Binding of aromatic donor molecules to lactoperoxidase: proton NMR and optical difference spectroscopic studies

The interaction of aromatic donor molecules with lactoperoxidase (LPO) was studied using 1H-NMR and optical difference spectroscopy techniques. pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with pKa of 6.1) which is presumably a distal histidine. Dissociation constants evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements were found to be an order of magnitude larger than those for binding to horse radish peroxidase (HRP), indicating relatively weak binding of the donors to LPO. The dissociation constants evaluated in presence of excess of I- and SCN- showed a considerable increase in their values, indicating that the iodide and thiocyanate ions compete for binding at the same site. The dissociation constant of the substrate binding was, however, not affected by cyanide binding to the ferric centre of LPO. All these results indicate that the organic substrates bind to LPO away from the ferric center. Comparison of the dissociation constants between the different substrates suggested that hydrogen bonding of the donors with the distal histidine amino acid, and hydrophobic interaction between the donors and the active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the LPO-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative and relatively low compared to those for binding to HRP. The distances of the substrate protons from the ferric center were found to be in the range 9.4-11.1 A which are 2-3 A larger than those reported for the HRP-substrate complexes. These structural informations suggest that the heme in LPO may be more deeply buried in the heme crevice than that in the HRP.     

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.     

Interaction of aromatic donor molecules with lactoperoxidase probed by optical difference spectra

On the basis of optical difference spectra, lactoperoxidase (LPO) was shown to form a 1:1 complex with aromatic donor molecules: resorcinol, hydroquinone, phenol, p-cresol, guaiacol, aniline, and benzohydroxamic acid. As compared with horseradish peroxidase (HRP), the values of the dissociation constant, Kd, of LPO-donor complexes were found to be 4-720-fold larger and were not greatly changed in the presence of KCN and by changes in pH in the range 4-9. The apparent enthalpy and entropy of the binding reactions were found to be -13 kJ mol-1 and -29 J mol-1 K-1, respectively, somewhat smaller (in absolute value) than the corresponding values of HRP. The difference spectra of LPO-donor complexes resembled each other, in contrast to the case of HRP, and the bindings of the donors to LPO occurred in a competitive fashion between the donors. Incubation of LPO with phenylhydrazine and hydrogen peroxide markedly depressed donor binding, the intensity of the Soret band, and the catalytic activity, probably as the result of formation of meso-phenyl derivatives of the heme. These findings suggest that the binding of aromatic donors to LPO occurs at a specific site, probably near the heme edge, where the electron transfer in the peroxidase reaction may take place.     

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.     

Nuclear magnetic relaxation study on the interaction of glycyl-L-tyrosine with manganese-carboxypeptidase A in solution.

The spin-lattice and spin-spin relaxation rates were measured of the Gly C alpha and Tyr aryl protons of glycyl-L-tyrosine (Gly-Tyr) bound to manganese(II)-substituted carboxypeptidase A (MnCPA) in aqueous solution. The temperature and frequency dependences of the relaxation rates were analyzed using the Solomon-Bloembergen-Morgan equations. The binding modes of MnCPA with Gly-Tyr in solution are different from that of ZnCPA in crystals. 1. Mn(II)-coordinated water of MnCPA is not excluded by the binding of Gly-Tyr substrate molecules. 2. The Gly carbonyl group does not coordinate tightly to the metal ion of MnCPA. The Gly C alpha protons of Gly-Tyr in the productive binding site are appreciably mobile. 3. A non-productive loose binding of another Gly-Tyr molecule is suggested by simulation of the temperature and frequency dependences of the proton relaxation rates.     

H NMR investigation of the influence of interacting sites on the dynamics and thermodynamics of substrate and ligand binding to horseradish peroxidase.

