pH-induced conformational changes of the Fe(2+)-N epsilon (His F8) linkage in deoxyhemoglobin trout IV detected by the Raman active Fe(2+)-N epsilon (His F8) stretching mode.

To investigate heme-protein coupling via the Fe(2+)-N epsilon (His F8) linkage we have measured the profile of the Raman band due to the Fe(2+)-N epsilon (His F8) stretching mode (nu Fe-His) of deoxyHb-trout IV and deoxyHbA at various pH between 6.0 and 9.0. Our data establish that the band of this mode is composed of five different sublines. In deoxyHb-trout IV, three of these sublines were assigned to distinct conformations of the alpha-subunit (omega alpha 1 = 202 cm-1, omega alpha 2 = 211 cm-1, omega alpha 3 = 217 cm-1) and the other two to distinct conformations of the beta-subunit (omega beta 1 = 223 cm-1 and omega beta 2 = 228 cm-1). Human deoxyHbA exhibits two alpha-chain sublines at omega alpha 1 = 203 cm-1, omega alpha 2 = 212 cm-1 and two beta-chain sublines at omega beta 1 = 217 cm-1 and omega beta 2 = 225 cm-1. These results reveal that each subunit exists in different conformations. The intensities of the nu Fe-His sublines in deoxyHb-trout IV exhibit a significant pH dependence, whereas the intensities of the corresponding sublines in the deoxyHbA spectrum are independent on pH. This finding suggests that the structural basis of the Bohr effect is different in deoxyHbA and deoxyHb-trout IV. To analyse the pH dependence of the deoxyHb-trout IV sublines we have applied a titration model describing the intensity of each nu Fe-His subline as an incoherent superposition of the intensities from sub-sublines with the same frequency but differing intrinsic intensities due to the different protonation states of the respective subunit. The molar fractions of these protonation states are determined by the corresponding Bohr groups (i.e., pK alpha 1 = pK alpha 2 = 8.5, pK beta 1 = 7.5, pK beta 2 = 7.4) and pH. Hence, the intensities of these sublines reflect the pH dependence of the molar fractions of the involved protonation states. Fitting this model to the pH-dependent line intensities yields a good reproduction of the experimental data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermal fluctuations between conformational substates of the Fe(2+)-HisF8 linkage in deoxymyoglobin probed by the Raman active Fe-N epsilon (HisF8) stretching vibration.

We have measured the VFe-His Raman band of horse heart deoxymyoglobin dissolved in an aqueous solution as a function of temperature between 10 and 300 K. The minimal model to which these data can be fitted in a statistically significant and physically meaningful way comprises four different Lorentzian bands with frequencies at 197, 209, 218, and 226 cm-1, and a Gaussian band at 240 cm-1, which exhibit halfwidths between 10 and 12.5 cm-1. All these parameters were assumed to be independent of temperature. The temperature dependence of the apparent total band shape's frequency is attributed to an intensity redistribution of the subbands at omega 1 = 209 cm-1, omega 2 = 218 cm-1, and omega 3 = 226 cm-1, which are assigned to Fe-N epsilon (HisF8) stretching modes in different conformational substrates of the Fe-HisF8 linkage. They comprise different out-of-plane displacements of the heme iron. The two remaining bands at 197 and 240 cm-1 result from porphyrin modes. Their intensity ratio is nearly temperature independent. The intensity ratio I3/I2 of the vFe-His subbands exhibits a van't Hoff behavior between 150 and 300 K, bending over in a region between 150 and 80 K, and remains constant between 80 and 10 K, whereas I2/I1 shows a maximum at 170 K and approaches a constant value at 80 K. These data can be fitted by a modified van't Hoff expression, which accounts for the freezing into a non-equilibrium distribution of substates below a distinct temperature Tf and also for the linear temperature dependence of the specific heat of proteins. The latter leads to a temperature dependence of the entropic and enthalpic differences between conformational substates. The fits to the intensity ratios of the vFe-His subbands yield a freezing temperature of Tf = 117 K and a transition region of delta T = 55 K. In comparison we have utilized the above thermodynamic model to reanalyze earlier data on the temperature dependence of the ratio Ao/A1 of two subbands underlying the infrared absorption band of the CO stretching vibration in CO-ligated myoglobin (A. Ansari, J. Berendzen, D. Braunstein, B. R. Cowen, H. Frauenfelder, M. K. Kong, I. E. T. Iben, J. Johnson, P. Ormos, T. B. Sauke, R. Scholl, A. Schulte, P. J. Steinbach, R. D. Vittitow, and R. D. Young, 1987, Biophys. Chem. 26:237-335).(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR local range investigations in amorphous starchy substrates I. Structural heterogeneity probed by (13)C CP-MAS NMR

