title: Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans.
abstract: The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2--C94S, C127S, and C130S--and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif. In contrast, electronic absorption and resonance Raman spectra of the C94S variant are most consistent with a mixture of five-coordinate high-spin and six-coordinate low-spin Fe(III) heme, neither of which are ligated by a Cys(thiolate) ligand. The EPR spectrum of C94S is dominated by a large, axial high-spin Fe(III) signal, confirming that the native ligation motif is not maintained in this variant. Together, these data reveal that Cys(94) is the distal Fe(III) heme ligand in BxRcoM-2; by sequence alignment, Cys(94) is also implicated as the distal Fe(III) heme ligand in BxRcoM-1, another homologue found in the same organism.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Smith
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Marvin
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Freeman
authors.forename: Robert L
authors.initials: RL
authors.lastname: Kerby
authors.forename: Gary P
authors.initials: GP
authors.lastname: Roberts
authors.forename: Judith N
authors.initials: JN
authors.lastname: Burstyn
mesh_terms: D000595:Amino Acid Sequence; D019117:Burkholderia / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D014644:Genetic Variation; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D015394:Molecular Structure; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D016415:Sequence Alignment; D013059:Spectrum Analysis, Raman
chemical_list: D006420:Hemeproteins; D008024:Ligands; D003545:Cysteine