The influence of substrate benzhydroxamic acid (BHA) and iron ligand (cyanide) on the thermodynamics and dynamics of each of the two binding sites of horseradish peroxidase (HRP) isozyme C has been investigated by 1H NMR spectroscopy. A combination of line-width analysis and saturation transfer spectroscopy has allowed the direct determination of the off-rate of substrate and ligand in the absence or presence of the other. These off-rates, together with available dissociation constants obtained by optical spectroscopy (Schonbaum, 1973), provide estimates for kon. The dissociation constant for cyanide binding to the BHA.HRP complex was also directly determined by NMR. In all cases the 1H NMR determined dynamic and thermodynamic data agree well with those values available in the literature. BHA binding leads to a 200-fold decrease in CN- affinity that arises from a factor greater than 10 decrease in koff(CN-) and greater than 2 x 10(3) decrease in kon(CN-). While a portion of the decrease in kon(CN-) can be rationalized by water coordination of the iron in the BHA.HRP complex, the additional decrease in kon(CN-) and that in koff(CN-) indicates that BHA in the binding pocket blocks the CN- ligation channel and serves as a "gate" to CN- exchange. This view is supported by observing a factor greater than 4 decrease in distal His labile proton exchange with bulk water in HRP-CN upon BHA binding. The ternary complex BHA.HRP-CN is shown to be heterogeneous. While the thermodynamics of BHA and CN- binding appear similar in the two ternary complexes, the BHA on- and off-rates for the two complexes differ by a factor of approximately 10. The two heterogeneous forms interconvert at 25 degrees C at approximately 2 x 10(2) s-1, precluding the determination of any difference in the CN- binding rates by saturation transfer. The greater lability of one of the two ternary complexes is attributed to an alternate orientation of some distal residue that blocks the substrate binding channel in one of the forms. Transferred nuclear Overhauser effects from the heme to BHA in the ternary complex reveal that the BHA substrate is in contact not only with the heme pyrrole D substituents but also with the distal His 42, indicating that the polar side chain of BHA extends well into the distal heme pocket.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromium(III)-adenosine triphosphate as a paramagnetic probe to determine intersubstrate distances on pyruvate kinase. Detection of an active enzyme-metal-ATP-metal complex.

The interaction of CrATP, a stable, substitution-inert, paramagnetic tridentate complex of ATP, with muscle pyruvate kinase has been studied by measuring the effects of CrATP on the kinetics of pyruvate enolization and on the longitudinal nuclear magnetic relaxation rate (1/T1) of the protons of water and the protons and carbon atoms of pyruvate to investigate the existence and activity of bimetallic enzyme-M(II)-CrATP complexes and to determine intersubstrate distances on a kinase. The paramagnetic effect of CrATP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP yielded characteristic enhancements of 1/T1 for the binary enzyme-CrATP, ternary enzyme-Mg(II)-crATP, and quaternary enzyme-Mg(II)-crATP-pyruvate complexes of 3.5, 1.7, and 1.2 and dissociation constants of CrATP of 400, 200, and 200 muM, respectively. From the frequency dependence of 1/T1, the number of fast exchanging water protons in the coordination spheres of Cr(III) is approximately 6 in CrATP and in both the ternary enzyme-Mg(II)-CrATP complex and the quaternary enzyme-Mg(II)-CrATP-pyruvate complex. The paramagnetic effect of enzyme-bound Mn(II) on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn(II) complex with CrATP decreases the characteristic enhancement due to Mn(II) from 24 +/- 3 to 6 +/- 1. Titration of the ternary eznyme-Mn(II)-pyruvate complex with CrATP decreases the enhancement from 6 +/- 1 to 0.5 +/- 0.1. The affinity of the enzyme for Mn(II) is increased 2-fold upon binding of CrATP as indicated by decreases in the amplitude of the EPR spectrum of free Mn(II). The dissociation constants of CrATP from the enzyme-Mn(II)-CrATP complex, the enzyme-CrATP-pyruvate complex, and the enzyme-Mn(II)-CrATP-pyruvate complex are all 200 muM. The observed titration behavior, the characteristic enhancement values, the tightening by Mg(II) of the binding of CrATP to the enzyme, and the tightening of the binding of Mn(II) to the enzyme by CrATP establish the existence of enzyme-M(II)-CrATP and enzyme-M(II)-CrATP-pyruvate complexes containing two cations, Mg(II) or Mn(II) and Cr(III), at the active site.     

