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The Heme‐Binding PAS Domain Mediates Dimerization in the CO‐Sensing Transcription Factor Bx RcoM‐1
Author(s) -
Pinhancos Rebeca C.,
Bowman Hannah E.,
Dent Matthew R.,
Young Byron H.,
Berndsen Christopher E.,
Burstyn Judith N
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.908.15
Subject(s) - heme , chemistry , allosteric regulation , effector , transcription factor , hemeprotein , transcription (linguistics) , ligand (biochemistry) , circular dichroism , binding site , dna , stereochemistry , biochemistry , biophysics , biology , enzyme , gene , receptor , linguistics , philosophy
The gases NO, O 2 , CO and H 2 S are small‐molecule effectors involved in signaling pathways. Heme serves as an effector‐binding site in many proteins that sense these molecules, including gas‐sensing transcription factors, and the effector‐binding signal is transduced through varying allosteric mechanisms. Bacterial transcription factors are excellent systems in which to study gas‐mediated allostery due to their simplicity relative to the multi‐protein complexes involved in eukaryotic transcription. RcoM‐1 is a heme‐containing, CO‐responsive transcription factor that appears to regulate aerobic CO oxidation in the non‐pathogenic bacterium Burkholderia xenovorans . The RcoM‐1 heme plays a key role in CO‐sensing. Under reducing conditions, the Fe(III) heme undergoes a redox‐mediated ligand switch in which a neutral donor replaces an axial cysteine(thiolate) ligand, Cys 94 . The neutral donor ligand Met 104 was identified through a spectroscopic study of three Met‐to‐Leu variants. RcoM‐1 variants in which Met 104 is replaced with a non‐coordinating Leu residue exhibited electronic absorption and magnetic circular dichroism spectra that, under reducing conditions, are indicative of a five‐coordinate, high‐spin ( S = 2) Fe(II) heme center. CO replaces Met 104 , binds to the Fe(II) heme, and presumably modulates DNA‐binding activity. The structure of RcoM‐1 is still unknown; however, sequence homology suggests that RcoM‐1 is a unique, single‐component signaling protein that contains both a PAS‐like heme‐binding domain and a LytTR‐like DNA‐binding domain. We probed the oligomeric status and topology of this novel single‐component transcription factor. Size exclusion chromatography (SEC), SAXS, and analytical ultracentrifugation data (M W /M S = 2.25 ± 0.01) are consistent with a dimeric structure for RcoM‐1. Further SEC data show that an RcoM‐1 truncate containing only the heme domain is dimeric and that variants lacking the His 74 ligand, which bear no heme, are also dimeric. Taken together, these data suggest that RcoM‐1 dimerizes via its heme domain, but dimerization does not require heme. A stable, dimeric RcoM‐1 homology model that dimerizes via the heme domain was built that fits well within the molecular envelope predicted by SAXS. Support or Funding Information The National Science Foundation supported these studies under CHE‐1213739 (to J.N.B.)