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The Effects of Heme Pocket Hydrophobicity on the Ligand Binding Dynamics in Myoglobin as Studied with Leucine 29 Mutants

1997; Elsevier BV; Volume: 272; Issue: 48 Linguagem: Inglês

10.1074/jbc.272.48.30108

1083-351X

Takeshi Uchida, Koichiro Ishimori, Isao Morishima,

Neonatal Health and Biochemistry

To examine the effects of heme pocket hydrophobicity on the ligand binding in myoglobin, some artificial mutants of human myoglobin have been prepared, in which less hydrophobic amino acid residue (Ala, Gly, Ser) is located at the Leu29 (10th residue of the B helix) position. CO rebinding rates for the mutants were markedly decelerated, while the1H, and 15N NMR spectra of the mutants show that the structural changes around the heme iron for these mutants are rather small. The kinetic and structural properties of the mutants indicate that the ligand binding rate depends on the hydrophobicity inside the heme cavity for these mutants in addition to the volume of the side chain at the 29-position. On the basis of the IR stretching frequency of liganded CO, invasion of water molecules into the heme pocket in the mutants is suggested, which would be induced by the decrease in the hydrophobicity due to the amino acid substitution. A slight red shift of the position of the Soret peak for the serine mutant L29S also supports the reduced hydrophobicity inside the heme cavity. We can concluded that, together with the kinetic properties of the mutants, the hydrophobicity of the heme pocket is one of the key factors in regulating the ligand binding to the heme iron. To examine the effects of heme pocket hydrophobicity on the ligand binding in myoglobin, some artificial mutants of human myoglobin have been prepared, in which less hydrophobic amino acid residue (Ala, Gly, Ser) is located at the Leu29 (10th residue of the B helix) position. CO rebinding rates for the mutants were markedly decelerated, while the1H, and 15N NMR spectra of the mutants show that the structural changes around the heme iron for these mutants are rather small. The kinetic and structural properties of the mutants indicate that the ligand binding rate depends on the hydrophobicity inside the heme cavity for these mutants in addition to the volume of the side chain at the 29-position. On the basis of the IR stretching frequency of liganded CO, invasion of water molecules into the heme pocket in the mutants is suggested, which would be induced by the decrease in the hydrophobicity due to the amino acid substitution. A slight red shift of the position of the Soret peak for the serine mutant L29S also supports the reduced hydrophobicity inside the heme cavity. We can concluded that, together with the kinetic properties of the mutants, the hydrophobicity of the heme pocket is one of the key factors in regulating the ligand binding to the heme iron. Various elegant techniques to elucidate the molecular mechanism of the ligand binding process in hemoproteins have provided us with abundant information about the structural and functional factors that regulate the ligand binding dynamics (1Gibson Q.H. Olson J.S. McKinnie R.E. Rohlfs R.J. J. Biol. Chem. 1986; 261: 10228-10239Abstract Full Text PDF PubMed Google Scholar, 2Rohlfs R.J. Mathews A.J. Carver T.E. Olson J.S. Springer B.A. Egeberg K.D. Sligar S.G. J. Biol. Chem. 1990; 265: 3168-3176Abstract Full Text PDF PubMed Google Scholar, 3Carver T.E. Rohlfs R.J. Olson J.S. Gibson Q.H. Blackmore R.S. Springer B.A. Sligar S.G. J. Biol. Chem. 1990; 265: 20007-20020Abstract Full Text PDF PubMed Google Scholar). Site-directed mutagenesis, which is one of the potent techniques recently developed, has been applied to investigate functional roles of key residues in the active site of myoglobin (3Carver T.E. Rohlfs R.J. Olson J.S. Gibson Q.H. Blackmore R.S. Springer B.A. Sligar S.G. J. Biol. Chem. 1990; 265: 20007-20020Abstract Full Text PDF PubMed Google Scholar, 4Egeberg K.D. Springer B.A. Sligar S.G. Carver T.E. Rohlfs R.J. Olson J.S. J. Biol. Chem. 1990; 265: 11788-11795Abstract Full Text PDF PubMed Google Scholar, 5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar, 6Lambright D.G. Balasubramanian S. Decatur S.M. Boxer S.G. Biochemistry. 