Magnetic resonance studies of Mn(II)-, Mn(III)-, and Fe(III)-conalbumin complexes.

Apoconalbumin binds Mn(II) at two sites with association constants of K1 = 7 (+/- 1) X 10(4) and K2 = 0.4 (+/- 0.25) X 10(4) M-1. The binding is tighter in the presence of excess bicarbonate resulting in K1 = 1.8 (+/- 0.2) X 10(5) and K2 = 3 (+/- 2) X 10(4) M-1. The electron paramagnetic resonance spectrum (at both 9 and 35 GHz) of Mn(II) bound at the tight site reveals a rhombic distortion (lambda = E/D approximately equal to 0.25-0.31) in the protein ligand environment of the mental ion. An evaluation of the 1/pT1p, paramagnetic contribution to the longitudinal relaxation rate of solvent protons with Mn(II)-, Mn(III)-, and Fe(III)-derivatives of conalbumin revealed that the mental ion in each site of conalbumin is accessible to one water molecule. For Mn(II)-conalbumin and Mn(III)-conalbumin species, inner coordination sphere protons are rapidly exchanging with the bulk solvent, while slow exchange conditions prevail for Fe(III)-conalbumin.     

Conformational changes of S-1 related to its dissociation from actin.

The peptide pattern obtained after proteolysis of S-1 with trypsin was different in the absence or presence of anions. The affinity of tryptic and undigested S-1 for anions (CN-, SCN- or HCO3-) was different, as reflected by the altered values of Ki or Ka obtained from ATPase activity measurements. Anions CN-, SCN-, HCO3-, or PPi induced dissociation of actomyosin when added to acto-S-1 or acto-heavy-meromyosin. Among nucleoside di- and triphosphates, only triphosphates were effective with regard to the dissociation. The results suggest the existence of a regulatory site of cationic nature on S-1, which might be involved in the dissociation of actin from myosin.     

Manganese (II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli w). II. Electron paramagnetic resonance and nuclear magnetic resonance studies of enzyme-bound manganese(II) with substrates and a potential transition-state analogue, methionine sulfoximine.

The enhancement of the longitudinal proton relaxation rate of solvent water protons which occurs when Mn(II) is bound to the "tight" metal ion site of unadenylylated glutamine synthetase (GS) was used to determine the binding constant of L-methionine (SR)-sulfoximine to GS-Mn(II) complexes. The binary enhancement for GS-Mn(II) is 22 at 24 MHz, 25 degrees C. The enhancement is lowered in the presence of the sulfoximine and the computed dissociation constant is 30 muM with epsilont, the enhancement for the ternary complex, equal to 3.0. Titration curves for the sulfoximine were also obtained in the presence of Mg-ADP, Mg-ADP plus Pi, and Mg-ATP. The dissociation constants were 9, 5, and 0.8 muM, respectively. The progressive tightening of the dissociation constants is symptomatic of conformational changes at the active site as the total subsite occupied by ATP is filled. The number of rapidly exchanging water molecules drops from 2 to approximately 0.1 when saturating concentrations of L-methionine (SR)-sulfoximine and nucleotide are present. The kinetically determined KI value of approximately 4 muM for the sulfoximine is about three orders of magnitude tighter than thee Km' value of approximately 3 mM for L-glutamate. The previously mentioned dissociation constants obtained by enhancement titrations are also orders of magnitude tighter than Km'. These data suggest that L-methionine (SR)-sulfoximine is a "transition-state" analogue for the glutamine synthetase reaction. ...     

