Title
Met104 is the CO-replaceable ligand at Fe(II) heme in the CO-sensing transcription factor BxRcoM-1
Author
Hannah E. Bowman, Matthew R. Dent, Judith N. Burstyn
Year
2016
Journal
Journal of Biological Inorganic Chemistry
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.
Instrument
J-715
Keywords
Magnetic circular dichroism, Coordination chemistry, Ligand binding, Biochemistry