1994; 33: 5518-5525Crossref PubMed Scopus (50) Google Scholar, 7Springer B.A. Sligar S.G. Olson J.S. Phillips Jr., G.N. Chem. Rev. 1994; 94: 699-714Crossref Scopus (719) Google Scholar), since it is a small molecular weight protein and has been used as a model for hemoproteins.In myoglobin, some amino acid residues essential for the ligand binding have already been suggested (3Carver T.E. Rohlfs R.J. Olson J.S. Gibson Q.H. Blackmore R.S. Springer B.A. Sligar S.G. J. Biol. Chem. 1990; 265: 20007-20020Abstract Full Text PDF PubMed Google Scholar, 4Egeberg K.D. Springer B.A. Sligar S.G. Carver T.E. Rohlfs R.J. Olson J.S. J. Biol. Chem. 1990; 265: 11788-11795Abstract Full Text PDF PubMed Google Scholar, 6Lambright D.G. Balasubramanian S. Decatur S.M. Boxer S.G. Biochemistry. 1994; 33: 5518-5525Crossref PubMed Scopus (50) Google Scholar, 7Springer B.A. Sligar S.G. Olson J.S. Phillips Jr., G.N. Chem. Rev. 1994; 94: 699-714Crossref Scopus (719) Google Scholar, 8Case D.A. Karplus M. J. Mol. Biol. 1979; 132: 343-368Crossref PubMed Scopus (390) Google Scholar, 9Olson J.S. Mathews A.J. Rohlfs R.J. Springer B.A. Egeberg K.D. Sligar S.G. Tame J. Renaud J.-P. Nagai K. Nature. 1988; 336: 265-266Crossref PubMed Scopus (224) Google Scholar, 10Elber R. Karplus M. J. Am. Chem. Soc. 1990; 112: 9161-9175Crossref Scopus (476) Google Scholar, 11Carver T.E. Olson J.S. Smerdon S.J. Krzywda S. Wilkinson A.J. Gibson Q.H. Blackmore R.S. Ropp J.D. Sligar S.G. Biochemistry. 1991; 30: 4697-4705Crossref PubMed Scopus (65) Google Scholar, 12Lambright D.G. Balasubramanian S. Boxer S.G. Biochemistry. 1993; 32: 10116-10124Crossref PubMed Scopus (48) Google Scholar, 13Huang X. Boxer S.G. Nat. Struct. Biol. 1994; 1: 226-229Crossref PubMed Scopus (95) Google Scholar, 14Lai H.H. Li T.S. Lyons D.S. Phillips G.N. Olson J.S. Gibson Q.H. Protein Struct. Funct. Genet. 1995; 22: 322-339Crossref PubMed Scopus (49) Google Scholar). Among these amino acid residues, Leu29 at B10 1Alphanumeric codes (e.g. B10) refer to the position of the residue within the helices and loops of the myoglobin folding pattern, i.e. B10 denotes the 10th residue in the B helix. 1Alphanumeric codes (e.g. B10) refer to the position of the residue within the helices and loops of the myoglobin folding pattern, i.e. B10 denotes the 10th residue in the B helix. has been one of the crucial amino acid residues that controls the ligand binding process and geometry of ligand (5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar, 15Carver T.E. Brantley Jr., R.E. Singleton E.W. Arduini R.M. Quillin M.L. Phillips Jr., G.N. Olson J.S. J. Biol. Chem. 1992; 267: 14443-14450Abstract Full Text PDF PubMed Google Scholar, 16Gibson Q.H. Regan R. Elber R. Olson J.S. Carver T.E. J. Biol. Chem. 1992; 267: 22022-22034Abstract Full Text PDF PubMed Google Scholar, 17Gibson Q.H. Regan R. Olson J.S. Carver T.E. Dixon B. Pohajdak B. Sharma P.K. Vinogradov S.N. J. Biol. Chem. 1993; 268: 16993-16998Abstract Full Text PDF PubMed Google Scholar). The x-ray structure of myoglobin has shown that leucine 29 forms a hydrophobic cluster with other distal hydrophobic residues to restrict the movement of the distal side chains (Fig. 1) (18Phillips S.E.V. J. Mol. Biol. 1980; 142: 531-554Crossref PubMed Scopus (618) Google Scholar). A simulation study (10Elber R. Karplus M. J. Am. Chem. Soc. 1990; 112: 9161-9175Crossref Scopus (476) Google Scholar) has revealed that most CO molecules undergo many collisions with the residues forming walls of the heme pocket including the hydrophobic cluster. The number of collisions of Leu29 was second to that of Val68 in myoglobin (10Elber R. Karplus M. J. Am. Chem. Soc. 1990; 112: 9161-9175Crossref Scopus (476) Google Scholar), which has been also supported by picosecond and nanosecond geminate recombination studies (16Gibson Q.H. Regan R. Elber R. Olson J.S. Carver T.E. J. Biol. Chem. 1992; 267: 22022-22034Abstract Full Text PDF PubMed Google Scholar). On the basis of a detailed analysis of kinetics, Gibson