Kinetic evidence supports the existence of two halide binding sites that have a distinct impact on the heme iron microenvironment in myeloperoxidase

Myeloperoxidase (MPO) structural analysis has suggested that halides and pseudohalides bind to the distal binding site and serve as substrates or inhibitors, while others have concluded that there are two separate sites. Here, evidence for two distinct binding sites for halides comes from the bell-shaped effects observed when the second-order rate constant of nitric oxide (NO) binding to MPO was plotted versus Cl- concentration. The chloride level used in the X-ray structure that produced Cl- binding to the amino terminus of the helix halide binding site was insufficient to populate either of the two sites that appear to be responsible for the two phases. Biphasic effects were also observed when the I-, Br-, and SCN- concentrations were plotted against the NO combination rate constants. Interestingly, the trough concentrations obtained from the bell-shaped curves are comparable to normal plasma levels of halides and pseudohalides, suggesting the potential relevance of these molecules in modulating MPO function. The second-order rate constant of NO binding in the presence of plasma levels of I-, Br-, and SCN- is 1-2-fold lower compared to that obtained in the absence of these molecules and remains unaltered through the Cl- plasma level. When Cl- exceeded the plasma level, the NO combination rate becomes indistinguishable from the second phase of the bell-shaped curve that was obtained in the absence of halides. Our results are consistent with two halide binding sites that could be populated by two halides in which both display distinct effects on the MPO heme iron microenvironment.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K_D = 25 ± 4 μM and K′_D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ?_b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ?_b = 3.5 ± 0.4 for tight binding sites and an average ?_b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s^?1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.     

Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli.

The interaction of Mn2+, substrates and initiators with RNA polymerase have been studied by kinetic and magnetic resonance methods. As determined by electron paramagnetic resonance, Mn2+ binds to RNA polymerase at one tight binding site with a dissociation constant less than 10 muM and at 6 +/- 1 weak binding sites with dissociation constants 100-fold greater. The binding of Mn2+ to RNA polymerase at both types of sites causes an order of magnitude enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons, indicating the presence of residual water ligands on the enzyme-bound Mn2+. A kinetic analysis of the Mn2+-activated enzyme with poly(dT) as template indicates the substrate to be MnATP under steady-state conditions in the presence or absence of the initiator ApA. ATP and UTP interact with the tightly bound Mn2+ to form ternary complexes with approximately 50% greater enhancement factors. The dissociation constant of MnATP from the tight Mn2+ site as determined by longitudinal proton relaxation rate (PRR) titration (4.7 muM) is similar to the KM of MnATP in the ApA-initiated RNA polymerase reaction (10 +/- 3 muM) but not in the ATP-initiated reaction (160 +/- 30 muM). Similarly, the dissociation constant of the substrate MnUTP from the tight Mn2+ site (90 muM) is in agreement with the KM of MnUTP (101 +/- 13 muM) when poly[d(A-T)]-poly[d(A-T)] is used as template, indicating the tight Mn2+ site to be the catalytic site for RNA chain elongation. Manganese adenylyl imidodiphosphate (MnAMP-PNP) has been found to be a substrate for RNA polymerase. It has the same affinity as MnATP for the tight site but, unlike the results obtained with MnATP, the enhancement is decreased by 43% in the enzyme Mn-AMP-PNP complex. These results suggest that the enzyme-bound Mn2+ interacts with the leaving pyrophosphate group. The initiators ApA and ApU and the inhibitor rifamycin interact with the enzyme-Mn2+ complex producing small (15-20%) decreases in the enhancement. The dissociation constant of ApA estimated from PRR data (less than or equal to 1.5 muM) agrees with that determined kinetically (1.0 +/- 0.5 muM) as the concentration of ApA required to produce half-maximal change in the KM of MnATP. In the presence of the initiation specific reagents ApA, ApU, or rifamycin, the affinity of the enzyme-Mn complex for ATP or UTP shows little change. However, ATP and UTP no longer increase the enhancement factor of the tightly bound Mn2+ but decrease it by 30-55%, indicating a change in the environment of the Mn2+-substrate complex on the enzyme when the initiation site is either occupied or blocked. Although the role of the six weak Mn2+ binding sites is not clear, the presence of a single tightly bound Mn2+ at the catalytic site for chain elongation which interacts with the substrate reinforces the number of active sites as one per molecule of holoenzyme and provides a paramagnetic reference point for further structural studies.     

