Production and characterization of erythropoietic protoporphyric heterodimeric ferrochelatases

Najahi‐Missaoui, Wided; Dailey, Harry A.

doi:10.1182/blood-2004-12-4661

Cited by 24 publications

(20 citation statements)

References 41 publications

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“…Incidentally, none of the monomeric mutants we identified showed detectable enzymatic activity (Table 1), which is in agreement with earlier studies demonstrating that the dimerization of FECH is essential for its function (Wu et al, 2001;Najahi-Missaoui and Dailey, 2005;Medlock et al, 2007b). To investigate whether the amino acid residues Trp301 and Leu311 are directly involved in salicylic acid binding, we analyzed these mutants in the monomeric background.…”

Section: Resultssupporting

confidence: 68%

Salicylic Acid Induces Mitochondrial Injury by Inhibiting Ferrochelatase Heme Biosynthesis Activity

et al. 2013

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Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECHsalicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

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Section: Resultssupporting

confidence: 68%

Salicylic Acid Induces Mitochondrial Injury by Inhibiting Ferrochelatase Heme Biosynthesis Activity

et al. 2013

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“…The assay utilized is a direct assay and measures the disappearance of porphyrin substrate to determine heme production 77 . PGRMC1 alone did not bind porphyrin and showed no activity (Table 2).…”

Section: Resultsmentioning

confidence: 99%

A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase

Piel¹,

et al. 2016

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Heme is an iron containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was carried out to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both Progesterone Receptor Membrane Component 1 (PGRMC1) and Progesterone Receptor Membrane Component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in hemoglobinization relative to untreated cells. In vitro heme transfer experiments showed that purified PGRMC1 was able to donate heme to apo-cytochrome b5. In the presence of PGRMC1 in vitro measured FECH activity decreased in a dose dependent manner. Interactions between FECH and PGRMC1 were strongest for the conformation of FECH associated with product release suggesting that PGRMC1 may regulate FECH activity by controlling heme release. Overall, the data illustrate a role for PGRMC1 in regulating heme synthesis via interactions with FECH and suggest that PGRMC1 may be a heme chaperone or sensor.

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“…Enzyme activity of each variant was assessed first by rescue of a ferrochelatase deficient strain of Escherichia coli (Δ hemH ) (26, 27). Following rescue of Δ hemH , variant ferrochelatases were assayed using the continuous direct spectroscopic method (28). …”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Solvent-Filled Channels in Human Ferrochelatase

et al. 2012

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Ferrochelatase catalyzes the formation of protoheme from two potentially cytotoxic products, iron and protoporphyrin IX. While much is known from structural and kinetic studies on human ferrochelatase of the dynamic nature of the enzyme during catalysis and the binding of protoporphyrin IX and heme, little is known about how metal is delivered to the active site and how chelation occurs. Analysis of all ferrochelatase structures available to date reveals the existence of several solvent filled channels which originate at the protein surface and continue to the active site. These channels have been proposed to provide a route for substrate entry, water entry and proton exit during the catalytic cycle. In order to begin to understand the functions of these channels, a number of variants which line these solvent filled channels were investigated in vitro and in vivo. Data presented herein support the role of one of these channels, which originates at the surface residue H240, in iron delivery to the active site. Structural studies of the arginyl variant of the conserved residue F337, which resides at the back of the active site pocket, suggest that it not only regulates the opening and closing of active site channels, but also plays a role in regulating the enzyme mechanism. These data provide insight into the movement of substrate and water into and out of the active site and how this movement is coordinated with the reaction mechanism.

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Production and characterization of erythropoietic protoporphyric heterodimeric ferrochelatases

Cited by 24 publications

References 41 publications

Salicylic Acid Induces Mitochondrial Injury by Inhibiting Ferrochelatase Heme Biosynthesis Activity

Salicylic Acid Induces Mitochondrial Injury by Inhibiting Ferrochelatase Heme Biosynthesis Activity

A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase

Identification and Characterization of Solvent-Filled Channels in Human Ferrochelatase

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