1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.     

Evidence for N coordination to Fe in the [2Fe-2S] clusters of Thermus Rieske protein and phthalate dioxygenase from Pseudomonas

Rieske-type iron/sulfur proteins and several NADH-dependent oxygenases contain Fe/S clusters with similar spectral and magnetic properties. Purified Rieske iron/sulfur protein from Thermus thermophilus contains two apparently identical [2Fe-2S] clusters in a polypeptide having only four cysteine residues, and it has been proposed that each Fe/S cluster is coordinated to two cysteine S-atoms and to an unknown number of other non-sulfur atoms (Fee, J. A., Findling, K. L., Yoshida, T., Hille, R., Tarr, G. E., Hearshen, D. O., Dunham, W. R., Day, E. P., Kent, T. A., and Munck, E. (1984) J. Biol. Chem. 259, 124-133). We have examined the Rieske protein from Thermus and the phthalate dioxygenase from Pseudomonas cepacia with electron nuclear double resonance (ENDOR) and pulsed EPR methods and report here evidence for the direct coordination of nitrogenous ligands to the Fe/S clusters in these proteins. The electron nuclear double resonance signals arising from 14N have been interpreted in terms of a strongly coupled ligand with AN = approximately 26-28 MHz and a weakly coupled ligand with AN = approximately 9 MHz. The pulsed EPR spectrum shows a rich pattern of lines in the Fourier transformed data having peaks in the range of 0.8 to 6.7 MHz. The lower frequency resonances are tentatively associated with coupling of the unpaired spin to the remote N-atoms of coordinated imidazole rings.     

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii

beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I,while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s(-1) for the wild-type enzyme in the reaction with pentane-2,4-dione.The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.     

Iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26.1: characterization and reconstitution with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+

Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.1 were depleted of Fe by a simple procedure involving reversible dissociation of the H subunit. The resulting intact Fe-depleted RCs contained 0.1-0.2 Fe per RC as determined from atomic absorption and electron paramagnetic resonance (EPR) spectroscopy. Fe-depleted RCs that have no metal ion occupying the Fe site differed from native RCs in the following respects: (1) the rate of electron transfer from QA- to QB exhibited nonexponential kinetics with the majority of RCs having a rate constant slower by only a factor of approximately 2, (2) the efficiency of light-induced charge separation (DQA----D+QA-) produced by a saturating flash decreased to 63%, and (3) QA appeared readily reducible to QA2-. Various divalent metal ions were subsequently incorporated into the Fe site. The electron transfer characteristics of Fe-depleted RCs reconstituted with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+ were essentially the same as those of native RCs. These results demonstrate that neither Fe2+ nor any divalent metal ion is required for rapid electron transfer from QA- to QB. However, the presence of a metal ion in the Fe site is necessary to establish the characteristic, native, electron-transfer properties of QA. The lack of a dominant role of Fe2+ or other divalent metals in the observed rate of electron transfer from QA- to QB suggests that a rate-limiting step (for example, a protonation event or a light-induced structural change) precedes electron transfer.     

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.     

Spectrophotometric and ESR evidence for vanadium(IV) deferoxamine complexes.

Complexes of vanadium(IV), vanadyl, are reported to be formed with the trihydroxamic acid deferoxamine (H3DF+). One complex exhibits a reddish-violet color, with a major absorbance peak at 386 nm and a smaller peak at 520 nm. This complex is potentially useful for the microdetermination of vanadyl. The apparent molar absorptivity is 3.91 mM-1 cm-1, and the complex obeys Beer's law in the concentration range of 0.6-63 ppm. Electron spin resonance studies indicate the formation of two vanadyl complexes that are 1:1 in vanadyl and deferoxamine, but have two or three bound hydroxamate groups. ESR and spectrophotometric evidence indicate that the red, low pH form, involves an octahedral vanadium (4+) ion coordinated by three hydroxamate ligands. One of these hydroxamates is displaced by an oxygen at pH greater than 2.8 according to the following equilibria: VO2+ + H3DF+ in equilibrium with VIV(DF)2+ + H3O+, VIV(DF)2+ + H2O in equilibrium with VO(HDF)+ + H+, where pk2 = 2.8.     

Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme

The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.     

Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin

Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react with an unknown system component. Evidence is presented that identifies this component as amine buffers, including 3-N-morpholinopropanesulfonic acid (MOPS), which is widely used in ferritin studies. In the presence of non-amine buffers, the Fe2+ to O2 stoichiometry was approximately 4.0, but at high concentrations of amine buffers (0.10 M) the Fe2+ to O2 stoichiometry is approximately 2.5 for iron loadings of eight to 30 Fe2+ per HoSF. Decreasing the concentration of amine buffer to zero resulted in an Fe2+ to O2 stoichiometry of approximately 4. Direct evidence for amine buffer modification during Fe2+ deposition was obtained by comparing authentic and modified buffers using mass spectrometry, NMR, and thin layer chromatography. Tris(hydroxymethyl)aminomethane, MOPS, and N-methylmorpholine (a MOPS analog) were all rapidly chemically modified during Fe2+ deposition to form N-oxides. Under identical conditions no modification was detected when amine buffer, H2O2, and O2 were combined with Fe2+ or ferritin separately. Thus, a short-lived ferritin intermediate is required for buffer modification by H2O2. Variation of the Fe2+ to O2 stoichiometry versus the Fe2+ to HoSF ratio and the amine buffer concentration are consistent with buffer modification.     

Evaluation of intramolecular equilibria in complexes formed between substituted imidazole ligands and nickel (II), copper (II) or zinc (II)

The metal ion-binding properties of imidazole-4-acetate (ImA-), 4(5)-aminoimidazole-5(4)-carboxamide (AImC), 2,2'biimidazole(BiIm) (I. T?r?k et al., J. Inorg. Biochem. 71 (1998) 7-14), and bis (imidazol-2-yl)methane(BiImM) (K. Várnagy et al., J. Chem. Soc., Dalton Trans. (1994) 2939-2945) have been evaluated by using the recently published stability constants and by applying the recently established log K(ML)M versus pK(HL)H straight-line plots (L. E. Kapinos et al., Inorg. Chim. Acta 280 (1998) 50-56) which hold for simple imidazole-type ligands. The indicated analysis regarding the intramolecular equilibrium between a monodentatally imidazole-nitrogen-coordinated (open) species and a chelated isomer provides helpful insights, e.g., the formation degree of chelates is more favored if six-membered rings can be formed, as in the case with M(BiImM)2+ compared to M(BiIm)2+, though in both instances the formation degree of the chelates is large. The formation degree of chelates in the M(ImA)+ complexes increases in the series Zn(ImA)+ (87%)相似文献   

Sequential resonance assignments of oxidized high-potential iron-sulfur protein from Chromatium vinosum.

2D NMR spectra of the high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum have been used to obtain partial resonance assignments for the oxidized paramagnetic redox state of the protein. Sequence-specific assignments were made using NOESY and COSY spectra in H2O and D2O of the following backbone segments: Asn-5-Arg-33, Glu-39-Asp-45, Gly-55-Cys-63, Gly-68-Ala-78, and Leu-82-Gly-85. NOESY spectra with a spectral width wide enough to include the hyperfine-shifted resonances revealed numerous NOE contacts between these signals and those in the main envelope of the proton spectrum. With the aid of the X-ray crystal structure [Carter, C.W., Kraut, J., Freer, S. T., Xuong, N. H., Alden, R. A., & Bartsch, R. G. (1974) J. Biol. Chem. 249, 4212], these NOEs permitted seven of the nine hyperfine-shifted signals to be assigned to three of the cysteine residues liganded to the metal cluster (Cys-43, Cys-46, and Cys-77). The other two hyperfine-shifted signals produced no detectable NOEs to other resonances in the spectrum and were tentatively assigned to the remaining cysteinyl ligand (Cys-63). These assignments, in conjunction with recent theoretical models of the electronic structure of the Fe4S4 cluster [Noodleman, L. (1988) Inorg. Chem. 27, 3677; Bertini, I., Briganti, F., Luchinat, C., Scozzafava, A., & Sola, M. (1991) J. Am. Chem. Soc. 113, 1237], indicate that the iron atoms coordinated to Cys-63 and Cys-77 are those of the mixed-valence Fe(3+)-Fe2+ pair whereas Cys-43 and Cys-46 are ligands to the Fe(3+)-Fe3+ metal pair.     

Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.     

NMR studies of the active site of isopenicillin N synthase, a non-heme iron(II) enzyme.

