Activation mechanism of the CO sensor CooA. Mutational and resonance Raman spectroscopic studies

Abstract

CooA is a CO-dependent heme protein transcription factor of the bacterium Rhodospirillum rubrum. CO binding to its heme causes CooA to bind DNA and activate expression of genes for CO metabolism. To understand the nature of CO activation, several CooA mutational variants have been studied by resonance Raman spectroscopy, in vivo activity measurements, and DNA binding assays. Analysis of the Fe-C and C-O stretching Raman spectroscopy bands permits the conclusion that when CO displaces the Pro2 heme ligand, the protein forms a hydrophobic pocket in which the C-helix residues Gly117, Leu116, and Ile113 are close to the bound CO. The displaced Pro2 terminus is expelled from this pocket, unless the pH is raised above the pKa, in which case the terminus remains in H-bond contact. The pKa for this transition is 8.6, two units below that of aqueous proline, reflecting the hydrophobic nature of the pocket. The proximal Fe-His bond in Fe[II]CooA is as strong as it is in myoglobin but is weakened by CO binding, an effect attributable to loss of an H-bond from the proximal His77 ligand to the adjacent Asn42 side chain. A structural model is proposed for the position of the CO-bound heme in the active form of CooA, which has implications for the mechanism of CO activation.