Activation Mechanism of the CO Sensor CooA

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. 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. CooA is a member of the emerging class of heme sensor proteins that modulate biological activity in response to fluctuations in the concentration of the gaseous molecules CO, NO, or O2 (1Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (237) Google Scholar, 2Chan M.K. Nat. Struct. Biol. 2000; 7: 822-824Crossref PubMed Scopus (25) Google Scholar). Binding of these diatomic ligands to the heme induces the biological response. The mechanisms whereby the protein responds to the binding event are of great current interest. CooA is isolated from the bacterium Rhodospirillum rubrum, which grows anaerobically on CO as sole energy source (3Shelver D. Kerby R.L. He Y.P. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (193) Google Scholar). CooA regulates the expression of genes, termed coo for COoxidation, whose products are associated with CO metabolism. The protein is a homodimer (222 amino acids per monomer) containing two b-type hemes that reversibly bind CO. The CO binding enables the protein to bind a specific DNA sequence and induce transcription of the coo genes. CooA contains DNA-binding and signal domains analogous to those found in the catabolite gene activator protein CAP 1The abbreviations used are: CAP, catabolite gene activator protein; RR, resonance Raman; Mb, myoglobin; WT, wild-type; Mops, 4-morpholinepropanesulfonic acid. (or CRP) (1Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (237) Google Scholar, 2Chan M.K. Nat. Struct. Biol. 2000; 7: 822-824Crossref PubMed Scopus (25) Google Scholar) (Fig. 1). The heme is bound within the signal domain, where it is ligated by a histidine (His77) side chain (although this ligand is replaced by the Cys75 side chain when the Fe[II] is oxidized to Fe[III] (4Reynolds M.F. Shelver D. Kerby R.L. Parks R.B. Roberts G.P. Burstyn J.N. J. Am. Chem. Soc. 1998; 120: 9080-9081Crossref Scopus (56) Google Scholar)). The sixth Fe[II] ligand is unprecedented; it is the N-terminal proline residue (Pro2) from the opposite chain, which is displaced by CO binding (5Yamamoto K. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Nakajima H. S. J. Biol. Chem. PubMed Scopus (56) Google Scholar). The of CO-bound CooA is but on the of the CAP J. Biol. 2000; PubMed Scopus Google Scholar) it is to that the DNA-binding domains are position for DNA binding by of the which the of the protein and close to the heme (1Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (237) Google Scholar, 2Chan M.K. Nat. Struct. Biol. 2000; 7: 822-824Crossref PubMed Scopus (25) Google Scholar, H. Kerby R.L. Thorsteinsson M.V. J. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). has been that a of the the is a the hemes and the DNA-binding domains R.L. H. Thorsteinsson M.V. Roberts G.P. J. Biol. PubMed Scopus Google but the for this CO binding remains To the nature of the CO-bound form of CooA and the mechanism of its CO activation, have a of CooA variants by and in vivo specific DNA binding, and the and C–O stretching resonance Raman The residues for are in the of the heme as by the Fe[II]CooA a close of this The to the position of the in CooA and a specific model for that CooA and to the C-helix of the active form the of a that the an H-bond from the His77 proximal ligand to the side chain of structural a for the binding and of S. H. J. A. A. J. S. J. and in a structural model for the heme that is with the and as a for and in of CooA and CooA variants in an a in the R.L. H. Thorsteinsson M.V. Roberts G.P. J. Biol. PubMed Scopus Google and in vivo the in Scholar). the and CooA in a expression which of CooA expression M.V. Kerby R.L. H. W.N. Poulos T.L. J. Roberts G.P. J. Biol. Chem. 2000; PubMed Scopus Google Scholar). CooA of CooA and CooA variants with as D. Thorsteinsson M.V. Kerby R.L. Roberts G.P. M.F. Parks R.B. Burstyn J.N. PubMed Scopus Google Scholar). CooA variants as and CooA with which are D. Thorsteinsson M.V. Kerby R.L. Roberts G.P. M.F. Parks R.B. Burstyn J.N. PubMed Scopus Google Scholar, M.V. Kerby R.L. H. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar, H. Kerby R.L. Thorsteinsson M.V. Burstyn J.N. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). The of CooA and CooA variants to on the case of CooA, The heme of CooA the of Fe[III] CooA (3Shelver D. Kerby R.L. He Y.P. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (193) Google Scholar) or by a (3Shelver D. Kerby R.L. He Y.P. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (193) Google and protein concentration the DNA Binding DNA binding of CooA and CooA variants the M.V. Kerby R.L. H. W.N. Poulos T.L. J. Roberts G.P. J. Biol. Chem. 2000; PubMed Scopus Google Scholar). a a DNA containing with on of the and used the concentration of DNA used as the DNA by of the binding to a with of the as PubMed Scopus Google Scholar). CooA in pH to a heme concentration of pH pH pH and pH CooA with for and with CO for by of the Fe[III] to the Fe[II] form with concentration and CO binding by in the with of and from a and from a in a of the bound CO by the and by it with a a The and a with a and a K. with and The of this has been to understand the nature of the heme of CO-bound CooA and this to understand the mechanism of CooA activation. and activity are and in I. the that which on structural are to the of which the of the protein is the the two of that of in vivo activity and for the CooA for DNA Raman used for the in the of CO used for the in the Raman to CO in the Raman to to of measurements, pH pH pH pH pH pH used for the in the of CO used for the in the Raman to to of measurements, in a The of this are in the are two forms of with C–O stretching as by of has an effect on the of in this and a in of the that the Fe–His proximal bond is weakened in the form of to of the H-bond as the of this and is with a for this in CooA activation. The bound CO is in close to Leu116, and Gly117, which has implications for the of the heme and the protein CO by the of mutational variants these that and is with of these of pH of CooA, and of variants containing the terminus that that CO of Pro2 to its from the hydrophobic CO binding pocket, of its when The of the Fe[II]CooA heme is the of to from the strong ligand of the Pro2 of CO-bound have of a stretching and an CO stretching Shelver D. Thorsteinsson M.V. Roberts G.P. PubMed Scopus Google Scholar). Ishikawa H. Takahashi S. Ishimori K. Morishima I. K. Nakajima H. S. J. Biol. Chem. 1998; PubMed Scopus Google Scholar) for the as To this the and of the (Fig. to the bands as and the the has a this a The is a from the of (Fig. a which is that of the CO of myoglobin in that the molecules and have the The by Ishikawa H. Takahashi S. Ishimori K. Morishima I. K. Nakajima H. S. J. Biol. Chem. 1998; PubMed Scopus Google is to the of the two but a with this is the in Nakajima H. S. S. K. J. Am. Chem. Soc. PubMed Scopus Google Scholar) for molecules has two of and in but the The of the to the is the is that the by is the active form and that is a form that has to the DNA binding an active and or a of the molecules are in the of an energy of of CooA CO Binding the by the of the form of is by a weakened in a of (Fig. of and are in heme proteins J. Am. Chem. Soc. PubMed Scopus Google Scholar, J. Am. Chem. Soc. Scopus Google as for a of variants in in the of the binding pocket or and the the J. Am. Chem. Soc. PubMed Scopus Google Scholar, J. Am. Chem. Soc. Scopus Google Scholar). the is displaced when the ligand bond is of the proximal ligand and the CO J. Am. Chem. Soc. PubMed Scopus Google Scholar, 2000; Scopus Google Scholar). The in for Fe[II] CO ligand 2000; Scopus Google Scholar). the are on the the is to variants with hydrophobic in which the histidine is replaced by hydrophobic residues of in the of the it above the by a weakened bond from the to the proximal ligand, His77 in CooA from the in Shelver D. Thorsteinsson M.V. Roberts G.P. PubMed Scopus Google a of heme proteins of the The to in the CO binding pocket Shelver D. Thorsteinsson M.V. Roberts G.P. PubMed Scopus Google Scholar). of the pocket by the to to residue with a or a of that a R.B. PubMed Scopus Google Scholar) with the bound CO, but this is the residues that form the binding pocket are of a The of a weakened bond in is by the by Ishikawa H. Ishimori K. Morishima I. Nakajima H. S. 2000; PubMed Scopus Google which the Fe–His stretching in the of a of the Fe–His bond is in Fe[II] and is found in of Raman Scholar). The found by Ishikawa H. Ishimori K. Morishima I. Nakajima H. S. 2000; PubMed Scopus Google Scholar) that the bond is in for the with a bond in the CO The which on the (Fig. have a weakened bond and is to have an its of the strong is the bond in Fe[II]CooA Fe[II]CooA is the Fe–His is of Raman and its of a in the of Fe[II]CooA H. Kerby R.L. Thorsteinsson M.V. J. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google in which the side chain causes of the Pro2 ligand The heme is from the to to the (Fig. of the is by the position of the strong the of Raman Scholar). The is the as in the Fe–His bond is as strong in of Fe[II]CooA as in that the Fe–His bond is strong in Fe[II]CooA is that of Pro2 the proximal Fe–His and that it in the form of as as its the position to as the protein to the Fe[II]CooA but this in of of CO Nakajima H. S. S. K. J. Am. Chem. Soc. PubMed Scopus Google Scholar, Ishikawa H. Ishimori K. Morishima I. Nakajima H. S. 2000; PubMed Scopus Google Scholar). that CO binding the the mechanism for this of has been that H-bond the of the and the Fe–His bond J. Am. Chem. Soc. Scopus Google Scholar). the Fe–His is in Fe[II] forms of the that have a side chain in position to the proximal H-bond of Raman Scholar). the proximal is to a of a as as to a (Fig. the Fe[II]CooA (Fig. the H-bond is the side chain of Asn42 (Fig. The is (1Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (237) Google Scholar, 2Chan M.K. Nat. Struct. Biol. 2000; 7: 822-824Crossref PubMed Scopus (25) Google it that the H-bond is to the proximal in the Fe–His bond on the H-bond the of the bond in of the H-bond when CO is by the or when Asn42 is replaced by or which have of H-bond (Fig. that Asn42 its are within of His77 in the Asn42 to His77 in its by which the have raised and by a H-bond have The of effect that His77 has from residue in to the that the on the is to the a for the a weakened H-bond in the Asn42 with His77 in the Fe[II] form and the CO-bound the nature of the residue is of to activation. and CooA DNA in the of CO CooA for this residue in the active The for that effect is that the activity that DNA are to the in vivo the DNA binding, the with and has R.L. H. Thorsteinsson M.V. Roberts G.P. J. Biol. PubMed Scopus Google Scholar). that an of the mechanism is the of the The and below that the bond is by of the heme CO binding, which is for CooA activation. C-helix but the CO of CooA for heme pocket has an in ligand in heme proteins Chem. PubMed Scopus Google Scholar). CooA is specific to CO, and an of the CO binding pocket in and the in response to CO. have that hydrophobic residues and are for CO H. Kerby R.L. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar) and that is for this response H. Kerby R.L. Thorsteinsson M.V. J. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). the of these residues with to the bound CO the of a of CooA variants that in of activity and in of the bound CO or to bound CO on the of Pro2 is the ligand of Fe[II]CooA displaced by CO, the that the displaced Pro2 form the CO pocket. in of the Pro2 terminus by of the two residues effect on the or that Pro2 in CooA form the CO pocket and is with the that Pro2 is for of CooA by CO M.V. Kerby R.L. H. W.N. Poulos T.L. J. Roberts G.P. J. Biol. Chem. 2000; PubMed Scopus Google Scholar). that the DNA binding of these two Pro2 variants are CooA, a for the displaced Pro2 in the active form of of the Pro2 variants are Pro2 is the bound CO, the of Fe[II]CooA that the for CO pocket residues are on the with Leu116, and (1Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (237) Google Scholar, H. Kerby R.L. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). The of CooA variants these on and with a of these residues close to the bound CO and these residues are replaced by H-bond histidine and and from the position in CooA, as for a is that these the in of Pro2 and that CO displaces His77 is to that the His77 is replaced by the are and is a CO-bound of ligands Shelver D. Thorsteinsson M.V. Roberts G.P. PubMed Scopus Google Scholar). The effect is for the CooA an of and an of are opposite in and in to those when the histidine of is replaced by hydrophobic residues J. Am. Chem. Soc. PubMed Scopus Google Scholar, J. Am. Chem. Soc. Scopus Google Scholar). CooA a effect on the by and that the or to a close to the CO to a H-bond the in CooA is and a effect on the CO as by and bands (Fig. with the of position effect on and the CO remains The of to CooA has been H. Kerby R.L. Thorsteinsson M.V. J. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar) and is in the activity of variants in I. is for and its the it to that its is to the bound CO. this it is that is an of the C-helix per and to the bound CO. position the effect of on is as and in CooA and the effect in and A is that in these molecules with the CO, the that from the or to the side chain. that Ile113 is close to the bound CO but as close as the case of CooA, the a of and effect from to the of the to the CO. position have on as H. Kerby R.L. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar) and in I. The is and have that residues and H. Kerby R.L. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). is that the effect is the hydrophobic pocket is for the in the of the Pro2 ligand by CO. position and H-bond have been to CooA of of the hydrophobic pocket H. Kerby R.L. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). CooA it DNA binding activity with that of CooA in the of and CO-dependent DNA binding H. Kerby R.L. Thorsteinsson M.V. Burstyn J.N. Roberts G.P. J. Biol. Chem. PubMed Scopus Google and in the of position an effect on the as by and by is with the that the is a the CO. CooA and activity is by the but is a of the active The activity of CooA in the of CO has been and the that the residue as a ligand in the Fe[III] and Fe[II] have that this to a as that in CooA when bound to CO H. Kerby R.L. Thorsteinsson M.V. Burstyn J.N. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). The of or in is with of a hydrophobic for the bound CO. in the and CooA the and to the in the (Fig. is for but remains displaced from the by the as that the bond is by the residue is by the CO binding the bond in these as in that these to the of molecules as the of the is to the the of its signal and the of the a binding pocket is for the by its position in the (Fig. close to those of variants with the that Gly117, Leu116, and Ile113 are in or the CO binding pocket. the that of the Ile113 and side are with to CO of CooA are those A is that the CO the and that the DNA binding of the it is that induces a in the which is by residues for effect on C-helix variants and and variants and is the on the of CooA as in 1). M.V. Kerby R.L. H. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar) of its on of The of to the heme in these variants effect on that these residues are the bound CO and with the pocket by Gly117, Leu116, and The has effect on the activity but a is the CooA the is The is and is and is from the heme are Gly117, Leu116, and it is that the side chain the of the bound CO in a of the the of the in Fe[II]CooA M.V. Kerby R.L. H. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar) two of the side chain, the with the of the heme or with the of is that in the of the with the CO. The Pro2 from the CO in the CO-bound of CooA mutational have that Pro2 is for the response of CooA to CO M.V. Kerby R.L. H. W.N. Poulos T.L. J. Roberts G.P. J. Biol. Chem. 2000; PubMed Scopus Google Scholar). the Pro2 ligand is a of in CO binding by the of CooA CO binding is by the of the Pro2 ligand by CO, it to the of the displaced have above that Pro2 to the bound CO but when the pH above the and of (Fig. The replaced by the replaced by the is close to that of the is in the pH is an in the but a is in The pH and are to those by the and variants and The remains above the (Fig. in the that the effect of the pH is to a H-bond with the bound CO. in and the pH and bands are in the H-bond this H-bond from the CooA whose and bands are when the pH is to (Fig. that the H-bond is the N-terminal with the bound CO its the two and are the terminus is from with the CO. The Pro2 in CooA pH the when it is displaced from the Fe[II] pH the CO binding pocket is the terminus expelled from the binding it is as the histidine is expelled from the binding pocket pH D. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the displaced in with the CO and as the histidine in is the pKa of the displaced aqueous the pKa of the is but it in of the energy of Pro2 from the hydrophobic pocket. Mb, the pKa of the is D. PubMed Scopus Google Scholar, J. PubMed Scopus Google units the aqueous the pH of by the and and the pH (Fig. The a pKa of 8.6, two units the aqueous pKa of the of the pKa to Pro2 is the energy for the H-bond is as in CooA as it is in that the is to the pKa The which is pH when the pH is raised to pH the is found that the for whose pH raised to or in the protein the from pH that is a mechanism for of the that Pro2 the bound CO and the of Pro2 in the active form of CooA is The DNA of the CO-bound forms of and CooA is to that of CooA, of the active when the terminus is the of Pro2 to to the form of CooA in the of CO R.L. H. Thorsteinsson M.V. Roberts G.P. J. Biol. PubMed Scopus Google Scholar) and to a mechanism for the binding of CO when it is The of Pro2 is of a and is to a strong to the for strong is in the of which a for the stretching of Raman Scholar) (Fig. used to resonance with the heme bands of Raman Scholar)). is to to the of the ligand in Fe[II] the from to when is replaced by in Fe[II] PubMed Scopus Google Scholar) and from to of the of or of Fe[II] S. A. A. PubMed Scopus Google Scholar, A. J. Scopus Google Scholar). The to the of Shelver D. Thorsteinsson M.V. Roberts G.P. PubMed Scopus Google Scholar) that His77 or in His77 is by the to have a H-bond with and the position of is when Asn42 is replaced by the (Fig. that the is to His77 but the strong ligand of is by the (Fig. that by in the and Pro2 is replaced by the is replaced by a the a ligand the the of residues is the The Fe[II] protein remains but a of the heme is as by a on the the of Raman Scholar). Pro2 the ligand in but the of the chain the the is with the binding of CO to the The of this to the nature of the CO-bound form of CooA and its implications for the that to bind its DNA from the heme and its ligands for whose activity in vivo and in a DNA binding is that and activity the it is an of this that of the CooA are by the and activity activity that the protein has the to bind its DNA in of the the The are to the of the heme and its is this that to a model of the binding of CO the heme the from to active are to The C-helix residues Gly117, and of the CO binding pocket in is by the when side are replaced with H-bond of the hemes to the residues are from the heme in the Fe[II]CooA (1Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (237) Google Scholar). are for Leu116, for Gly117, and for The to the residue on the as the the to the residue on the opposite this of the is the from the heme are within of those from the is that in the case of the residue on the as the Ile113 and Gly117, the residue from the opposite is is the residue from the a of hemes and is to residues close to H-bond to with the bound CO. The which is in the Fe[II]CooA to CO The is that the and bands are when Asn42 is replaced by the residue (or by the of these bands to the H-bond of the proximal histidine ligand in J. Am. Chem. Soc. PubMed Scopus Google Scholar, J. Am. Chem. Soc. Scopus Google Scholar). the position of the and on the that the Fe–His bond is in in with His77 H-bond the Fe–His bond is strong in and in Fe[II]CooA as by Fe–His stretching for Fe[II]CooA in the heme of the the of proximal CO binding to loss of the H-bond in are the that from the with the Fe[II]CooA to a model for the to of The proposed are as in binding CO, the heme is to the to the side chain of on the the is active in the of CO, that a this position induces the heme by Pro2 as the a side chain in the of the side chain and a from the to the a of the heme to form a an for loss of the His77 remains bound to the heme and is displaced with is that this heme is in the ligand that is to to (4Reynolds M.F. Shelver D. Kerby R.L. Parks R.B. Roberts G.P. Burstyn J.N. J. Am. Chem. Soc. 1998; 120: 9080-9081Crossref Scopus (56) Google the heme position Cys75 heme and ligand has been for J. 1997; PubMed Scopus Google Scholar). is are a of that induce activity in the Fe[III] form of the protein M.V. Kerby R.L. H. J. Roberts G.P. J. Biol. Chem. PubMed Scopus Google Scholar). is that when the heme is displaced it is by the C-helix from the whose and Ile113 residues form the hydrophobic binding pocket for the CO. A of the C-helix H-bond these to the CO, a of the C-helix to this the that the to the active is as a of hemes and in the of CooA molecules this The by the to have an H-bond on the that the heme is from its position in the Fe[II]CooA these molecules are active in binding are in a with the active molecules or are from by of energy is the for the proposed heme and C-helix that hydrophobic are H. Kerby R.L. Roberts G.P. J. Biol. Chem. PubMed Scopus Google by the of the heme that from a ligand a ligand of the Fe[II]CooA the heme to to it by the (Fig. by the of the opposite C-helix the The hydrophobic of the CO binding pocket is by the pKa of the displaced to the aqueous pKa spectroscopy, with activity measurements, on variants of CooA has the nature of the CO C-helix Leu116, and Gly117, are to close to the bound CO, a hydrophobic pocket. The displaced Pro2 ligand is expelled from this pocket, unless the is the pKa for this is The to of the proximal H-bond when CO the of the Fe[II]CooA these to the that when CO the heme is displaced an adjacent and is by the C-helix of the opposite are to the protein for DNA