The (13)C CP-MAS (Cross Polarization and Magic Angle Spinning) NMR signatures of a series of amorphous and semi-crystalline samples prepared from various starchy substrates (native potato starch, amylopectin, amylose) following different techniques of preparation (casting, freeze drying, solvent exchange) are compared. Decompositions of the C1 resonance spectra reveal the existence of four or five main types of alpha(1-4) linkages, which can be quantified. The influence of the intrinsic primary structure (linear or branched) and of the preparation procedure on conformational changes and resulting crystallinity are interpreted in terms of distributions of average glycosidic linkages dihedral angles (Phi, Psi). The role of hydration is also considered. An improved understanding at different structural levels is obtained in relation to local and intermediate range orders. Such information may be useful for the understanding of the structural evolution of a large variety of starchy substrates before or after treatments widely used in industrial processes.     

Redox control of calcineurin by targeting the binuclear Fe(2+)-Zn(2+) center at the enzyme active site.

The interaction of protein serine/threonine phosphatase calcineurin (CaN) with superoxide and hydrogen peroxide was investigated. Superoxide specifically inhibited phosphatase activity of CaN toward RII (DLDVPIPGRFDRRVSVAAE) phosphopeptide in tissue and cell homogenates as well as the activity of the enzyme purified under reducing conditions. Hydrogen peroxide was an effective inhibitor of CaN at concentrations several orders of magnitude higher than superoxide. Inhibition by superoxide was calcium/calmodulin-dependent. Nitric oxide (NO) antagonized superoxide action on CaN. We provide kinetic and spectroscopic evidence that native, catalytically active CaN has a Fe(2+)-Zn(2+) binuclear center in its active site that is oxidized to Fe(3+)-Zn(2+) by superoxide and hydrogen peroxide. This oxidation is accompanied by a gain of manganese dependence of enzyme activity. CaN isolated by a conventional purification procedure was found in the oxidized, ferric enzyme form, and it became increasingly dependent on divalent cations. These results point to a complex redox regulation of CaN phosphatase activity by superoxide, which is modified by calcium, NO, and superoxide dismutase.     

Inhibition of the erythrocyte (Ca2+ + Mg2+)-ATPase by nonheme iron.

The erythrocyte calmodulin-stimulated (Ca2+ + Mg2+)-ATPase (CaM-ATPase), an integral membrane protein, is inhibited in different types of congenital hemolytic anemias for which oxidative processes appear as a common feature. The oxidation of hemoglobin and its degradation lead to the accumulation of ferric heme (hemin) and nonheme iron in the red cell. We have shown previously that hemin inhibits the activity of the enzyme of normal erythrocyte (Leclerc et al. (1988) Biochim. Biophys. Acta, 946, 49-56) involving an oxidation of thiol groups. The present study demonstrates that nonheme iron also inhibits the CaM-ATPase activity. In contrast with hemin, the inhibition of the enzyme induced by the nonheme treatment is prevented by butylated hydroxytoluene, a protecting agent of unsaturated phospholipid peroxidations, while dithiothreitol, a reducing agent of protein disulfide bridges, does not restore the activity of the enzyme. We conclude that nonheme iron inhibits the enzyme at least in part, through the peroxidation of phospholipids of the membrane bilayer.     

Changes in the gamma chain heterogeneity of hemoglobin F of the baboon (Papio cynocephalus) postnatally and after partial switching to hemoglobin F production by various stimuli

The postnatal switch from hemoglobin (Hb) F to Hb A in the baboon (Papio cynocephalus) occurs somewhat more rapidly than in humans. Minor components which are related to Hb F and Hb A are also present and show reciprocal rise and fall. The baboon produces two types of gamma chain presumably from nonallelic genes. These have either an isoleucyl (I gamma) or a valyl (V gamma) residue in position 75. As in the human case with G gamma and A gamma chains, the ratio I gamma to V gamma chains changes during the postnatal switch. Production of Hb F in the baboon may be stimulated by phenylhydrazine or more effectively by 5-azacytidine. With phenylhydrazine, the ratio of I gamma to V gamma chains in the Hb F is the same as in the traces of Hb F in the juvenile or adult baboon. However, with 5-azacytidine, at least some of the Hb F that is produced probably has been synthesized with an I gamma to V gamma ratio that is present prenatally and in the newborn baboon.     

Properties of hemoglobin A and hemoglobin Zurich (beta63 histidine replaced by arginine): quantitative evaluation of functional abnormalities in hemoglobins.

The abnormal hemoglobin Zurich (β₆₃ his→arg) exhibits abnormal properties. Thus, ν_CO occurs at 1951 cm^?1 for HbACO while HbZCO shows bands at 1950 cm^?1 and 1958 cm^?1 for CO bound in α and β chains respectively (the βCOs are displaced less readily by O₂). Acid catalyzed reductive displacement of superoxide by azide is slower on the β chain of HbZO₂ than on the α chain under conditions where with HbAO₂ both chains appear equally reactive. The one electron donor hydroquinone produces metHb and peroxide more rapidly from HbZO₂ than from HbAO₂. These property differences can be related to the β₆₃ residue. Such studies provide generally useful probes of the structural basis for hemoglobin diseases.     

