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

Gonzalo Gonzalez, Elhadji M. Dioum, Craig M. Bertolucci, Takeshi Tomita, Masao Ikeda-Saito, Myles R. Cheesman, Nicholas J. Watmough, Marie Alda Gilles-Gonzalez

Research output: Contribution to journalArticle

59 Scopus citations

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 (M951 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 of O2, CO, and NO. As a result, the affinity for O2 was enhanced 10-fold in M951 EcDosH, but the partition constant M = [Kd(O2)/Kd(CO)] between CO and O2 was raised to about 30 from the extraordinarily low EcDosH value of 1. Thus a major consequence of the increased O2 affinity of this sensor was the loss of its unusually strong ligand discrimination.

Original languageEnglish (US)
Pages (from-to)8414-8421
Number of pages8
JournalBiochemistry
Volume41
Issue number26
DOIs
StatePublished - Jul 2 2002

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli'. Together they form a unique fingerprint.

  • Cite this

    Gonzalez, G., Dioum, E. M., Bertolucci, C. M., Tomita, T., Ikeda-Saito, M., Cheesman, M. R., Watmough, N. J., & Gilles-Gonzalez, M. A. (2002). Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli. Biochemistry, 41(26), 8414-8421. https://doi.org/10.1021/bi025845x