Drosophila Nuclear Receptor E75 Is a Thiolate Hemoprotein
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- 作者：Eve de Rosny ; Arjan de Groot ; Celine Jullian-Binard ; Jacques Gaillard ; Franck Borel ; Eva Pebay-Peyroula ; Juan Carlos Fontecilla-Camps ; Hé ; lè ; ne M. Jouve
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：August 15, 2006
- 年：2006
- 卷：45
- 期：32
- 页码：9727 - 9734
- 全文大小：441K
- 年卷期：v.45,no.32(August 15, 2006)
- ISSN：1520-4995
文摘
Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcriptionfactors are involved in almost all physiological processes. They regulate transcription in response to bindingof rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormoneligand 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) spectroscopiesto characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of heminto the growth medium during expression of the protein in Escherichia coli were necessary to producegood yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containinga strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequentlyshown by single and double mutations. Using a similar approach, we have also established that the sixthiron ligand of a well-defined coordination conformation, which accounts for approximately half of thetotal species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 isa new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.