Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.     

Modulating carbon monoxide binding affinity and kinetics in myoglobin: the roles of the distal histidine and the heme pocket docking site

 Myoglobin has long served as a model system for understanding the relations between protein structure, dynamics, and function. Its ability to discriminate between toxic CO and vital O₂, two small ligands that are almost equivalent in size and dipole moment, has attracted much attention. To understand discrimination and reversible ligand-binding in Mb, both the bound state and the "docked" state that leads to binding need to be studied. We have reported previously the nearly linear Fe–C–O geometry of bound CO and the nearly orthogonal geometry of docked CO [Lim et al. (1995), Science 269 : 962]. With the exception of X-ray structures, a preponderance of evidence points to a nearly linear Fe–C–O geometry and calls into question the proposal that the highly conserved distal histidine forces CO to bind in a nonoptimal geometry. The differences between the bound CO structures determined using IR and X-ray methods might arise from a water molecule hydrogen bonded to the distal histidine in some of the unit cells. Discrimination by Mb is manifested not only thermodynamically but also kinetically. Time-resolved CO rebinding studies that compare Mb with microperoxidase suggest that the heme pocket docking site in Mb exerts steric control of the ligand rebinding rate, slowing the rate of CO binding by a factor of more than 10⁴. Received, accepted: 23 May 1997     

Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin.

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.     

Thin-layer microcalorimetric studies of oxygen and carbon monoxide binding to hemoglobin and myoglobin.

A thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand binding macromolecules. We have measured the enthalpy of binding oxygen and carbon monoxide to horse myoglobin, human hemoglobin A0 and sperm whale myoglobin in phosphate buffer at pH 7.6, with the enzyme reducing system of Hayashi. Reactions of human hemoglobin were also done under various buffer conditions in order to elucidate the Bohr effect. These binding reactions were found not to exhibit a detectable enthalpy change over the temperature range of 10 degrees C to 25 degrees C. The enzyme reducing system was shown to react with oxygen in a manner that releases a substantial amount of heat. This problem was corrected by using a minimum amount and by placing the buffer and enzyme system in the reference cell effectively cancelling the oxygen enzyme reaction heat as well as the heat of gas dissolution. It was also demonstrated that glucose-6-phosphate, one of the reducing system components, in 50 mM concentrations can influence the heat of binding oxygen and carbon monoxide to hemoglobin. This effect was shown to be absent in the myoglobins and also with hemoglobin at glucose-6-phosphate concentrations less than 5 mM.     

Theoretical characterization of carbon monoxide vibrational spectrum in sperm whale myoglobin distal pocket

In this article we use the perturbed matrix method and an extended molecular dynamics sampling of the carbon monoxide (CO) in the myoglobin distal pocket to characterize the CO vibrational spectrum and hence to relate its spectroscopic features with the atomic-molecular behavior. Results show the accuracy of the method employed and confirm the assignment of the spectroscopic B1 and B2 states proposed by Lim et al.     

Assignment of heme and distal amino acid resonances in the 1H-NMR spectra of the carbon monoxide and oxygen complexes of sperm whale myoglobin

Assignments of resonances of the heme and distal amino acid protons in spectra of the CO and O2 complexes of sperm whale myoglobin are reported. These resonances provide information on the conformation of the heme pocket. For oxymyoglobin, the assignments of the heme meso protons disagree with those proposed previously on the basis of partial deuteration experiments. Rapid ring flips about the C beta-C gamma bond are detected for Phe-CD1. Recent claims for two conformational substates of valine-E11 in carbonmonoxymyoglobin (Bradbury, J.H. and Carver, J.A. (1984) Biochemistry 23, 4905-4913) are shown to be in error. The pK of His-97 (FG3) in carbonmonoxymyoglobin has been determined (pK = 5.9). This residue appears to influence many spectroscopic properties of myoglobin. The distal His-E7 in carbonmonoxymyoglobin has pK less than 5.0. Differences in the heme pocket conformation in the CO complexes of myoglobin and leghemoglobin are discussed. These differences may be influential in O2 and CO association reactions.     