The Fe(2+)-His(F8) Raman band shape of deoxymyoglobin reveals taxonomic conformational substates of the proximal linkage

The band shape of the Raman line attributed to the Fe(2+)-N(epsilon)(His(F8)) stretching mode in deoxymyoglobin contains significant information on the nature of the Fe-His proximal linkage. Raman lines appearing close to it, however, obscure the true line profile. To isolate this from its accompanying lines we use its isotopic shift of approximately 1 cm(-1) when (56)Fe in natural-abundance deoxymyoglobin is substituted by (54)Fe. This enables us to isolate the true line shape. We have measured this line shape in sperm whale myoglobin dissolved in a 66% vol/vol glycerol/water solution for nine temperatures from 10 K to 300 K. The nu(Fe-His) band shows a complex temperature-dependent profile, with a shoulder on its high-frequency wing, which becomes more prominent with increasing temperature. Detailed analysis reveals that the band is composed of five distinct lines attributable to taxonomic conformational substates of the nu(Fe-His) linkage. These are in thermodynamic equilibrium above the glass transition temperature T(f) but freeze in into the thermodynamic distribution at T(f) for lower temperatures. Alternative models that try to explain the nu(Fe-His) band shape by either an anharmonic coupling of the nu(Fe-His) to a low-frequency heme doming mode or by conformational substates related to a Gaussian distribution of iron out-of-plane displacements are at variance with the distinct features observed experimentally.     

Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage.

Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I.     

Fe(II) oxidation and Fe(III) incorporation by the M(r) 66,000 microsomal iron protein that stimulates NADPH oxidation.

In a previous study (Minotti, G., and Ikeda-Saito, M. (1991) J. Biol. Chem. 266, 20011-20017) we demonstrated the existence of a M(r) 66,000 microsomal iron protein (MIP) which stimulates NADPH oxidation by shunting electrons from NADPH-cytochrome P-450 reducase to its bound Fe(III). In the present study, purified MIP was depleted of iron and the apoMIP was examined for its ability to incorporate Fe(III) upon an incubation with Fe(II). It was found that apoMIP had an oxygen-dependent ferroxidase activity coupled with the incorporation of Fe(III). The reconstituted MIP exhibited a Fe(III) content and an NADPH oxidation activity similar to those of native MIP. However, the reconstitution of MIP from apoMIP and Fe(II) had to be performed in the presence of detergents to prevent the formation of protein aggregates and the oxidative incorporation of an iron which could not react with NADPH-cytochrome P-450 reductase. This redox inactive iron was probably bound nonspecifically to artifactual sites formed by the protein aggregates.     

The high-resolution X-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe(3+) and Zn(2+) derivatives

The high-resolution structure of the non-haem ferritin from Escherichia coli (EcFtnA) is presented together with those of its Fe(3+) and Zn(2+) derivatives, this being the first high-resolution X-ray analysis of the iron centres in any ferritin.The binding of both metals is accompanied by small changes in the amino acid ligand positions. Mean Fe(A)(3+)-Fe(B)(3+) and Zn(A)(2+)-Zn(B)(2+) distances are 3.24 A and 3.43 A, respectively. In both derivatives, metal ions at sites A and B are bridged by a glutamate side-chain (Glu50) in a syn-syn conformation. The Fe(3+) derivative alone shows a third metal site (Fe( C)( 3+)) joined to Fe(B)(3+) by a long anti-anti bidentate bridge through Glu130 (mean Fe(B)(3+)-Fe(C)(3+) distance 5.79 A). The third metal site is unique to the non-haem bacterial ferritins.The dinuclear site lies at the inner end of a hydrophobic channel connecting it to the outside surface of the protein shell, which may provide access for dioxygen and possibly for metal ions shielded by water. Models representing the possible binding mode of dioxygen to the dinuclear Fe(3+) pair suggest that a gauche micro-1,2 mode may be preferred stereochemically.Like those of other ferritins, the 24 subunits of EcFtnA are folded as four-helix bundles that assemble into hollow shells and both metals bind at dinuclear centres in the middle of the bundles. The structural similarity of EcFtnA to the human H chain ferritin (HuHF) is remarkable (r.m.s. deviation of main-chain atoms 0.66 A) given the low amino acid sequence identity (22 %). Many of the conserved residues are clustered at the dinuclear centre but there is very little conservation of residues making inter-subunit interactions.     

Observations on the (Ca2+ plus Mg2+)-ATPase activator found in various mammalian erythrocytes.

