Met104 is the CO-replaceable ligand at Fe(II) heme in the CO-sensing transcription factor BxRcoM-1

  • Hannah E. Bowman
  • Matthew R. Dent
  • Judith N. Burstyn
Original Paper

Abstract

Both Met104 and Met105 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 Cys94 as the thiolate ligand in the Fe(III) state of the heme cofactor. Upon reduction, a neutral donor replaces Cys94 trans to His74. Homology modelling implicated either Met104 or Met105 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. Met104 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 Met105 is important to the stability of the Fe(III) heme pocket during oxidation.

Keywords

Carbon monoxide Electronic absorption spectroscopy Electron paramagnetic resonance Heme Magnetic circular dichroism spectroscopy 

Abbreviations

CO

Carbon monoxide

CooA

CO oxidation activator

DTT

Dithiothreitol

EcDOS

Escherichia coli direct oxygen sensor

EPR

Electron paramagnetic resonance

MCD

Magnetic circular dichroism

MOPS

3-(N-Morpholino)propanesulfonic acid

NO

Nitric oxide

PAS

Per-Arnt-Sim domain

RcoM

Regulator of CO metabolism

VTVH

Variable temperature variable field

WT

Wild type

Supplementary material

775_2016_1368_MOESM1_ESM.pdf (220 kb)
Supplementary material 1 (PDF 219 kb)

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Copyright information

© SBIC 2016

Authors and Affiliations

  • Hannah E. Bowman
    • 1
  • Matthew R. Dent
    • 1
  • Judith N. Burstyn
    • 1
  1. 1.Department of ChemistryUniversity of Wisconsin-MadisonMadisonUSA

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