Site-directed mutagenesis of the temperature-sensing histidine protein kinase CorS from Pseudomonas syringae

Several plant pathogenic bacteria belonging to the species Pseudomonas syringae produce the phytotoxin coronatine to enhance their virulence. Pseudomonas syringae pv. glycinea PG4180 synthesizes coronatine at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, its optimal growth temperature. In contrast, temperature has virtually no effect on coronatine synthesis in P. syringae pv. tomato strain DC3000. A modified two-component system controlling coronatine synthesis and consisting of the histidine protein kinase (HPK), CorS, the response regulator, CorR, and a third essential component, CorP, had been identified previously in both strains. CorS had been identified previously as a potential thermo-sensor. Comparison of the amino acid sequences of the HPKs from the two organisms revealed distinct differences. Site-directed mutagenesis of CorS from PG4180 was used to identify amino acyl residues potentially important for temperature signal perception. Point mutations and combinations of these were introduced into corS of PG4180 to generate corS variants with increased similarities to the respective allele from strain DC3000. These mutations resulted in either loss of activity, increase of thermoresponsiveness, or had no effect on CorS activity. Although none of the introduced mutations resulted in a clear conversion of CorS activity from thermo-responsive to temperature-independent, amino acyl residues important for temperature-dependent CorS activity and coronatine biosynthesis were identified.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Site-directed mutagenesis of the FAD-binding histidine of 6-hydroxy-D-nicotine oxidase. Consequences on flavinylation and enzyme activity

In 6-hydroxy-D-nicotine oxidase (6-HDNO) FAD is covalently bound to His71 of the polypeptide chain by an 8 alpha-(N3-histidyl)-riboflavin linkage. The FAD-binding histidine was exchanged by site-directed mutagenesis to either a Cys- or Tyr-residue, two amino acids known to be involved in covalent binding of FAD in other enzymes, or to a Ser-residue. None of the amino acid replacements for His71 allowed covalent FAD incorporation into the 6-HDNO polypeptide. Thus, the amino acid residues involved in covalent FAD-binding require a specific polypeptide surrounding in order for this modification to proceed and cannot be replaced with each other. Enzyme activity was completely abolished with Tyr in place of His71. 6-HDNO activity with non-covalently bound FAD was found with 6-HDNO-Cys and to a lesser extent also with 6-HDNO-Ser. However, the Km values for 6-HDNO-Cys and 6-HDNO-Ser were increased approximately 20-fold as compared to 6-HDNO-His. Both mutant enzymes, in contrast to the wild-type enzyme, needed additional FAD in the enzymatic assay (50 microM for 6-HDNO-Ser and 10 microM for 6-HDNO-Cys) for maximal enzyme activity.     

Site-directed mutagenesis of the distal basic cluster of pancreatic bile salt-dependent lipase

Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg(423), Lys(429), Arg(454), Arg(458), and Lys(462), distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr(427) and Tyr(453), associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.     

Stereochemistry of carbon monoxide binding to myoglobin and hemoglobin

The binding of carbon monoxide to myoglobin and hemoglobin is examined to determine the origin of the deviation of the FeCO geometry from that found in model systems. Possible distortions due to protein-ligand interactions are analyzed with special attention to protein relaxation. It is estimated that the protein can support a strain of less than 10 kcal per mole; this may be sufficient to produce a displacement of a linear FeCO unit from the heme normal.     

Heme-linked proton dissociation of carbon monoxide complexes of myoglobin and peroxidase.

It was found from spectrophotometric titration and proton balance measurement that the pKa value of a heme-linked protonation group of horseradish ferro-peroxidase C (donor:H2O2 oxidoreductase, EC 1.11.1.7) shifted from 7.25 to 8.25 upon combination with CO. The spectrophotometric titration experiment with myoglobin also revealed the presence of a heme-linked protonation group, the pKa value being 5.57 in myoglobin and 5.67 in the CO-myoglobin complex. It was concluded that the distinct shift of the pKa value in the case of peroxidase was attributable to the presence of a hydrogen bond between the sixth ligand and the distal base. The difference in the strength of such hydrogen bonding between peroxidase and myoglobin was discussed.     

The rate of entry of dioxygen and carbon monoxide into myoglobin.

The model for carbon monoxide or dioxygen recombination with heme proteins developed by the group at the University of Illinois is reexamined. We propose that the carbon monoxide or dioxygen molecule enters the protein at essentially a diffusion-limited rate determined by the solvent viscosity and that the protein offers no important barriers to this entry. The viscosity dependence of the entry rate k(ED), its magnitude (1 x 10(10) M(-1)s(-1), and the rate of quenching of triplet states of protoprophyrin IX in apomyoglobin by dioxygen are used as supporting evidence. Comparison is made to the model of a fluctuating protein developed by G. Weber.     

