Met(104) is the CO-replaceable ligand at Fe(II) heme in the CO-sensing transcription factor BxRcoM-1.
Both Met(104) and Met(105) are involved, either directly or indirectly, in the redox mediated ligand switch of the heme-dependent transcription factor, RcoM-1. Recent studies of Burkholderia xenovorans RcoM identified Cys(94) as the thiolate ligand in the Fe(III) state of the heme cofactor. Upon reduction, a neutral donor replaces Cys(94) trans to His(74). Homology modelling implicated either Met(104) or Met(105) as the possible ligand in the Fe(II) state. We spectroscopically compared wild type (WT) RcoM-1 to three Met-to-Leu variants (M104L, M105L, and M104L/M105L) to identify which Met residue acts as the ligand. All proteins were isolated as admixtures of Fe(III) and Fe(II)-CO heme; oxidation by ferricyanide enables study of homogeneous oxidation and coordination states. Met(104) is the CO-replaceable Fe(II) heme ligand. The magnetic circular dichroism (MCD) spectrum of Fe(II) M105L resembled WT. M104L and M104L/M105L, however, showed spectra arising from the formation of a high-spin, five-coordinate species indicating the loss of the ligand. The electron paramagnetic resonance (EPR) spectra of WT Fe(III) RcoM-1, oxidized Fe(III) M104L, and as-isolated M105L exhibited narrow, rhombic low-spin signals typical of thiolate-bound hemes. In contrast, oxidized Fe(III) M105L and oxidized Fe(III) M104L/M105L revealed a broad, rhombic low-spin, six-coordinate signal indicative of replacement of the thiolate by a neutral ligand. Thus, we conclude that Met(105) is important to the stability of the Fe(III) heme pocket during oxidation.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706, USA.
Hannah E
HE
Bowman
Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706, USA.
Matthew R
MR
Dent
Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706, USA. burstyn@chem.wisc.edu.
Judith N
JN
Burstyn
D019117:Burkholderia / Q000737:chemistry / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D005296:Ferrous Compounds; D008024:Ligands; D014157:Transcription Factors; D006418:Heme; D002248:Carbon Monoxide; D008715:Methionine
Carbon monoxide; Electron paramagnetic resonance; Electronic absorption spectroscopy; Heme; Magnetic circular dichroism spectroscopy