The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

Abstract

CooA from Rhodospirillum rubrum is a heme-containing transcriptional activator that becomes activated only upon binding CO. The basis for this specificity has been probed in a CooA variant, termed ⌬P3R4 CooA, lacking two residues adjacent to the Pro 2 heme ligand, which weakens that ligand. ⌬P3R4 CooA can bind imidazole and CN ؊ , as well as CO, and form a 6-coordinate low spin adduct with each. However, in contrast to the case with CO, imidazole and CN ؊ do not stimulate the DNA binding activity of ⌬P3R4 CooA. This result indicates that the CO-specific activation of CooA is not merely the result of creation of a 6-coordinate CooA adduct but that there must be another element to this response. One feature of CooA activation is modest repositioning of the C-helices upon CO binding, so we altered a portion of the C-helix (residues Ile 113 and Leu 116 ) located near the heme-bound CO in wild type CooA, and we investigated the effect on CO-specific activation. Surprisingly, the sizes of Ile 113 and/or Leu 116 positions are not critical for CooA activation by CO, disproving a precise interaction between these residues and the CO-bound heme as a basis for the CO activation mechanism and CO ligand specificity. In contrast, hydrophobic residues at these positions contribute to the activation. Some CooA variants altered at these positions in the background of ⌬P3R4 were also found to show low but reproducible activation in response to imidazole binding to the heme. A model for the role of hydrophobicity in CooA activation and specificity is suggested.

Dynamics of Carbon Monoxide Binding to CooA

“…3) (23). This variant is fully active in the presence of CO, and the Fe(II) form is predominantly six-coordinate and low spin, suggesting that Pro-2 remains bound to the heme.…”

Molecular Biology of Microbial Hydrogenases

“…Thus, the CooA dimer functions both as a redox sensor and as a specifi c CO sensor (Aono et al, 2000; reviewed by Roberts et al, 2001;Aono, 2003). Structural models for the heme movement during CO-specifi c activation of CooA have been proposed, based on mutational and spectroscopic studies (Nakajima et al, 2001;Coyle et al, 2003;Youn et al, 2003). c) Induction under N limitation.…”

Changing the Ligand Specificity of CooA, a Highly Specific Heme-based CO Sensor

“…Although FixL shows significant discrimination for O 2 by some in vitro assays, it does respond to a variety of small molecules to some detectable extent (6). In contrast, CooA, the CO-oxidation activator protein from Rhodospirillum rubrum, is highly selective toward CO and fails to respond detectably to other small molecules (7,8).…”

“…1). In the CO-bound form, the C-helix residues Ile 113 , Leu 116 , and Gly 117 have been identified to be near the bound CO by resonance Raman analysis (15), and the roles of these residues in CooA function in response to CO have been evaluated (8,16). However, the active form of CooA has not been solved structurally and therefore the molecular basis of CO specificity is unknown.…”

“…It has been shown that hydrophobicity along this region of the C helices is important for activity, leading to the notion that CO binding displaces the N terminus, which in turn exposes a hydrophobic surface of the C helices. This might provide the driving force for repositioning these C helices (8). A central problem in understanding CooA function is therefore to explain the extreme CO specificity in terms of the mechanism that repositions the C helices.…”