The inhibition by anion binding of reactions of inorganic radical anions with bovine carbonic anhydrase B

Reactions of the inorganic radical anions, Br(2) and (SCN)2, with bovine carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) have been studied by pulse radiolysis. Reaction is almost completely inhibited by the binding of Br-, SCN- and ClO4- to an electrophilic site at the active centre of the enzyme. Dissociation constants for anion binding calculated from the reduction in free radical reactivity agree well with inhibition constants for these anions. The anions OCN- and CN-, although potent inhibitors of carbonic anhydrase activity, have relatively little effect on the reactivity of radical anions with the enzyme. Reaction of radical anions occurs mainly with tryptophan and tyrosine residues in the hydrophobic core of the enzyme, through a channel at the active site. This channel is closed by the anions in accord with their position in the lyotropic series.     

Interactions of iron bleomycin, phosphate or cyanide, and DNA: sequence-dependent conformations and reactions

The hypothesis was investigated that axial ligands bound to Fe(III)-bleomycin [Fe(III)Blm] are destabilized at specific 5'-guanine-pyrimidine-3' binding sites but are stable at nonselective dinucleotides. DNA oligomers and calf-thymus DNA were used in reactions with L-Fe(III)Blm, where phosphate and cyanide served as examples of large and small ligands (L). Both ligands underwent dissociation when L-Fe(III)Blm was bound to d(GGAAGCTTCC)2 (I) but not d(GGAAATTTCCC)2 (II) and at large ratios of calf-thymus DNA to drug. Fe(III)Blm is high spin in 20 mM phosphate buffer, signifying the presence of a phosphate adduct. In the titration of HPO4-Fe(III)Blm with calf-thymus DNA, a large excess of DNA was needed to reach the low-spin state, consistent with an equilibrium competition between phosphate and DNA for Fe(III)Blm. Equilibrium constants for binding Fe(III)Blm and CN-Fe(III)Blm to calf-thymus DNA (6.8x10(5) M(-1) and 5.9x10(4) M(-1), respectively, in HEPES buffer at 25 degrees C and pH 7.4) showed that the CN- ligand also reduced the affinity of DNA for the drug. The kinetics of dissociation of CN- from CN-Fe(III)Blm-DNA were slow and first order in bound drug. The reversible nature of these dissociation reactions was shown using 1H NMR spectroscopy of Fe(III)Blm-I in the absence and presence of large excesses of CN- or phosphate. The results are discussed in terms of a two-state hypothesis for the binding of L-Fe(III)Blm to specific and nonspecific dinucleotides. It is proposed that steric restrictions at specific sites inhibit binding of these ligands.     

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S(0) and S(2) states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D(2)O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S(0) and S(2) state multiline disappeared after 3 h of D(2)O incubation in the S(0) and S(1) states, respectively. The signal with 4.0 MHz hyperfine constants in S(0) state multiline disappeared after 3 h of D(2)O incubation in the S(0) state, while the similar signal in S(2) state multiline disappeared only after 24 h of D(2)O incubation in the S(1) state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 A, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.     

A proton-relaxation enhancement study of the interaction of manganous ions with phospholipids in aqueous dispersions.

An interaction of dipalmitoylphosphatidylcholine (PC) and phosphatidylserine (PS) with manganous ions has been investigated by measuring the effect of bound manganese upon the longitudinal relaxation rate, 1/T1, of the solvent water protons and evaluating the enhancement factor epsilon b. The observed enhancement values were used to determine the number of interacting sites per polar head group, n, and the values of association constants, KA, of manganese to PC and PS. Changes in epsilon b correlate with structural changes at the interacting site. By increasing the temperature one can see an abrupt decrease in epsilon b within the temperature interval from 40 to 50 degrees C indicating the thermal phase transition of PC as established by calorimetry, fluorescence and high-resolution NMR measurements. That an enhancement of 1/T1 of the solvent-water protons occurs at all is explained by assuming a restricted rotation of the Mn2+-aquo complex in the bound state. In addition we suppose that the rotation of the Mn2+-aquo complex is the mechanism which dominates the relaxation of the water protons in teh bulk solvent when phospholipids are present.     