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.     

Nitric oxide and carbon monoxide equilibria of horse myoglobin and (N-methylimidazole)protoheme. Evidence for steric interaction with the distal residues

The Soret absorption maxima and extinction coefficients of the CO and NO complexes of horse myoglobin and (NMeIm)protoheme (NMeIm = 1-methylimidazole) have been determined. The partition coefficient N, equal to the ratio P1/2 (CO)/P1/2(NO), has been determined spectrophotometrically for horse myoglobin and (NMeIm)protoheme. P1/2-(NO) values calculated from the partition coefficients are 5.7 x 10(7) mmHg for (NMeIm)protheme and 1.1 x 10(6) mmHg for horse myoglobin. The ratio of P1/2(NO) values for protein and model is 1.9 which is similar to a value of 1.6 reported for the ratio of P1/2(O2) values. These values may be compared to a ratio of 15 for CO binding to protein and model complexes. This different ratio for CO provides further evidence for steric interaction of the bound CO with the protein based on a consideration of the preferred nonlinear geometry of Fe-NO and Fe-O2 and the linear geometry of Fe-CO.     

Site-directed mutagenesis of glutathione S-transferase YaYa: functional studies of histidine, cysteine, and tryptophan mutants.

The rat cytosolic glutathione S-transferase Ya subunit contains three histidine residues (at positions 8, 143, and 159), two cysteine residues (at positions 18 and 112), and a single tryptophan residue (at position 21). Histidine, cysteine, and tryptophan have been proposed to be present either near or at the active site of other glutathione S-transferase subunits. The functional role of these amino acids at each of the positions was evaluated by site-directed mutagenesis in which valine or asparagine, alanine, and phenylalanine were substituted for histidine, cysteine, and tryptophan, respectively. Mutant enzymes H8V, H143V, H159N, C112A, and W21F retained either full or better catalytic efficiencies (k(cat)/Km) toward 1-chloro-2,4-dinitrobenzene and glutathione. Lower but significant k(cat)/Km values were observed for H159V and C18A toward 1-chloro-2,4-dinitrobenzene. Some mutants displayed different thermal stabilities and intrinsic fluorescence intensities, but all retained the ability to bind heme. These results indicate that histidine, cysteine, and tryptophan in the glutathione S-transferase Ya subunit are not essential for catalysis nor are they involved in the binding of heme to the YaYa homodimer.     

Peroxynitrite-mediated oxidation of ferrous carbonylated myoglobin is limited by carbon monoxide dissociation

Peroxynitrite-mediated oxidation of ferrous nitrosylated myoglobin (Mb(II)-NO) involves the transient ferric nitrosylated species (Mb(III)-NO), followed by NO dissociation and formation of ferric myoglobin (Mb(III)). In contrast, peroxynitrite-mediated oxidation of ferrous oxygenated myoglobin (Mb(II)-O₂) involves the transient ferrous deoxygenated and ferryl derivatives (Mb(II) and Mb(IV)O, respectively), followed by Mb(III) formation. Here, kinetics of peroxynitrite-mediated oxidation of ferrous carbonylated horse heart myoglobin (Mb(II)-CO) is reported. Values of the first-order rate constant for peroxynitrite-mediated oxidation of Mb(II)-CO (i.e., for Mb(III) formation) and of the first-order rate constant for CO dissociation from Mb(II)-CO (i.e., for Mb(II) formation) are h = (1.2 ± 0.2) × 10⁻² s⁻¹ and k_off(CO) = (1.4 ± 0.2) × 10⁻² s⁻¹, respectively, at pH 7.2 and 20.0 °C. The coincidence of values of h and k_off(CO) indicates that CO dissociation represents the rate limiting step of peroxynitrite-mediated oxidation of Mb(II)-CO.     

Inhibition of dimethylnitrosamine-induced mutagenesis in Arabidopsis thaliana by diethyldithiocarbamate and carbon monoxide

Diethyldithiocarbamate and carbon monoxide markedly inhibited the frequency of embryonic and chlorophyll mutations induced by the metabolism-requiring mutagen dimethylnitrosamine in the higher plant Arabidopsis thaliana. In contrast, the monoamine oxidase substrates, tryptamine, benzylamine and 2-phenylethylamine, had no such effect. The mutagenicity of a direct-acting mutagen, N-methyl-N-nitrosourea, was not altered by these inhibitors or substrates.