The C-helix in CooA rolls upon CO binding to ferrous heme.

CooA is a homodimeric transcriptional activator from Rhodospirillum rubrum containing one heme in each subunit. CO binding to the heme in its sensor domain activates CooA, facilitating the binding to DNA by its DNA-binding domain. The C-helix links the two domains and shapes an interface between the subunits. To probe the nature of CO activation, residues at positions 112-121 on the C-helix were replaced by Asn or Gln and their effects were evaluated by resonance Raman spectroscopy and by the measurements of CO binding affinity. The nu(Fe-CO) stretching Raman line in CO-bound wild-type CooA was up-shifted by 6 cm(-1) in the L116Q, G117N, and L120Q mutants, indicating unequivocally that these residues are close to the bound CO. Residues Leu116 and Leu120 from each subunit form contacts with the corresponding residues in the opposite subunit, enabling hydrophobic interactions in the inactive ferrous form. Thus, in the CO-bound activated form, both C-helices appear to roll to direct these residues toward the heme, forming a hydrophobic pocket for the bound CO. The CO affinity is approximately one order of magnitude higher in the L112Q, I115Q, L116Q, G117N, L120Q, and T121N mutants but reduced in A114N mutant. The variation indicates that these residues are close to the heme in the ferrous and/or CO-bound forms and are responsible for CooA activation. A roll-and-slide mechanism is proposed for CO activation of CooA.