and co-workers (16Gibson Q.H. Regan R. Elber R. Olson J.S. Carver T.E. J. Biol. Chem. 1992; 267: 22022-22034Abstract Full Text PDF PubMed Google Scholar, 17Gibson Q.H. Regan R. Olson J.S. Carver T.E. Dixon B. Pohajdak B. Sharma P.K. Vinogradov S.N. J. Biol. Chem. 1993; 268: 16993-16998Abstract Full Text PDF PubMed Google Scholar) concluded that the initial movements of ligand after dissociation are toward the back of the distal pocket with the side chain of Leu29 acting as a part of the physical barrier that restricts the ligand movement away from and back toward the heme iron atom.In our previous study (5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar), we prepared two mutants that replaced Leu29 with alanine (L29A) or isoleucine (L29I). These substitutions caused a 3–5-fold decrease in the rate constants for CO and O2 association. Based on the remarkable decrease in the association constants, we have concluded that the leucine residue is an important constituent of the hydrophobic cluster for maintaining myoglobin's ligand binding properties. Since the mutation of the amino acids forming other hydrophobic clusters in the distal pocket affects a slight alteration of the ligand binding process (5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar), the hydrophobicity of the leucine residue seems to be essential for the large alteration of the ligand binding rates. To gain further insights into the functional and structural roles of the hydrophobicity at the position of 29 in the ligand binding dynamics, we prepared some more Leu29 mutants in which the hydrophobicity of the amino acid substituted for leucine is decreased.One of the mutants we have prepared here has a glycine residue at position 29 (L29G). The hydrophobic index of glycine is 0, which is lower than that of leucine (+2.31) (19Fauchère J.L. Pliska V. Eur. J. Med. Chem. 1983; 18: 369-375Google Scholar). The other amino acid we substituted for the leucine is serine (L29S). Serine is less hydrophobic due to its hydroxy group (hydrophobic index is −0.05), while its steric hindrance is similar to that of alanine. We also tried to introduce some other hydrophilic amino acid residue such as threonine and asparagine. Unfortunately, however, the mutants having a hydrophilic amino acid residue at the 29-position are highly unstable and fail to keep the heme inside the heme pocket in the cyano-met form. To discriminate effects of the hydrophobicity on the ligand binding from those of the steric difference in the side chain of the substituted amino acid residue, we prepared an additional mutant in which a phenylalanine residue is introduced into position 29 (L29F). The hydrophobicity of phenylalanine (its hydrophobic index is +2.43) is similar to that of leucine, whereas the steric hindrance is quite different (15Carver T.E. Brantley Jr., R.E. Singleton E.W. Arduini R.M. Quillin M.L. Phillips Jr., G.N. Olson J.S. J. Biol. Chem. 1992; 267: 14443-14450Abstract Full Text PDF PubMed Google Scholar).In this study, we utilized the laser photolysis technique to characterize the ligand binding properties of the mutants. Also, we examined the structural changes around the active site by using1H and 15N NMR, IR, and electronic absorption spectroscopies to elucidate the relationship between ligand binding properties and the hydrophobicity of heme pocket. Various elegant techniques to elucidate the molecular mechanism of the ligand binding process in hemoproteins have provided us with abundant information about the structural and functional factors that regulate the ligand binding dynamics (1Gibson Q.H. Olson J.S. McKinnie R.E. Rohlfs R.J. J. Biol. Chem. 1986; 261: 10228-10239Abstract Full Text PDF PubMed Google Scholar, 2Rohlfs R.J. Mathews A.J. Carver T.E. Olson J.S. Springer B.A. Egeberg K.D. Sligar S.G. J. Biol. Chem. 1990; 265: 3168-3176Abstract Full Text PDF PubMed Google Scholar, 3Carver T.E. Rohlfs R.J. Olson J.S. Gibson Q.H. Blackmore R.S. Springer B.A. Sligar S.G. J. Biol. Chem. 1990; 265: 20007-20020Abstract Full Text PDF PubMed Google Scholar). Site-directed mutagenesis, which is one of the potent techniques recently developed, has been applied to investigate functional roles of key residues in the active site of myoglobin (3Carver T.E. Rohlfs R.J. Olson J.S. Gibson Q.H. Blackmore R.S. Springer B.A. Sligar S.G. J. Biol. Chem. 1990; 265: 20007-20020Abstract Full Text PDF PubMed Google Scholar, 4Egeberg K.D. Springer B.A. Sligar S.G. Carver T.E. Rohlfs R.J. Olson J.S. J. Biol. Chem. 1990; 265: 11788-11795Abstract Full Text PDF PubMed Google Scholar, 5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar, 6Lambright D.G. Balasubramanian S. Decatur S.M. Boxer S.G. Biochemistry. 