Redox-controlled ligand exchange of the heme in the CO-sensing transcriptional activator CooA.
The transcriptional activator CooA from Rhodospirillum rubrum contains a b-type heme that acts as a CO sensor in vivo. CooA is the first example of a transcriptional regulator containing a heme as a prosthetic group and of a hemeprotein in which CO plays a physiological role. In this study, we constructed an in vivo reporter system to measure the transcriptional activator activity of CooA and prepared some CooA mutants in which a mutation was introduced at Cys, His, Met, Lys, or Tyr. Only the mutations of Cys75 and His77 affected the electronic absorption spectra of the heme in CooA. The electronic absorption spectra, EPR spectra, and the transcriptional activator activity of the wild-type and mutant CooA proteins indicate that 1) the thiolate derived from Cys75 is the axial ligand in the ferric heme, but it is not coordinated to the CO-bound ferrous heme; 2) Cys75 is protonated or displaced in the ferrous heme; and 3) His77 is the proximal ligand in the CO-bound ferrous heme and probably also in the ferrous heme, but it is not coordinated to the ferric heme. NMR spectra reveal that the conformational change around the heme, which will trigger the activation of CooA by CO, takes place upon the binding of CO to the heme.
Aono
S
S
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Nomi-gun, Ishikawa 923-1292, Japan. ano@jaist.ac.jp
Ohkubo
K
K
Matsuo
T
T
Nakajima
H
H
D001426:Bacterial Proteins / Q000737:chemistry; D002248:Carbon Monoxide / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D017930:Genes, Reporter; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008024:Ligands; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry; D012247:Rhodospirillum rubrum / Q000378:metabolism*; D013053:Spectrophotometry; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics; D015533:Transcriptional Activation / Q000502:physiology
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D015534:Trans-Activators; D006418:Heme; D002248:Carbon Monoxide