DNA binding by an imidazole-sensing CooA variant is dependent on the heme redox state.
CooA is a transcription factor from Rhodospirillum rubrum that is regulated by the binding of the small molecule effector, CO, to a heme moiety in the protein. The heme in CooA is axially ligated by two endogenous donors in the Fe(III) and Fe(II) states of the protein, and CO binding to the Fe(II) state results in replacement of the distal ligand. Reduction of the heme in the absence of CO results in a ligand switch on the proximal side, in which a cysteine thiolate in the Fe(III) state is replaced by a histidine in the Fe(II) state. Recently, a variant, termed RW CooA, was designed to respond to a new effector; Fe(II) RW CooA shows high specificity and induced DNA-binding activity in the presence of imidazole. Spectroscopic characterization of the imidazole adducts of RW CooA revealed that, unlike CO, imidazole binds to both Fe(III) RW CooA and Fe(II) RW CooA. The spectral characteristics are consistent with normal function of the redox-mediated ligand switch; Fe(III)-imidazole RW CooA bears a thiolate ligand and Fe(II)-imidazole RW CooA bears a neutral donor ligand. Since the effector binds to both redox states, RW CooA was used to probe the role of the redox-mediated ligand switch in the CooA activation mechanism. Functional studies of Fe(III)-imidazole and Fe(II)-imidazole ligated RW CooA demonstrate that only the Fe(II)-imidazole form is active for DNA binding. Thus, the ligand switch is essential for the activating conformational change and may prevent aberrant activation of CooA by other neutral diatomic molecules.
Clark
Robert W
RW
Department of Chemistry, Hope College, Holland, MI 49423, USA.
Youn
Hwan
H
Lee
Andrea J
AJ
Roberts
Gary P
GP
Burstyn
Judith N
JN
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D012247:Rhodospirillum rubrum / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D004268:DNA-Binding Proteins; D006420:Hemeproteins; D007093:Imidazoles; D015534:Trans-Activators; D006418:Heme; D006639:Histidine; C029899:imidazole; D002248:Carbon Monoxide