Resonance Raman evidence for a novel charge relay activation mechanism of the CO-dependent heme protein transcription factor CooA.
Resonance Raman spectra of the CO-responsive transcription factor CooA from Rhodospirillum rubrum provides evidence on the nature of heme ligation and its CO activation mechanism. The Fe(III) form gives standard low-spin heme spectrum, while the Fe(II) form is low spin for wild-type (WT) CooA and mixed spin for a CooA variant, H77Y, with an His77Tyr substitution. The Fe(II) porphyrin skeletal mode nu11 is at a value (1541 cm-1) indicative of a neutral donor ligand for the H77Y variant but is at an unusually depressed frequency (1529 cm-1) for the WT protein, indicating a strongly donating ligand. This ligand is proposed to be His77 imidazolate, formed by proton transfer to a nearby acceptor. The WT CO adduct has FeCO and CO stretching frequencies that indicate a neutral trans ligand and negative polarity in the CO binding pocket, while the CO adduct of His77Tyr has both 6- and 5-coordinate signals and a nonpolar CO environment. Photolysis of the WT CO adduct by the Raman laser produced a low-spin product at steady state, indicating fast recombination of the displaced ligand. These data suggest a novel YH- - -His- charge relay mechanism for CooA activation by CO. In this mechanism, His77 is reprotonated upon ligand displacement by CO; CO displaces either His77 or the trans ligand, X. The resulting charge on Y- may induce the protein conformation change required for site-selective DNA binding.
Department of Chemistry, Princeton University, New Jersey 08544, USA.
K M
KM
Vogel
T G
TG
Spiro
D
D
Shelver
M V
MV
Thorsteinsson
G P
GP
Roberts
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D012247:Rhodospirillum rubrum; D013059:Spectrum Analysis, Raman; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006420:Hemeproteins; D008024:Ligands; D015534:Trans-Activators; D002248:Carbon Monoxide