The active site structure of isopenicillin N synthase (IPNS) has been previously studied by the use of M?ssbauer, EPR, electronic absorption, and NMR spectroscopies [Chen, V.J., Frolik, C.A., Orville, A.M., Harpel, M.R., Lipscomb, J.D., Surerus, K.K., & Münck, E. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C.A., & Chen, V.J. (1990) Inorg. Chem. 26, 1111-1112]. These studies have revealed three coordinated His residues along with three sites for substrate [delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine, ACV], NO, and water binding on the active Fe(II) of IPNS. We report here NMR studies of Fe(II)IPNS and its Co(II)-substituted derivative [Co(II)IPNS]. By the use of NOE techniques on the Co(II)IPNS-ACV complex, we have recognized a -CH2-CH less than spin system at 14.6, 24.3, and 38.6 ppm that is assigned to the alpha and beta protons of a coordinated Asp residue. Corresponding solvent nonexchangeable features are found near 40 ppm in Fe(II)IPNS and the Fe(II)IPNS-ACV complex, but the peaks are too broad for NOE effects to be observed. The binding of NO to the Fe(II) center results in a significant change in the configuration of the metal site: (a) The C beta H2 resonances due to the coordinated Asp residue disappear. The loss of the signal may indicate a change of the carboxylate configuration from syn-like to anti-like or, less likely, its displacement by NO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Critical role of specific ions for ligand-induced changes regulating pyruvate dehydrogenase kinase isoform 2

In the complete absence of K+ and phosphate (Pi), pyruvate dehydrogenase kinase isoform 2 (PDHK2) was catalytically very active but with an elevated Km for ATP, and this activity is insensitive to effector regulation. We find that K+ or 5-fold lower levels of NH4+ markedly enhanced quenching of Trp383 fluorescence of PDHK2 by ADP and ATP. K+ binding caused an approximately 40-fold decrease in the equilibrium dissociation constants (Kd) for ATP from approximately 120 to 3.0 microM and an approximately 25-fold decrease in Kd for ADP from approximately 950 to 38 microM. Linked reductions in Kd of PDHK2 for K+ were from approximately 30 to approximately 0.75 mM with ATP bound and from approximately 40 to approximately 1.7 mM with ADP bound. Without K+, there was little effect of ADP on pyruvate binding, but with 100 mM K+ and 100 microM ADP, the L0.5 of PDHK2 for pyruvate was reduced by approximately 14 fold. In the absence of K+, Pi had small effects on ligand binding. With 100 mM K+, 20 mM Pi modestly enhanced binding of ADP and hindered pyruvate binding but markedly enhanced the binding of pyruvate with ADP; the L0.5 for pyruvate was specifically decreased approximately 125-fold with 100 microM ADP. Pi effects were minimal when NH4+ replaced K+. We have quantified coupled binding of K+ with ATP and ADP and elucidated how linked K+ and Pi binding are required for the potent inhibition of PDHK2 by ADP and pyruvate.     

Wheat germ phosphoglycerate mutase: evidence for a metalloenzyme

Wheat germ phosphoglycerate mutase, exposed to 3.4 M guanidinium chloride at 22 degrees C and pH 7.8, slowly undergoes time-dependent inactivation which can be fully reversed by adding excess Co2+ or Mn2+ to a 50-fold dilution of the denaturing medium. Titration of the denatured enzyme with either Co2+ or Mn2+ shows that wheat germ mutase preferentially binds Co2+. Assuming 1:1 complexation between metal atom and protein, the apparent dissociation constants (Kd) for E Co2+ and E Mn2+ at 22 degrees C and pH 8.7 are approximately 1.06 and 1.84, respectively. Other metal atoms (e.g., Cr2+, Cu2+, Fe2+, Fe3+, Mg2+, and Ni2+) have no effect in restoring the apoenzyme's catalytic activity. At low concentrations (0.11-0.23 mM) Zn2+ partially restores activity, but promotes protein precipitation at elevated concentrations. Evidence suggests that all bisphosphoglycerate-independent phosphoglycerate mutases require either an intra- or an extramolecular metal atom in order to function. Attempts to characterize wheat germ mutase as a glycoprotein have yielded negative results.     

X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for Fe-S interaction in the enzyme-substrate complex.

Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]Ethylpropylamiloride, a ligand to analyze the properties of the Na+/H+ exchange system in the membranes of normal and hypertrophied kidneys

[3H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system (Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E. J., and Lazdunski, M. (1984) EMBO J. 3, 2647-2651). This paper extends the analysis of the properties of interaction of [3H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [3H]Ethylpropylamiloride-binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [3H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [3H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers.     

