title: Activation mechanism of the CO sensor CooA. Mutational and resonance Raman spectroscopic studies.
abstract: CooA is a CO-dependent heme protein transcription factor of the bacterium Rhodospirillum rubrum. CO binding to its heme causes CooA to bind DNA and activate expression of genes for CO metabolism. To understand the nature of CO activation, several CooA mutational variants have been studied by resonance Raman spectroscopy, in vivo activity measurements, and DNA binding assays. Analysis of the Fe-C and C-O stretching Raman spectroscopy bands permits the conclusion that when CO displaces the Pro2 heme ligand, the protein forms a hydrophobic pocket in which the C-helix residues Gly117, Leu116, and Ile113 are close to the bound CO. The displaced Pro2 terminus is expelled from this pocket, unless the pH is raised above the pKa, in which case the terminus remains in H-bond contact. The pKa for this transition is 8.6, two units below that of aqueous proline, reflecting the hydrophobic nature of the pocket. The proximal Fe-His bond in Fe[II]CooA is as strong as it is in myoglobin but is weakened by CO binding, an effect attributable to loss of an H-bond from the proximal His77 ligand to the adjacent Asn42 side chain. A structural model is proposed for the position of the CO-bound heme in the active form of CooA, which has implications for the mechanism of CO activation.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
authors.forename: Candace M
authors.initials: CM
authors.lastname: Coyle
authors.forename: Mrinalini
authors.initials: M
authors.lastname: Puranik
authors.forename: Hwan
authors.initials: H
authors.lastname: Youn
authors.forename: Steen Brøndsted
authors.initials: SB
authors.lastname: Nielsen
authors.forename: Robert D
authors.initials: RD
authors.lastname: Williams
authors.forename: Robert L
authors.initials: RL
authors.lastname: Kerby
authors.forename: Gary P
authors.initials: GP
authors.lastname: Roberts
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Spiro
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D015374:Biosensing Techniques; D002248:Carbon Monoxide / Q000032:analysis*; D006418:Heme / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman / Q000379:methods; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D015534:Trans-Activators; D006418:Heme; D002248:Carbon Monoxide