1. A soluble activator of membrane (Ca2+ plus Mg2)-ATPase is present in hemolysates of the newborn calf and cow, the new born and adult pig as well as human erythrocytes. 2. The activator is also found in reticulocytes of the adult pig. 3. The activator obtained from any of the above species is capable of stimulating the membrane (Ca2+ plus Mg2+)-ATPases of the other species, regardless of the age of the animals. 4. The results obtained from density fractionation of human erythrocytes revealed that the soluble factor has little simulatory effect on membranes of young erythrocytes from which it is derived but caused a marked stimulation on (Ca2+ plus Mg2+)-ATPase activity of the intermediate aged and old erythrocyte membranes. 5. The above observations support the following conclusions: (a) the extremely low levels of (Ca2+ plus Mg2+)-ATPase in cow erythrocytes is not due to the lack of a (Ca2+ plus Mg2+)-ATPase activator; (b) the distribution of (Ca2+ plus Mg2+)-atpase activator is not species specific and the differences in the level of membrane (Ca2+ plus Mg2+)-ATPase activity in various species of cells is an inherent property of that particular membrane (c) the (Ca2+ plus Mg2+)-ATPase activator is present at least from the time of reticulocyte formation and remain during tthe life span of the erythrocyte.     

Structural characterization of Ca(2+)-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.     

Structural transition from the random coil to quadruplex of AG(3)(T(2)AG(3))(3) induced by Zn(2+)

Structures of G-quadruplex DNAs can be typically stabilized by monovalent cations such as K(+), Na(+). Some divalent and trivalent cations, such as Sr(2+), Pb(2+), Tb(3+) and Eu(3+), can also induce the formation of G-quadruplex DNA. Here we show that Zn(2+) can induce the human telomeric sequence AG(3)(T(2)AG(3))(3) to fold the G-quadruplex structure by UV absorbance difference spectra and circular dichroism (CD) spectroscopy. At micromolar concentrations, the Zn(2+)-induced changes in the UV absorbance difference spectra and CD spectra are the characteristics of antiparallel G-quadruplexes although the long wavelength CD maximum is around 285 nm rather than the typical value of 295 nm. The binding stoichometry of Zn(2+) per one AG(3)(T(2)AG(3))(3) molecule is four. To our knowledge, the structural transition of human telomeric sequence induced by Zn(2+) was observed for the first time.     

The origin of the split S3 EPR signal in Ca(2+)-depleted photosystem II: histidine versus tyrosine.

The radical formed as the formal S3 charge storage state in Ca(2+)-depleted photosystem II and detected as a split EPR signal was previously assigned to an oxidized histidine radical on the basis of its UV spectrum. In a recent paper [Hallahan, B. J., Nugent, J. H. A., Warden, J. T., & Evans, M. C. W. (1992) Biochemistry 31, 4562-4573], this assignment was challenged, and it was suggested that the signal arises instead from the well-known tyrosine radical Tyrz., the electron carrier between the photooxidized chlorophyll and the Mn cluster. Here, we provide evidence that the measurements of the Tyr., on which the new interpretation was based, are artifactual due to the use of saturating microwave powers. Other than a relaxation-enhancement effect, the formation of the split S3 signal is accompanied by no change in the Tyr. signal. Although essentially unrelated to the origin of the S3 radical, several other experimental and interpretational problems in the work of Hallahan et al. (1992) are pointed out and rationalized. For example, the inability of Hallahan et al. (1992) to observe the split S3 signal in samples containing DCMU or without a chelator, in contrast to our observations, is attributed to a number of technical problems including the incomplete inhibition of the enzyme. We thus conclude that the assignment of the split S3 signal as His., although not proven, remains the most reasonable on the basis of current data.     

Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures

Acid glycosaminoglycans (GAGs) antioxidant activity was assessed in a fibroblast culture system by evaluating reduction of oxidative system-induced damage. Three different methods to induce oxidative stress in human skin fibroblast cultures were used. In the first protocol cells were treated with CuSO4 plus ascorbate. In the second experiment fibroblasts were exposed to FeSO4 plus ascorbate. In the third system H2O2 was utilised. The exposition of fibroblasts to each one of the three oxidant systems caused inhibition of cell growth and cell death, increase of lipid peroxidation evaluated by the analysis of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and superoxide dismutase (SOD) levels, and rise of lactate dehydrogenase activity (LDH). The treatment with commercial GAGs at different doses showed beneficial effects in all oxidative models. Hyaluronic acid (HA) and chondroitin-4-sulphate (C4S) exhibited the highest protection. However, the cells exposed to CuSO4 plus ascorbate and FeSO4 plus ascorbate were better protected by GAGs compared to those exposed to H2O2. These outcomes confirm the antioxidant properties of GAGs and further support the hypothesis that these molecules may function as metal chelators.