1994; 33: 5518-5525Crossref PubMed Scopus (50) Google Scholar, 7Springer B.A. Sligar S.G. Olson J.S. Phillips Jr., G.N. Chem. Rev. 1994; 94: 699-714Crossref Scopus (719) Google Scholar), since it is a small molecular weight protein and has been used as a model for hemoproteins. In myoglobin, some amino acid residues essential for the ligand binding have already been suggested (3Carver T.E. Rohlfs R.J. Olson J.S. Gibson Q.H. Blackmore R.S. Springer B.A. Sligar S.G. J. Biol. Chem. 1990; 265: 20007-20020Abstract Full Text PDF PubMed Google Scholar, 4Egeberg K.D. Springer B.A. Sligar S.G. Carver T.E. Rohlfs R.J. Olson J.S. J. Biol. Chem. 1990; 265: 11788-11795Abstract Full Text PDF PubMed Google Scholar, 6Lambright D.G. Balasubramanian S. Decatur S.M. Boxer S.G. Biochemistry. 1994; 33: 5518-5525Crossref PubMed Scopus (50) Google Scholar, 7Springer B.A. Sligar S.G. Olson J.S. Phillips Jr., G.N. Chem. Rev. 1994; 94: 699-714Crossref Scopus (719) Google Scholar, 8Case D.A. Karplus M. J. Mol. Biol. 1979; 132: 343-368Crossref PubMed Scopus (390) Google Scholar, 9Olson J.S. Mathews A.J. Rohlfs R.J. Springer B.A. Egeberg K.D. Sligar S.G. Tame J. Renaud J.-P. Nagai K. Nature. 1988; 336: 265-266Crossref PubMed Scopus (224) Google Scholar, 10Elber R. Karplus M. J. Am. Chem. Soc. 1990; 112: 9161-9175Crossref Scopus (476) Google Scholar, 11Carver T.E. Olson J.S. Smerdon S.J. Krzywda S. Wilkinson A.J. Gibson Q.H. Blackmore R.S. Ropp J.D. Sligar S.G. Biochemistry. 1991; 30: 4697-4705Crossref PubMed Scopus (65) Google Scholar, 12Lambright D.G. Balasubramanian S. Boxer S.G. Biochemistry. 1993; 32: 10116-10124Crossref PubMed Scopus (48) Google Scholar, 13Huang X. Boxer S.G. Nat. Struct. Biol. 1994; 1: 226-229Crossref PubMed Scopus (95) Google Scholar, 14Lai H.H. Li T.S. Lyons D.S. Phillips G.N. Olson J.S. Gibson Q.H. Protein Struct. Funct. Genet. 1995; 22: 322-339Crossref PubMed Scopus (49) Google Scholar). Among these amino acid residues, Leu29 at B10 1Alphanumeric codes (e.g. B10) refer to the position of the residue within the helices and loops of the myoglobin folding pattern, i.e. B10 denotes the 10th residue in the B helix. 1Alphanumeric codes (e.g. B10) refer to the position of the residue within the helices and loops of the myoglobin folding pattern, i.e. B10 denotes the 10th residue in the B helix. has been one of the crucial amino acid residues that controls the ligand binding process and geometry of ligand (5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar, 15Carver T.E. Brantley Jr., R.E. Singleton E.W. Arduini R.M. Quillin M.L. Phillips Jr., G.N. Olson J.S. J. Biol. Chem. 1992; 267: 14443-14450Abstract Full Text PDF PubMed Google Scholar, 16Gibson Q.H. Regan R. Elber R. Olson J.S. Carver T.E. J. Biol. Chem. 1992; 267: 22022-22034Abstract Full Text PDF PubMed Google Scholar, 17Gibson Q.H. Regan R. Olson J.S. Carver T.E. Dixon B. Pohajdak B. Sharma P.K. Vinogradov S.N. J. Biol. Chem. 1993; 268: 16993-16998Abstract Full Text PDF PubMed Google Scholar). The x-ray structure of myoglobin has shown that leucine 29 forms a hydrophobic cluster with other distal hydrophobic residues to restrict the movement of the distal side chains (Fig. 1) (18Phillips S.E.V. J. Mol. Biol. 1980; 142: 531-554Crossref PubMed Scopus (618) Google Scholar). A simulation study (10Elber R. Karplus M. J. Am. Chem. Soc. 1990; 112: 9161-9175Crossref Scopus (476) Google Scholar) has revealed that most CO molecules undergo many collisions with the residues forming walls of the heme pocket including the