Proton and iodine-127 nuclear magnetic resonance studies on the binding of iodide by lactoperoxidase

Interaction of an iodide ion with lactoperoxidase was studied by the use of 1H NMR, 127I NMR, and optical difference spectrum techniques. 1H NMR spectra demonstrated that a major broad hyperfine-shifted signal at about 60 ppm, which is ascribed to the heme peripheral methyl protons, was shifted toward high field by adding KI, indicating the binding of iodide to the active site of the enzyme; the dissociation constant was estimated to be 38 mM at pH 6.1. The binding was further detected by 127I NMR, showing no competition with cyanide. Both 1H NMR and 127I NMR revealed that the binding of iodide to the enzyme is facilitated by the protonation of an ionizable group with a pKa value of 6.0-6.8, which is presumably the distal histidyl residue. Optical difference spectra showed that the binding of an aromatic donor molecule to the enzyme is slightly but distinctly affected by adding KI. On the basis of these results, it was suggested that an iodide ion binds to lactoperoxidase outside the heme crevice but at the position close enough to interact with the distal histidyl residue which possibly mediates electron transport in the iodide oxidation reaction.     

Interaction of thiocyanate with horseradish peroxidase. 1H and 15N nuclear magnetic resonance studies

Interaction of thiocyanate with horseradish peroxidase (HRP) was investigated by relaxation rate measurements (at 50.68 MHz) of the 15N resonance of thiocyanate nitrogen and by following the hyperfine shifted ring methyl proton resonances (at 500 MHz) of the heme group of SCN-.HRP solutions. At pH 4.0, the apparent dissociation constant (KD) for thiocyanate binding to HRP was deduced to be 158 mM from the relaxation rate measurements. Chemical shift changes of 1- and 8-ring methyl proton resonances in the presence of various amounts of thiocyanate at pH 4.0 yielded KD values of 166 and 136 mM, respectively. From the pH dependence of KD and the 15N resonance line width, it was observed that thiocyanate binds to HRP only under acidic conditions (pH less than 6). The binding was found to be facilitated by protonation of an acid group on the enzyme with pKa 4.0. The pH dependence of the 15N line width as well as the apparent dissociation constant were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. The KD for thiocyanate binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as resorcinol, cyanide, and iodide ions. It was found that the presence of cyanide (which binds to heme iron at the sixth coordination position) and resorcinol did not have any effect on the binding of thiocyanate, indicating that the binding site of the thiocyanate ion is located away from the ferric center as well as from the aromatic donor binding site. The KD in the presence of iodide, however, showed that iodide competes with thiocyanate for binding at the same site. The distance of the bound thiocyanate ion from the ferric center was deduced from the 15N relaxation time measurements and was found to be a 6.8 A. From the distance as well as the change in the chemical shifts and line width of 1- and 8-methyl proton resonances, it is suggested that the binding site of thiocyanate may be located near heme, placed symmetrically with respect to 1- and 8-methyl groups of the heme of HRP. Similarity in the modes of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2 may also proceed via the two-electron transfer pathway under acidic conditions, as is the case for iodide.     

Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies

The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 +/- 5, 173 +/- 20, and 83 +/- 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 +/- 0.2 A from the 15N T1 measurements.     

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.     

Oxidation-reduction potential measurements of cytochrome c peroxidase and pH dependent spectral transitions in the ferrous enzyme.

The redox potential of the ferrous/ferric couple in cytochrome c peroxidase has been measured as a function of pH between pH 4.5 and 8. The redox potential decreases linearly as a function of pH between pH 4.5 and 7 with a slope of --57 +/- 2 mV per pH unit. Above pH 7, there is a positive inflection in the midpoint potential versus pH plot attributed to an ionizable group in the ferrous enzyme with pKa of 7.6 +/- 0.1. The midpoint potential at pH 7 is--0.194 V relative to the standard hydrogen electrode at 25 degree C. Ferrocytochrome c peroxidase undergoes a reversible spectral transition as a function of pH. Below pH 7, the enzyme has a spectrum typical of high spin ferroheme proteins while above pH 8, the spectrum is typical of low spin ferroheme proteins. The transition is caused by a co-operative, two proton ionization with an apparent pKa of 7.7 +/- 0.2. Two other single proton ionizations cause minor perturbations to the spectrum of ferrocytochrome c peroxidase. One has a pKa of 5.7 +/- 0.2 while the second has a pKa of 9.4 +/- 0.2.     

