Analysis of the L116K variant of CooA, the heme-containing CO sensor, suggests the presence of an unusual heme ligand resulting in novel activity.
CooA is the CO-sensing transcriptional activator from Rhodospirillum rubrum, in which CO binding to its heme prosthetic group triggers a conformational change of CooA that allows the protein to bind its cognate target DNA sequence. By a powerful in vivo screening method following the simultaneous randomization of the codons for two C-helix residues, 113 and 116, near the distal heme pocket of CooA, we have isolated a series of novel CooA variants. In vivo, these show very high CO-independent activities (comparable with that of wild-type CooA in the presence of CO) and diminished CO-dependent activities. Sequence analysis showed that this group of variants commonly contains lysine at position 116 with a variety of residues at position 113. DNA-binding analysis of a representative purified variant, L116K CooA, revealed that this protein is competent to bind target DNA with K(d) values of 56 nm for Fe(III), 36 nm for Fe(II), and 121 nm for Fe(II)-CO CooA forms. Electron paramagnetic resonance and electronic absorption spectroscopies, combined with additional mutagenic studies, showed that L116K CooA has a new ligand replacing Pro(2) in both Fe(III) and Fe(II) states. The most plausible replacement ligand is the substituted lysine at position 116, so that the ligands of Fe(III) L116K CooA are Cys(75) and Lys(116) and those in the Fe(II) form are His(77) and Lys(116). A possible explanation for CO-independent activity in L116K CooA is that ligation of Lys(116) results in a repositioning of the C-helices at the CooA dimer interface. This result is consistent with that repositioning being an important aspect of the activation of wild-type CooA by CO.
Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA.
Hwan
H
Youn
Robert L
RL
Kerby
Marc V
MV
Thorsteinsson
Robert W
RW
Clark
Judith N
JN
Burstyn
Gary P
GP
Roberts
D001426:Bacterial Proteins; D002248:Carbon Monoxide / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008024:Ligands; D011487:Protein Conformation; D012247:Rhodospirillum rubrum; D015534:Trans-Activators / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D006420:Hemeproteins; D008024:Ligands; D015534:Trans-Activators; D006418:Heme; D002248:Carbon Monoxide; D004247:DNA; D007501:Iron