title: Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli.
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 of O(2), CO, and NO. As a result, the affinity for 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.
authors.affiliation: Department of Biochemistry, The Ohio State University, 1060 Carmack Road, Columbus, Ohio 43210-1002, USA. gilles-gonzalez.1@osu.edu
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Gonzalez
authors.forename: Elhadji M
authors.initials: EM
authors.lastname: Dioum
authors.forename: Craig M
authors.initials: CM
authors.lastname: Bertolucci
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tomita
authors.forename: Masao
authors.initials: M
authors.lastname: Ikeda-Saito
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Watmough
authors.forename: Marie-Alda
authors.initials: MA
authors.lastname: Gilles-Gonzalez
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013059:Spectrum Analysis, Raman
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli