Roles of Heme Axial Ligands in the Regulation of CO Binding to CooA

Abstract

CooA is a CO-dependent transcription factor of the bacterium Rhodospirillum rubrum that contains a six-coordinate heme. It has as its heme axial ligands Pro2 and Cys75 in the ferric state and Pro2 and His77 in the ferrous state. To probe the regulation of CO binding and the ligand switching mechanism in CooA, we have prepared site-directed mutants in which the residues contributing the axial ligands are substituted. The properties of these mutants were investigated by resonance Raman and CO titration methods. Wild-type CooA binds CO with a modest dissociation constant (Kd) of 11 μm, this value being typical for gas-sensing heme proteins. The Kd value was greatly decreased in the P2H mutant, indicating that Pro2 coordination fine tunes CO sensing in CooA. The bound CO in P2H gives rise to a ν(Fe-CO) stretching Raman line at 490 cm-1, which is similar to that in wild-type CooA. Thus, Pro2 is the ligand that is replaced by exogenous CO. In the H77A mutant, equilibrium CO binding is biphasic, and at high CO pressures two CO molecules occupy both axial sites. The ν(Fe-CO) stretching Raman line for the first CO molecule was observed at 497 cm-1. Some of the His77 mutants showed an additional ν(Fe-CO) line at 525 cm-1. The binding affinity of the second CO molecule correlates with the five-coordinate component in the ferrous His77 mutants and also with the acidity of the side chain at position 77. Thus, we propose the Cys75-His77 ligand switch is controlled by His77 acting as a proton reservoir. CooA is a CO-dependent transcription factor of the bacterium Rhodospirillum rubrum that contains a six-coordinate heme. It has as its heme axial ligands Pro2 and Cys75 in the ferric state and Pro2 and His77 in the ferrous state. To probe the regulation of CO binding and the ligand switching mechanism in CooA, we have prepared site-directed mutants in which the residues contributing the axial ligands are substituted. The properties of these mutants were investigated by resonance Raman and CO titration methods. Wild-type CooA binds CO with a modest dissociation constant (Kd) of 11 μm, this value being typical for gas-sensing heme proteins. The Kd value was greatly decreased in the P2H mutant, indicating that Pro2 coordination fine tunes CO sensing in CooA. The bound CO in P2H gives rise to a ν(Fe-CO) stretching Raman line at 490 cm-1, which is similar to that in wild-type CooA. Thus, Pro2 is the ligand that is replaced by exogenous CO. In the H77A mutant, equilibrium CO binding is biphasic, and at high CO pressures two CO molecules occupy both axial sites. The ν(Fe-CO) stretching Raman line for the first CO molecule was observed at 497 cm-1. Some of the His77 mutants showed an additional ν(Fe-CO) line at 525 cm-1. The binding affinity of the second CO molecule correlates with the five-coordinate component in the ferrous His77 mutants and also with the acidity of the side chain at position 77. Thus, we propose the Cys75-His77 ligand switch is controlled by His77 acting as a proton reservoir. A new class of heme proteins is emerging that senses small gaseous molecules such as NO, O2, and CO (1Rodgers K.R. Curr. Opin. Chem. Biol. 1999; 3: 158-167Crossref PubMed Scopus (201) Google Scholar, 2Chan M.K. Curr. Opin. Chem. Biol. 2001; 5: 216-222Crossref PubMed Scopus (99) Google Scholar, 3Aono S. Acc. Chem. Res. 2003; 36: 825-831Crossref PubMed Scopus (91) Google Scholar). Binding of these diatomic ligands to the heme triggers the biological response. Soluble guanylyl cyclase binds NO and exerts a variety of physiological responses including neurotransmission and smooth muscle vasodilation (4Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar, 5Ignarro L.J. Biochem. 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Science. 2001; 293: 510-514Crossref PubMed Scopus (780) Google Scholar, 11Dioum E.M. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.A. McKnight S.L. Science. 2002; 298: 2385-2387Crossref PubMed Scopus (381) Google Scholar). The heme moieties in these sensor proteins are directly involved in binding their respective effector molecules and thereby regulating the activity of the proteins. Thus, the mechanisms whereby these proteins respond to the binding of gaseous molecules are currently of great interest. CooA is isolated from the photosynthetic bacterium Rhodospirillum rubrum, which can utilize CO as a sole energy source (12Kerby R.L. Ludden P.W. Roberts G.P. J. Bacteriol. 1995; 177: 2241-2244Crossref PubMed Google Scholar). CooA regulates the expression of genes, termed coo for CO oxidation, in which products are associated with CO metabolism (13Kerby R.L. Hong S.S. Ensign S.A. Coppoc L.J. Ludden P.W. Roberts G.P. J. Bacteriol. 1992; 74: 5284-5294Crossref Google Scholar). CooA is a homodimer (221 amino acids/monomer) containing two b-type hemes that reversibly bind CO. The signal of CO binding to the heme is transmitted to the DNA binding domain, enabling the protein to bind its target DNA sequence and induce transcription of the coo genes. The DNA binding and signaling domains are analogous to those found in the catabolite gene activator protein CAP (or CRP) (14Lanzilotta 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 (235) Google Scholar, 15Chan M.K. Nat. Struct. Biol. 2000; 7: 822-824Crossref PubMed Scopus (24) Google Scholar). The heme in CooA is bound within the signaling domain where it is ligated by a histidine (His77) side chain in the ferrous state (14Lanzilotta 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 (235) Google Scholar). This ligand is replaced by the Cys75 side chain in the ferric state (16Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 17Aono S. Ohkubo K. Matsuo T. Nakajima H. J. Biol. Chem. 1998; 273: 25757-25764Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Although ligand switching indicates that the position of the heme cofactor is flexible relative to the surrounding protein matrix of CooA, the physiological role and exact mechanism of the ligand switch remains elusive. The crystal structure of ferrous CooA revealed unexpectedly that the heme ligand trans to His77 is the amino-terminal proline residue (Pro2) from the opposite chain in the homodimer (see Fig. 1) (14Lanzilotta 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 (235) Google Scholar). Previous studies (16Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 18Reynolds 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), including electron paramagnetic resonance analysis and site-directed mutagenesis showed that one of the ligands in the ferric state is Cys75 in the thiolate form, and Pro2 is proposed indirectly to be the trans ligand (19Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 20Nakajima H. Honma Y. Tawara T. Kato T. Park S.-Y. Miyatake H. Shiro Y. Aono S. J. Biol. Chem. 2001; 276: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Because the ferrous heme in CooA is six-coordinate and low spin with Pro2/His77 ligands, the CO-sensing mechanism of CooA necessarily involves the displacement of one of these two ligands. The displaced ligand in the CO-bound ferrous CooA is suggested to be Pro2 by nuclear magnetic resonance and time-resolved resonance Raman studies (21Yamamoto K. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Nakajima H. Aono S. J. Biol. Chem. 2001; 276: 11473-11476Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22Uchida T. Ishikawa H. Ishimori K. Morishima I. Nakajima H. Aono S. Mizutani Y. Kitagawa T. Biochemistry. 2000; 39: 12747-12752Crossref PubMed Scopus (63) Google Scholar). However, it has been argued elsewhere (23Vogel K.M. Spiro T.G. Shelver D. Thorsteinsson M.V. Roberts G.P. Biochemistry. 1999; 38: 2679-2687Crossref PubMed Scopus (72) Google Scholar, 24Dhawan I.K. Shelver D. Thorsteinsson M.V. Roberts G.P. Johnson M.K. Biochemistry. 1999; 38: 12805-12813Crossref PubMed Scopus (49) Google Scholar) that CO displaces His77 instead of Pro2 and that the changes in His77 ligation trigger DNA binding. Although the release of Pro2 from the heme iron by incoming CO triggers the sequential events that result in the activation of CooA, a variety of Pro2 variants was found to be CO-responsive, indicating that Pro2 is not critical for activation of CooA in response to CO (19Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 20Nakajima H. Honma Y. Tawara T. Kato T. Park S.-Y. Miyatake H. Shiro Y. Aono S. J. Biol. Chem. 2001; 276: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Thus, the role of the Pro2 ligand in the CO binding event remains to be clarified. In this paper, we resonance Raman and CO titration analysis of wild-type and a of mutants of CooA in their and bound the analysis of it is that the Pro2 and His77 residues regulate CO binding to CooA. Pro2 is revealed to be the ligand that is replaced by exogenous and the His77 ligand is to the CO to the Pro2 side of the heme. The of these ligands are in the of CO and mechanisms in CooA. and of high a gene CooA was a of the 1992; 5: PubMed Scopus Google Scholar, D.R. Biochemistry. 1995; PubMed Scopus Google Scholar, T. D. H. Y. Ishikawa Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the amino sequence of rubrum CooA, and with from to were (see Fig. and Because the sequence is chain by DNA was for two of A and in which of and by The of A was in a second with a which and and a which a The of was also with a which a and a which a and a The products were at the and of the and at the and the ligation products were to E. coli The DNA sequence of the was with a DNA The was to the CooA The containing the CooA sequence was from the and ligated a expression T. D. H. Y. Ishikawa Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The ligation products were E. coli and protein were as (16Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). were to as by CooA were from the heme which was by a and were a Raman were a with a of cm-1, by a A was and the were with A Raman was the The protein in proteins were prepared by the of with The were prepared by the of of CO. The of the CO were with a CO titration of ferrous CooA was in at A was with a CooA which was with and with a and the protein was by an of which was by a Full was by of CO in and were to the containing and the were The CO were P2H and Fig. resonance Raman of ferric and P2H are Raman is a for the spin and coordination of heme iron T.G. Spiro T.G. of Raman Scholar, J.R. K.M. K.M. The Scholar). In the for and H77A CooA, the and are observed at and cm-1, respectively. are typical of six-coordinate ferric low spin hemes and similar to those for the CooA (23Vogel K.M. Spiro T.G. Shelver D. Thorsteinsson M.V. Roberts G.P. Biochemistry. 1999; 38: 2679-2687Crossref PubMed Scopus (72) Google Scholar). The of and H77A are the the that His77 is not the iron in ferric CooA. In the of P2H is with the line to with a line cm-1. The indicates the of a ferric five-coordinate indicating that Pro2 is an axial ligand in the A the side of the line indicates that is a six-coordinate low spin component The also the of In Fig. resonance Raman of ferrous and P2H are In the for CooA, the and are observed at and cm-1, respectively. are typical of six-coordinate ferrous low spin hemes and similar to those for the CooA (23Vogel K.M. Spiro T.G. Shelver D. Thorsteinsson M.V. Roberts G.P. Biochemistry. 1999; 38: 2679-2687Crossref PubMed Scopus (72) Google Scholar). His77 and Pro2 are revealed to be the axial ligands of the ferrous CooA 1) (14Lanzilotta 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 (235) Google Scholar). a resonance Raman line for a ferrous five-coordinate high spin component is at in the for both H77A and However, are similar to those of CooA indicating that six-coordinate low spin in these ligand In the ferrous the low spin an is not for H77A where His77 is replaced by This that an ligand the ferrous heme in this This ligand be the of Cys75 as be CO is the of the residue that is displaced by exogenous CO. studies suggested that CO displaces the His77 ligand (23Vogel K.M. Spiro T.G. Shelver D. Thorsteinsson M.V. Roberts G.P. Biochemistry. 1999; 38: 2679-2687Crossref PubMed Scopus (72) Google Scholar, 24Dhawan I.K. Shelver D. Thorsteinsson M.V. Roberts G.P. Johnson M.K. Biochemistry. 1999; 38: 12805-12813Crossref PubMed Scopus (49) Google Scholar), recent studies that the Pro2 is the ligand that is replaced (21Yamamoto K. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Nakajima H. Aono S. J. Biol. Chem. 2001; 276: 11473-11476Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22Uchida T. Ishikawa H. Ishimori K. Morishima I. Nakajima H. Aono S. Mizutani Y. Kitagawa T. Biochemistry. 2000; 39: 12747-12752Crossref PubMed Scopus (63) Google Scholar). To this we the CO binding properties of the axial ligand mutants of CooA The of the of heme proteins is to the of the The of ferrous CooA and which are replaced by and CO binding. changes are associated with one of indicating that CO binding is a equilibrium Thus, the titration was in of a equilibrium the ferrous and CO-bound of the The changes at the of the ferrous protein were and the A that binding involves of a to the titration and an Kd value of 11 was for CO binding to CooA The P2H has affinity for its Kd value was to be The titration at a of indicating that of CO binds to heme in the P2H Thus, the Pro2 residue regulates the CO binding affinity of CooA the affinity by of of CO in ferrous CooA mutants with heme from from M.A. Gonzalez G. M.F. Biochemistry. 