ABSTRACT
A tyrosyl radical, as part of the amino acid chain of bovine liver catalase, supports dynamic proton spin polarization (DNP). Finding the position of the tyrosyl radical within the macromolecule relies on the accumulation of proton polarization close to it, which is readily observed by polarized neutron scattering. The nuclear scattering amplitude due to the polarization of protons less than 10â Å distant from the tyrosyl radical is ten times larger than the amplitude of magnetic neutron scattering from an unpaired polarized electron of the same radical. The direction of DNP was inverted every 5â s, and the initial evolution of the intensity of polarized neutron scattering after each inversion was used to identify those tyrosines which have assumed a radical state. Three radical sites, all of them close to the molecular centre and the haem, appear to be equally possible. Among these is tyr-369, the radical state of which had previously been proven by electron paramagnetic resonance.
ABSTRACT
Heme has been recently described as a regulating ligand for the activity of the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta and their Drosophila homologue E75. Here, we report the cloning, expression in Escherichia coli, purification, and screening for the heme-binding ability of 11 NR ligand-binding domains of Drosophila melanogaster (DHR3, DHR4, DHR39, DHR51, DHR78, DHR83, HNF4, TLL, ERR, FTZ-F1, and E78), of unknown structure. One of these NRs, DHR51, homologous to the human photoreceptor cell-specific nuclear receptor (PNR), specifically binds heme and exhibits a UV-visible spectrum identical to that of heme-bound E75-LBD. EPR and UV-visible absorption spectroscopy indicates that, like in E75, the heme contains a hexa-coordinated low spin ferric iron. One of its axial ligands is a tightly bound cysteine, while the other one is a histidine. A dissociation constant of 0.5 microM for the heme was measured by isothermal titration calorimetry. We show that DHR51 binds NO and CO and discuss the possibility that DHR51 may be either a gas or a heme sensor.
Subject(s)
Carrier Proteins/chemistry , Drosophila Proteins/chemistry , Hemeproteins/chemistry , PDZ Domains , Photoreceptor Cells, Vertebrate/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Structural Homology, Protein , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster , Heme-Binding Proteins , Hemeproteins/genetics , Hemeproteins/metabolism , Humans , Ligands , Molecular Sequence Data , PDZ Domains/genetics , Photoreceptor Cells, Vertebrate/metabolism , Protein Binding/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Sequence Homology, Amino Acid , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolismABSTRACT
Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. Here we have used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli were necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, we have also established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.