Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

Caillet‐Saguy, Célia; Piccioli, Mario; Turano, Paola; Lukat-Rodgers, Gudrun S.; Wolff, Nicolas; Rodgers, Kenton R.; Izadi-Pruneyre, Nadia; Delepierre, Muriel; Lecroisey, Anne

doi:10.1074/jbc.m112.366385

Cited by 33 publications

(44 citation statements)

References 45 publications

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“…The rR spectrum of H136A does not exhibit a pH dependence over the range of 5.0 to 10.0, as seen in Figure S9, suggesting that neither the 5cHS or the 6cHS H136A species binds hydroxide under alkaline conditions. The inability to form a hydroxide complex has also been reported for HasA(H32A), wherein tyrosinate is the lone amino acid ligand to the heme (82). In fact, there are very few examples of Fe(III) porphyrinates having two anionic axial RO − ligands, including OH − , because large overall positive charges are necessary to stabilize such axial ligand sets (83;84).…”

Section: Resultsmentioning

confidence: 77%

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment

et al. 2015

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The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicate that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A reduces readily in the presence of dithionite. Raman frequencies of the FeCO distortions in WT, H136A, and Y235A HmuT–CO complexes provide further evidence for the axial ligand assignments. Additionally, the se frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A–CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen-bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen-bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

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Section: Resultsmentioning

confidence: 77%

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment

et al. 2015

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“…However, spectroscopic and mutagenesis studies are consistent with PGRMC1 binding heme via tyrosine ligands rather than histidine 23, 24, 42 . This coordination motif is a feature shared with purported heme transporters such as ShuT 43 and PhuT 44 as well as the heme binding protein HasA 45, 46 .…”

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confidence: 94%

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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“…In both the H32A and Y75A mutants, coordination of a water molecule is sufficient to maintain the link between L1 and L2 loops and to hold the protein in a closed conformation. 62 The crystal structure of the HasA P (H32A)-ImH complex and NMR investigations of HasA P (H32A) and the HasA P (H32A)-ImH complex also confirm that the L1 loop remains in a closed conformation comparable to the holo WT protein. 66 The vibrational frequencies of coordinated CO constitute a sensitive probe of trans ligand field, FeCO structure, and the electrostatic landscape of the distal heme pockets of heme proteins.…”

Section: A Heme Environment/axial Coordination In Hasasmentioning

confidence: 64%

“…The roles of Tyr75, His32, and His83 in hemin-binding to HasA have been delineated by characterization of the electronic properties and the iron coordination sphere of site-directed mutant proteins. 16,62,63 Together with wild type (WT) HasA, HasA(Y75A), HasA(H32A), and HasA(H83A) have been examined in these studies via UV-visible (UV-vis), NMR, EPR, and rR spectroscopies and isothermal titration microcalorimetry (ITC). Based on ITC measurements, the two axial ligands make uneven contributions to hemin binding.…”

Section: A Heme Environment/axial Coordination In Hasasmentioning

confidence: 99%