Magnetic resonance studies of the manganese guanosine di- and triphosphate complexes with elongation factor Tu.

Analysis of titration data of EF-Tu-GDP with Mn(II) where free and bound Mn(II) were determined by proton relaxation rate of water (PRR) yields one tight Mn(II) binding site and a value of 2 muM for the dissociation constant of Mn(II) from the EF-Tu-MnGDP complex, K'A. The dissociation constant of manganese nucleotide from the ternary EF-Tu-MnGDP complex, K2, 0.2 muM, was derived from the known value of Ks, the dissociation constant for the binary EF-Tu-GDP complex, and the titration data of the ternary complex with excess GDP as titrant. The apparent number, n, of rapidly exchanging water ligands coordinated to bound Mn(II) in the ternary complex EF-Tu-MnGDP is estimated from the frequency dependence of the PRR of the complex to be approximately 1. The value of n and the values of PRR enhancements, epsilont = 4.3 for EF-Tu-MnGDP at 21 degrees, 24.3 MHZ and epsilont = 4.1 for the ternary GTP complex, are unusually low for protein-Mn-nucleotide complexes. The antibiotic X5108 which induces GTPase activity in EF-Tu-MgGTP was shown to bind stoichiometrically to EF-Tu-MnGDP and thereby change the PRR enhancement of the complex from 4.3 to 7.4. The characteristic broad lines in the EPR spectra of Mn(II) nucleotides are strikingly narrowed upon binding of Mn(II) nucleotides to EF-Tu. The long electron spin relaxation times inferred from the EPR spectra indicate a limited access of solvent water to the first coordination sphere of Mn(II) in its EF-Tu-nucleotide complexes. The frequency dependence of the PRR indicates that the electron spin relaxation time, T1e, is the dominant process modulating the Mn(II)-H2O interaction of the EF-Tu-MnGDP complex and consequently determines the correlation time. The value of T1e, estimated from the PRR experiments to be 2.5 ns at 21 degrees, is consistent with the lower limit of T1e obtained from the line widths of the EPR spectrum of the complex. Upon binding of a stoichiometric quantity of the antibiotic X5108, the EPR spectrum of EF-Tu-MnGDP is severely broadened indicating greater access of solvent water to the manganese coordination sphere, i.e. an opening of the nucleotide binding site as already suggested by the increased PRR enhancement.     

Binding of salicylhydroxamic acid and several aromatic donor molecules to Arthromyces ramosus peroxidase, investigated by X-ray crystallography, optical difference spectroscopy, NMR relaxation, molecular dynamics, and kinetics.

The X-ray crystal structure of the complex of salicylhydroxamic acid (SHA) with Arthromyces ramosus peroxidase (ARP) has been determined at 1.9 A resolution. The position of SHA in the active site of ARP is similar to that of the complex of benzhydroxamic acid (BHA) with ARP [Itakura, H., et al. (1997) FEBS Lett. 412, 107-110]. The aromatic ring of SHA binds to a hydrophobic region at the opening of the distal pocket, and the hydroxamic acid moiety forms hydrogen bonds with the His56, Arg52, and Pro154 residues but is not asscoiated with the heme iron. X-ray analyses of ARP-resorcinol and ARP-p-cresol complexes failed to identify the aromatic donor molecules, most likely due to the very low affinities of these aromatic donors for ARP. Therefore, we examined the locations of these and other aromatic donors on ARP by the molecular dynamics method and found that the benzene rings are trapped similarly by hydrophobic interactions with the Ala92, Pro156, Leu192, and Phe230 residues at the entrance of the heme pocket, but the dihedral angles between the benzene rings and the heme plane vary from donor to donor. The distances between the heme iron and protons of SHA and resorcinol are similar to those obtained by NMR relaxation. Although SHA and BHA are usually considered potent inhibitors for peroxidase, they were found to reduce compound I and compound II of ARP and horseradish peroxidase C in the same manner as p-cresol and resorcinol. The aforementioned spatial relationships of these aromatic donors to the heme iron in ARP are discussed with respect to the quantum chemical mechanism of electron transfer in peroxidase reactions.