The heme pocket afforded by Gly117 is crucial for proper heme ligation and activity of CooA.
CooA, a CO-sensing homodimeric transcription activator from Rhodospirillum rubrum, undergoes a conformational change in response to CO binding to its heme prosthetic group that allows it to bind specific DNA sequences. In a recent structural study (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880), it was suggested that CO binding to CooA results in a modest repositioning of the C-helices that serve as the dimer interface. Gly(117) is one of the residues on the C-helix within 7 A of the heme iron on the Pro(2) side of the heme in CooA. Analysis of a series of Gly(117) variants revealed altered CO-sensing function and heme ligation states dependent on the size of the substituted amino acid at this position; bulky substitutions perturbed CooA both spectrally and functionally. A combination of spectroscopic and mutagenic studies showed that a representative Gly(117) variant, G117I CooA, was specifically perturbed in its Pro(2) ligation in both Fe(III) and Fe(II) forms, but comparison with other CooA variants indicated that perturbation of Pro(2) ligation is not the basis for the lack of CO response in G117I CooA. These results have led to the hypothesis that (i) the heme and the C-helix region move toward each other following CO binding and the interaction of the heme with the C-helix is crucial for CooA activation, and (ii) this event occurs only when a properly sized heme pocket is afforded.
Youn
H
H
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Kerby
R L
RL
Thorsteinsson
M V
MV
Conrad
M
M
Staples
C R
CR
Serate
J
J
Beack
J
J
Roberts
G P
GP
D001426:Bacterial Proteins; D002248:Carbon Monoxide / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005998:Glycine; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D015534:Trans-Activators / Q000737:chemistry*
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D006420:Hemeproteins; D008024:Ligands; D015534:Trans-Activators; D006418:Heme; D002248:Carbon Monoxide; D004247:DNA; D005998:Glycine