insight into the function of active site residues in the catalytic mechanism of human ferrochelatase.

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically.

Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release.

5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA.

The mitochondrial heme metabolon: Insights into the complex(ity) of heme synthesis and distribution.

Heme is an essential cofactor in metazoans that is also toxic in its free state. Heme is synthesized by most metazoans and must be delivered to all cellular compartments for incorporation into a variety of hemoproteins. The heme biosynthesis enzymes have been proposed to exist in a metabolon, a protein complex consisting of interacting enzymes in a metabolic pathway.

Loss of hepatocyte β-catenin protects mice from experimental porphyria-associated liver injury.

Background & Aims: Porphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/β-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury.

Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis.

During erythroid differentiation, the erythron must remodel its protein constituents so that the mature red cell contains hemoglobin as the chief cytoplasmic protein component. For this, ∼10 molecules of heme must be synthesized, consuming 10 molecules of succinyl-CoA. It has long been assumed that the source of succinyl-coenzyme A (CoA) for heme synthesis in all cell types is the tricarboxylic acid (TCA) cycle.

Antibacterial photosensitization through activation of coproporphyrinogen oxidase.

Gram-positive bacteria cause the majority of skin and soft tissue infections (SSTIs), resulting in the most common reason for clinic visits in the United States. Recently, it was discovered that Gram-positive pathogens use a unique heme biosynthesis pathway, which implicates this pathway as a target for development of antibacterial therapies. We report here the identification of a small-molecule activator of coproporphyrinogen oxidase (CgoX) from Gram-positive bacteria, an enzyme essential for heme biosynthesis.

Erythropoietin signaling regulates heme biosynthesis.

Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, , regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane.

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process.

The HemQ coprohaem decarboxylase generates reactive oxygen species: implications for the evolution of classical haem biosynthesis.

Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes, including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates, including non-metallated porphyrins, such as protoporphyrin IX, which is well known to generate reactive oxygen species in the presence of light and oxygen. Staphylococcus aureus has an ancient haem biosynthetic pathway that proceeds via the formation of coproporphyrin III, a less reactive porphyrin.

Rapid and sensitive quantitation of heme in hemoglobinized cells.

Rapid and accurate heme quantitation in the research lab has become more desirable as the crucial role that intracellular hemoproteins play in metabolism continues to emerge. Here, the time-honored approaches of pyridine hemochromogen and fluorescence heme assays are compared with direct absorbance-based technologies using the CLARiTY spectrophotometer. All samples tested with these methods were rich in hemoglobin-associated heme, including buffered hemoglobin standards, whole blood from mice, and murine erythroleukemia (MEL) and K562 cells.

Identification of the Mitochondrial Heme Metabolism Complex.

Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis.

HemQ: An iron-coproporphyrin oxidative decarboxylase for protoheme synthesis in Firmicutes and Actinobacteria.

Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are iron-coproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX.

Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin.

It has been generally accepted that biosynthesis of protoheme (heme) uses a common set of core metabolic intermediates that includes protoporphyrin. Herein, we show that the Actinobacteria and Firmicutes (high-GC and low-GC Gram-positive bacteria) are unable to synthesize protoporphyrin. Instead, they oxidize coproporphyrinogen to coproporphyrin, insert ferrous iron to make Fe-coproporphyrin (coproheme), and then decarboxylate coproheme to generate protoheme.