Structural characterization of lactoperoxidase in the heme environment by proton NMR spectroscopy

The heme environmental structures of lactoperoxidase (LP) have been studied by the use of hyperfine-shifted proton NMR and optical absorption spectra. The NMR spectra of the enzyme in native and cyanide forms in H2O indicated that the fifth ligand of the heme iron is the histidyl imidazole with an anionic character and that the sixth coordination site is possibly vacant. These structural characteristics are quite similar to those of horseradish peroxidase (HRP), suggesting that these may be prerequisite to peroxidase activity. The pH dependences of the spectra of LP in cyanide and azide forms showed the presence of two ionizable groups with pK values of 6 and 7.4 in the heme vicinity, which is consistent with the kinetic results. The group with pK = 7.4 is associated with azide binding to LP in a slow NMR exchange limit, which is in contrast to the fast entry of azide to HRP.     

Nuclear magnetic resonance studies on the spatial relationship of aromatic donor molecules to the heme iron of horseradish peroxidase

The geometry of hydrogen donor molecules bound to horseradish peroxidase was investigated using nuclear magnetic resonance techniques. Between resorcinol and 2-methoxy-4-methylphenol which showed different optical difference spectra, little difference was observed in the orientation of the molecules bound to horseradish peroxidase: the minimal distances between the enzyme iron and the protons of the phenol rings are in the range of 8.4-11.0 A. This situation was not greatly different for the third compound studied in this paper, benzhydroxamic acid, providing evidence against the view that its side chain coordinates to the heme iron. Furthermore, it was found that transferred nuclear Overhauser effect for the signals of these compounds was observable only when the heme peripheral 8-methyl proton signal was irradiated. These results, together with a hypothetical model of the enzyme structure obtained by computer-aided simulation procedures, suggest that the binding of these donor molecules and competitive inhibitors occur in the vicinity of the heme peripheral 8-methyl group, with hydrophobic interactions probably with Tyr-185 and with hydrogen bond with adjacent amino acid residues such as Arg-183.     

Reaction of horseradish peroxidase with azide and some implications for the heme environmental structure. NMR and kinetic studies.

The azide complex of horseradish peroxidase was studied by high resolution 1H and 15N NMR spectroscopy and by the temperature-jump method. The heme peripheral methyl proton peaks and the ligand 15N resonance were resolved to show that binding of azide by horseradish peroxidase occurs only in acidic solution below pH 6.5. It was also found that the chemical exchange rate of azide with the ferric enzyme was much faster on the 1H and 15N NMR time scale. This was further substantiated by kinetics of azide binding by horseradish peroxidase where the chemical exchange rate was confirmed to be in the microseconds range at pH 5.0 and 23 degrees C. This rate is salient in usual ligand exchange reactions in hemoproteins so far reported. pH dependences of the first order association and dissociation rate constants were also studied by the temperature-jump method to suggest a strong linkage of the azide binding with a proton uptake of an amino acid residue on the enzyme. These results were compared with the case of horse metmyoglobin and were interpreted to indicate that a heme-linked ionizable group on the enzyme facilitates the fast entry of the ligand to the coordination site. A histidyl residue is a possible candidate for the ionizable group of the enzyme.     

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.     

Proton NMR study of the heme environment in bacterial quinol oxidases

The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy. The oxidized bo3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center. The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein. The oxidized hd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate. Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide. The proton signals near the low-spin heme b558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins. The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b595 and d) remain high-spin upon chemical reduction. The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins.     

A study of ligand binding to spleen myeloperoxidase

The ligand binding properties of spleen myeloperoxidase, a peroxidase formerly called "the spleen green hemeprotein", were studied as functions of temperature and pH, using chloride and cyanide as exogenous ligands. Ligand binding is influenced by a proton dissociable group with a pKa of 4. The protonated, uncharged form of cyanide binds to the unprotonated form of the enzyme, while chloride ion binds to the enzyme when this group is protonated. In both cyanide and chloride binding, the pH-dependent change in the apparent ligand affinity is due to a change in the apparent association rate with pH. The proton dissociable group on the enzyme involved in ligand binding has a delta H value of about 8 kcal . mol-1. The present results suggest that this ionizable group is the imidazole group of a histidine residue located near the ligand binding site.     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo-halide ion binding to chloroperoxidase

The proton nuclear magnetic resonance spectra of several chloroperoxidase-inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide-chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin.     

