Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli

Gonzalez, Gonzalo, Dioum, Elhadji M., Bertolucci, Craig M., Tomita, Takeshi, Ikeda-Saito, Masao, Cheesman, Myles R., Watmough, Nicholas J. and Gilles-Gonzalez, Marie-Alda (2002) Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli. Biochemistry, 41 (26). pp. 8414-8421. ISSN 0006-2960

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Abstract

The EcDos protein belongs to a group of heme-based sensors that detect their ligands with a heme-binding PAS domain. Among these various heme-PAS proteins, EcDos is unique in having its heme iron coordinated at both axial positions to residues of the protein. To achieve its high affinities for ligands, one of the axial heme-iron residues in EcDos must be readily displaceable. Here we present evidence from mutagenesis, ligand-binding measurements, and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopies about the nature of the displaceable residue in the heme-PAS domain of EcDos, i.e., EcDosH. The magnetic circular dichroism spectra in the near-infrared region establish histidine-methionine coordination in met-EcDos. To determine whether in deoxy-EcDos coordination of the sixth axial position is also to methionine, methionine 95 was substituted with isoleucine. This substitution caused the ferrous heme iron to change from an exclusively hexacoordinate low-spin form (EcDosH) to an exclusively pentacoordinate high-spin form (M95I EcDosH). This was accompanied by a modest acceleration of the dissociation rates of ligands but a dramatic increase (60-1300-fold) in the association rate constants for binding and NO. As a result, the affinity for of O-2, CO, O-2 was enhanced 10-fold in M95I EcDosH, but the partition constant M = [K-d(O-2)/K-d(CO)] between CO and O-2 was raised to about 30 from the extraordinarily low EcDosH value of 1. Thus a major consequence of the increased O-2 affinity of this sensor was the loss of its unusually strong ligand discrimination.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024) Faculty of Science > School of Biological Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017) Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Chemistry of Light and Energy
Depositing User:	Rachel Smith
Date Deposited:	10 May 2011 10:16
Last Modified:	24 Sep 2024 10:17
URI:	https://ueaeprints.uea.ac.uk/id/eprint/30096
DOI:	10.1021/bi025845x

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