hydrophobic cluster. The number of collisions of Leu29 was second to that of Val68 in myoglobin (10Elber R. Karplus M. J. Am. Chem. Soc. 1990; 112: 9161-9175Crossref Scopus (476) Google Scholar), which has been also supported by picosecond and nanosecond geminate recombination studies (16Gibson Q.H. Regan R. Elber R. Olson J.S. Carver T.E. J. Biol. Chem. 1992; 267: 22022-22034Abstract Full Text PDF PubMed Google Scholar). On the basis of a detailed analysis of kinetics, Gibson and co-workers (16Gibson Q.H. Regan R. Elber R. Olson J.S. Carver T.E. J. Biol. Chem. 1992; 267: 22022-22034Abstract Full Text PDF PubMed Google Scholar, 17Gibson Q.H. Regan R. Olson J.S. Carver T.E. Dixon B. Pohajdak B. Sharma P.K. Vinogradov S.N. J. Biol. Chem. 1993; 268: 16993-16998Abstract Full Text PDF PubMed Google Scholar) concluded that the initial movements of ligand after dissociation are toward the back of the distal pocket with the side chain of Leu29 acting as a part of the physical barrier that restricts the ligand movement away from and back toward the heme iron atom. In our previous study (5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar), we prepared two mutants that replaced Leu29 with alanine (L29A) or isoleucine (L29I). These substitutions caused a 3–5-fold decrease in the rate constants for CO and O2 association. Based on the remarkable decrease in the association constants, we have concluded that the leucine residue is an important constituent of the hydrophobic cluster for maintaining myoglobin's ligand binding properties. Since the mutation of the amino acids forming other hydrophobic clusters in the distal pocket affects a slight alteration of the ligand binding process (5Adachi S. Sunohara N. Ishimori K. Morishima I. J. Biol. Chem. 1992; 267: 12614-12621Abstract Full Text PDF PubMed Google Scholar), the hydrophobicity of the leucine residue seems to be essential for the large alteration of the ligand binding rates. To gain further insights into the functional and structural roles of the hydrophobicity at the position of 29 in the ligand binding dynamics, we prepared some more Leu29 mutants in which the hydrophobicity of the amino acid substituted for leucine is decreased. One of the mutants we have prepared here has a glycine residue at position 29 (L29G). The hydrophobic index of glycine is 0, which is lower than that of leucine (+2.31) (19Fauchère J.L. Pliska V. Eur. J. Med. Chem. 1983; 18: 369-375Google Scholar). The other amino acid we substituted for the leucine is serine (L29S). Serine is less hydrophobic due to its hydroxy group (hydrophobic index is −0.05), while its steric hindrance is similar to that of alanine. We also tried to introduce some other hydrophilic amino acid residue such as threonine and asparagine. Unfortunately, however, the mutants having a hydrophilic amino acid residue at the 29-position are highly unstable and fail to keep the heme inside the heme pocket in the cyano-met form. To discriminate effects of the hydrophobicity on the ligand binding from those of the steric difference in the side chain of the substituted amino acid residue, we prepared an additional mutant in which a phenylalanine residue is introduced into position 29 (L29F). The hydrophobicity of phenylalanine (its hydrophobic index is +2.43) is similar to that of leucine, whereas the steric hindrance is quite different (15Carver T.E. Brantley Jr., R.E. Singleton E.W. Arduini R.M. Quillin M.L. Phillips Jr., G.N. Olson J.S. J. Biol. Chem. 1992; 267: 14443-14450Abstract Full Text PDF PubMed Google Scholar). In this study, we utilized the laser photolysis technique to characterize the ligand binding properties of the mutants. Also, we examined the structural changes around the active site by using1H and 15N NMR, IR, and electronic absorption spectroscopies to elucidate the relationship between ligand binding properties and the hydrophobicity of heme pocket. We are grateful to Prof. S. G. Boxer and Dr. R. Varadarajan (Stanford University) for a gift of expression vector of human myoglobin gene. We are obliged to Dr. M. Unno (Tohoku University) for fruitful discussions.