Protein-structural heterogeneity in a non-allosteric monomeric insect hemoglobin monitored by proton magnetic resonance spectroscopy

Proton NMR has revealed two modes of structural heterogeneity in the monomeric hemoglobin I of Chironomus thummi thummi, CTT I; rotational disorder caused by a 180 degree rotation of the heme about the alpha, gamma-meso axis (primary heterogeneity), which varies for each preparation or reconstitution of this hemoglobin, and a 'silent' amino acid replacement [Thr/Ala exchange in position 98(FG4)] in the vicinity of the heme group, which is invariant under all experimental conditions. The heme rotational disorder (primary heterogeneity) can be removed by reconstitution of CTT I with the symmetrical protoheme III. The secondary splitting is not affected; the ratio of intensities of the two types of resonance remains constant. The 8-methyl and 3-methyl and one of the alpha-vinyl proton resonances for the major heme rotational component and the 5-methyl and 1-methyl and one of the alpha-vinyl proton resonances for the minor heme rotational component have been identified and assigned by reconstitution with deuterium-labeled heme. Decoupling experiments have been employed to assign vinyl beta protons in cis and trans position to the respective vinyl alpha protons. Hyperfine shifts for the heme protons exhibited no pH influence above pH 6, in accord with the lack of the alkaline Bohr effect. Below pH 6, pH effects are most strongly reflected by the 8-methyl and 5-methyl proton resonances possibly reflecting titration of the propionate groups.     

High-pressure nuclear magnetic resonance studies of hemoproteins. Pressure-induced structural changes in the heme environments of ferric low-spin metmyoglobin complexes

In order to gain an insight into nonbonded interactions in the heme microenvironments of hemoproteins, proton NMR spectra of the cyanide and methylamine complexes of metmyoglobin and its derivatives reconstituted with deutero- and meso-hemins in H2O were studied under high pressures. The exchangeable NH proton of distal histidyl imidazole exhibits substantial pressure-induced shift while the proximal histidyl NH proton shows no pressure effect for the cyanide complexes. The heme peripheral proton signals, especially 5- and 8-methyl and vinyl C alpha H resonances, were also affected by pressure. These observations are interpreted as arising from pressure-induced structural changes in the heme crevice in which the pressure effects are localized to the distal side rather than the proximal side and from possible changes in the van der Waals contacts at the heme periphery with nearby amino acid residues.     

Contribution of proton release to the B2 photocurrent of bacteriorhodopsin.

The contribution of proton release from the so-called proton release group to the microsecond B2 photocurrent from bacteriorhodopsin (bR) oriented in polyacrylamide gels was determined. The fraction of the B2 current due to proton release was resolved by titration of the proton release group in M. At pH values below the pKa of the proton release group in M, the proton release group cannot release its proton during the first half of the bacteriorhodopsin photocycle. At these pH values, the B2 photocurrent is due primarily to translocation of the Schiff base proton to Asp85. The B2 photocurrent was measured in wild-type bR gels at pH 4.5-7.5, in 100 mM KCl/50 mM phosphate. The B2 photocurrent area (proportional to the amount of charge moved) exhibits a pH dependence with a pKa of 6.1. This is suggested to be the pKa of the proton release group in M; the value obtained is in good agreement with previous results obtained by examining photocycle kinetics and pH-sensitive dye signals. In the mutant Glu204Gln, the B2 photocurrent of the mutant membranes was pH independent between pH 4 and 7. Because the proton release group is incapacitated, and early proton release is eliminated in the Glu204Gln mutant, this supports the idea that the pH dependence of the B2 photocurrent in the wild type reflects the titration of the proton release group. In wild-type bacteriorhodopsin, proton release contributes approximately half of the B2 area at pH 7.5. The B2 area in the Glu204Gln mutant is similar to that in the wild type at pH 4.5; in both cases, the B2 current is likely due only to movement of the Schiff base proton to Asp85.     

The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.     

Fluorescence titrations of residue 59 and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1

Fluorescence titrations of kynurenine and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1 were carried out by monitoring protein fluorescence through a pH change from 1.5 to 10.5. In the titration of kynurenine fluorescence at 455 nm, a few small but distinct quenching events occurred between pH 3.5 and 9.5. Three ionizable groups were found to be responsible for the individual steps of quenching observed. These groups are Glu 58 with pKa 4.6, His 40 or 92 with pKa 7.8 and Lys 41 with pKa 8.7. From this result, a subtle conformational change associated with the proton dissociation equilibria of Glu 58 and His 40 or 92 in the active site of Kyn 59-RNase T1 is suggested. The pH-titration behavior of tyrosine fluorescence in Kyn 59-RNase T1 was different from that of kynurenine fluorescence. Two acidic groups with pKa's 3.2 and 6.5 were detected as perturbants. In NFK 59-RNase T1, both N'-formylkynurenine and tyrosine showed almost the same fluorescence behavior during titration, which was characterized by two transitions between pH 3 and 8 in each titration curve. Two ionizable groups with pKa's 3.7-3.8 and 6.7-6.8 were determined. The role of the latter ionizable group is discussed in relation to the enzyme function of RNase T1. From the close similarity in structure and function between Kyn 59-RNase T1 and RNase T1, it is suggested that the same mechanism of conformational change linked to the ionization states of Glu 58 and His 40 or 92 exists in the native protein too.     

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.