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J. Biol. Chem., Vol. 278, Issue 4, 2333-2340, January 24, 2003

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

Hwan Youn, Robert L. Kerby, and Gary P. Roberts

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

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 Pro² 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 Ile¹¹³ and Leu¹¹⁶) located near the heme-bound CO in wild type CooA, and we investigated the effect on CO-specific activation. Surprisingly, the sizes of Ile¹¹³ and/or Leu¹¹⁶ 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.

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