3H]bumetanide binding to duck red cells. Correlation with inhibition of (Na + K + 2Cl) co-transport

Bumetanide is a potent inhibitor of cation-chloride co-transport systems in many cell types, including duck red cells. We studied equilibrium binding of [3H]bumetanide to intact duck red cells under a number of conditions known to affect (Na + K + 2Cl) co-transport in these cells. Saturable [3H]bumetanide binding to duck red cells is markedly stimulated by addition of norepinephrine or cell shrinkage, conditions which similarly stimulate co-transport. In the presence of norepinephrine and saturating concentrations of extracellular sodium, potassium, and chloride for the co-transporter, we found approximately 1000 [3H]bumetanide-binding sites/red cell, and measurement of 24Na+ influx on the same cells yielded a turnover number of approximately 4000/s for the co-transporter. 24Na+ influx was negatively correlated with the amount of bound [3H]bumetanide, and both saturable binding and inhibition of influx were half-maximal at approximately 10(-7) M [3H]bumetanide. Binding of [3H]bumetanide to duck red cells is stimulated in a saturable manner by increasing extracellular sodium and potassium. Chloride has a biphasic effect on [3H]bumetanide binding; increasing [Cl-]o (by replacement of methylsulfate) from 0 to 32 mM markedly enhances binding, whereas further increasing [Cl-]o to 160 mM inhibits binding. This behavior is similar to that reported for bumetanide inhibition of duck red cell (Na + K + 2Cl) co-transport (Haas, M., and McManus, T. J. (1983) Am. J. Physiol. 245, C235-C240; Haas, M., and McManus, T. J. (1982) Biophys. J. 37, 214a) and [3H]bumetanide binding to membranes from dog kidney outer medulla (Forbush, B. III, and Palfrey, H. C. (1983) J. Biol. Chem. 258, 11787-11792).     

Biochemical characterization of the structural Zn2+ site in the Bacillus subtilis peroxide sensor PerR

In Bacillus subtilis most peroxide-inducible oxidative stress genes are regulated by a metal-dependent repressor, PerR. PerR is a dimeric, Zn2+-containing metalloprotein with a regulatory metal-binding site that binds Fe2+ (PerR:Zn,Fe) or Mn2+ (PerR: Zn,Mn). Reaction of PerR:Zn,Fe with low levels of hydrogen peroxide (H2O2) leads to oxidation of two His residues thereby leading to derepression. When bound to Mn2+, the resulting PerR:Zn,Mn is much less sensitive to oxidative inactivation. Here we demonstrate that the structural Zn2+ is coordinated in a highly stable, intrasubunit Cys4:Zn2+ site. Oxidation of this Cys4:Zn2+ site by H2O2 leads to the formation of intrasubunit disulfide bonds. The rate of oxidation is too slow to account for induction of the peroxide stress response by micromolar levels of H2O2 but could contribute to induction under severe oxidative stress conditions. In vivo studies demonstrated that inactivation of PerR:Zn,Mn required 10 mM H2O2, a level at least 1000 times greater than that needed for inactivation of PerR:Zn,Fe. Surprisingly even under these severe oxidation conditions there was little if any detectable oxidation of cysteine residues in vivo: derepression was correlated with oxidation of the regulatory site. Because oxidation at this site required bound Fe2+ in vitro, we suggest that treatment of cells with 10 mM H2O2 released sufficient Fe2+ into the cytosol to effect a transition of PerR from the PerR:Zn,Mn form to the peroxide-sensitive PerR: Zn,Fe form. This model is supported by metal ion affinity measurements demonstrating that PerR bound Fe2+ with higher affinity than Mn2+.     

Gd3+ vibronic side band spectroscopy. New optical probe of Ca2+ binding sites applied to biological macromolecules.

A new spectroscopic technique is presented for obtaining infraredlike spectra of the binding sites of Ca2+ and other metals in biological macromolecules. The technique, based on the Ca(2+)-like binding properties of Gd3+, utilizes vibronic side bands (VSB) that appear in Gd3+ fluorescence. In the fluorescence spectrum of Gd3+, the separation in photon frequency between a VSB and its electronic origin at approximately 32,150 cm-1 (approximately 311 nm) is a direct measure of the vibrational frequency of a ligand coordinated to Gd3+ ion. As a consequence, the VSB are uncomplicated by molecular vibrations distant from the Gd3+ binding site. The vibrational spectra resulting from the VSB of Gd3+ coordinated to a Ca2+ binding protein, a phospholipid, and DNA are presented.