1994; PubMed Scopus Google V.M. Gonzalez G. Gilles-Gonzalez M.A. Biochemistry. 2000; 39: 2685-2691Crossref PubMed Scopus (235) Google from T. D. H. Y. Ishikawa Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google from J. Biol. Chem. Full Text PDF PubMed Google in a new In exogenous CO was observed to bind in two The first CO molecule binds with a to the H77A with a value of which is similar to that for This is the His77 ligand is replaced by a in this mutant, and a is to be at the coordination of The second CO molecule binds with the value being to be The of two binding indicates that His77 is for exogenous CO to the side of the heme. This is to be for changes in CooA that to binding to the target The Kd for the axial ligand mutants of CooA are in I. binding is the His77 and the Kd are by the of the at position 77. CO binding in the His77 mutants indicates that Pro2 be being replaced by CO in the second binding The second CO binding event was not observed in the P2H at of CO. This indicates that Pro2 is displaced by exogenous CO the properties of bound the ν(Fe-CO) stretching Raman line was The ν(Fe-CO) stretching is to the of the residues the bound CO and is an probe of the T. Y. M. T. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Biochemistry. 1994; PubMed Scopus Google Scholar, Spiro T.G. J. Am. Chem. Soc. 1994; Scopus Google Scholar, E. K. J. Am. Chem. Soc. 1991; Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar). in Fig. a Raman line is observed at 490 in CO-bound CooA. This line has been to the ν(Fe-CO) stretching in CO T. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Ohkubo K. Nakajima H. Aono S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Youn H. Kerby R.L. Roberts G.P. Spiro T.G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the Pro2 mutant, a similar line is observed at cm-1, indicating that the of the bound CO is the as that in CooA. the in the H77A the ν(Fe-CO) is observed at 497 cm-1, which is that in CooA and be to the from the heme proteins have a histidine ligand trans to CO in the ferrous CO-bound and the ν(Fe-CO) is a of a CO T. Biochemistry. 1994; PubMed Scopus Google Scholar, Spiro T.G. J. Am. Chem. Soc. 1994; Scopus Google Scholar, E. K. J. Am. Chem. Soc. 1991; Scopus Google Scholar). The in H77A that the bound CO is in a that in and P2H the ν(Fe-CO) in the has not been to In of the His77 an additional Raman line is observed at 525 in the of the ν(Fe-CO) The relative of this line the residue at position 77. side a proton and a This is in the The of two indicates two for the and this in be related to the two binding observed in the CO titration It be that the value for is the the mutants and this that the second CO binds to the ferrous heme in this which a ν(Fe-CO) stretching 525 cm-1, has a small value Thus, the proton is not a for the of the second ν(Fe-CO) and in His77 of the His77 mutants showed two ν(Fe-CO) the incoming CO molecule in the mutants in the ferrous state. in Fig. the ferrous high spin component of is observed at in His77 In a line to be in the His77 and this line can be to the of the ferrous high spin The relative of the the and those and rise to a line at cm-1. this proton is not a for the a similar to those of and To the the coordination in the ferric CooA, we the coordination structure by resonance Raman In the ferric the His77 mutants are not Thus, it is that coordination of the ferric heme by the is by the His77 resonance Raman of the ferric CooA. and The proteins in cm-1, at of CO titration and resonance Raman we were to that the heme axial ligand in CooA that is displaced by exogenous CO is Because the ν(Fe-CO) stretching in the and P2H proteins is similar the Pro2 ligand be from the ferrous heme. of the amino-terminal residues has the activation of CooA, and the amino-terminal residues are to be flexible (19Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 20Nakajima H. Honma Y. Tawara T. Kato T. Park S.-Y. Miyatake H. Shiro Y. Aono S. J. Biol. Chem. 2001; 276: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). in the resonance Raman of ferric and ferrous a six-coordinate component in this one of the axial ligands has been Although the of the ligand trans to His77 is at the the amino of in P2H be the In this the coordination be to the of a five-coordinate The CO binding affinity of heme proteins is from the are the of and is to the dissociation constant In the of CooA, the CO binding is T. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Ohkubo K. Nakajima H. Aono S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Nakajima H. T. E. H. K. Aono S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), and the of the Kd value has been In this we the CO affinity of CooA for the first an equilibrium titration and the Kd value is similar to those for proteins that gaseous ligands (8Delgado-Nixon V.M. Gonzalez G. Gilles-Gonzalez M.A. Biochemistry. 2000; 39: 2685-2691Crossref PubMed Scopus (235) Google Scholar, M.A. Gonzalez G. M.F. Biochemistry. 1994; PubMed Scopus Google Scholar). of the a six-coordinate and this to be in this class of proteins. coordination is for the binding of exogenous gaseous the ligand be to a to binding. in the of the Pro2 ligand greatly the CO and the Kd value that of J. Biol. Chem. Full Text PDF PubMed Google Scholar), which contains a five-coordinate heme. The of a five-coordinate component in the P2H is from the resonance Raman Thus, the of Pro2 as a ligand is a to fine the sensing of CO in the It be that a small Kd this protein contains a six-coordinate heme with axial ligands T. D. H. Y. Ishikawa Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. T. T. M. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This that is with a that the binding of gaseous the His77 the CO binds in a and the value is similar to that observed for P2H The are one of the axial ligands is replaced in these and the first CO molecule the coordination However, the second CO molecule the trans the binding of the first CO has with Thus, the coordination of His77 is both to the CO molecules to the coordination by and to the binding of a second CO This is in with the (21Yamamoto K. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Nakajima H. Aono S. J. Biol. Chem. 2001; 276: 11473-11476Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) that CO binds to the side of the heme in the His77 mutants both and five-coordinate ferrous hemes and the ligand trans to Pro2 in the six-coordinate is suggested to be the of Cys75 as be It be argued that the first CO the Pro2 in the six-coordinate His77 Pro2 is replaced by the first we the of a ν(Fe-CO) stretching line from the in the His77 ligands such as rise to a trans ν(Fe-CO) this Although such a line is observed at 525 in these mutants this line is related to the five-coordinate component in the ferrous protein Thus, and a five-coordinate component at cm-1, and these mutants also 525 Thus, the 525 line not be to the from the The CO-bound CooA was very and the to be to to the ν(Fe-CO) stretching Raman line However, the CO of the His77 mutants were and be with a This be related to the affinity of these mutants for CO as in the The second CO binds with affinity as revealed by the relative to CooA. Thus, the ν(Fe-CO) at 497 cm-1, which is observed in the His77 be to the first CO molecule bound to the ferrous heme. the His77 showed the and this value is the Kd for CooA This that the second CO molecule to be observed in the and showed with the Kd for CooA, and a Raman line is observed 525 in these Thus, the 525 component be to the second CO molecule that is bound to the ferrous heme trans to the first CO The CO binding for the His77 mutants is in Fig. The first CO molecule the ferrous heme with high affinity and as revealed by the CO titration The in these mutants CO rise to the 497 ν(Fe-CO) The second CO molecule the Pro2 ligand trans to the first and the gives rise to the 525 in Fig. the ferrous His77 mutants and five-coordinate and the of the component to be related to the of a proton that is of in the of a In the ferric Cys75 coordination is by the residue at position The ferric heme thiolate which the of the In the ferrous the iron is by the of the and is the is the ligand In the crystal structure of ferrous CooA, the residue of coordination in the of the ferrous heme is Cys75 (14Lanzilotta 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 (235) Google Scholar). a proton is the of Cys75 a with the side chain of residue the the ferrous heme In this His77 is a proton the state of the Cys75 side In the ferric the of His77 a proton and the of in the ferrous the proton is to the and the binding to the ferrous heme. In we have been to the CO binding affinity of CooA, and the affinity was found to be similar to Pro2 is to the ligand that is replaced by exogenous CO. Pro2 and His77 ligands are for regulating CO binding to CooA and for sensing the of CO in the His77 a role in the ligand switch in CooA the state in that the is a proton the state of the Cys75 side of for and J. of for