Transformation of industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn3+-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn2+. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn2+-independent manner. The reactions with the dyes were characterized by apparent Km values ranging from 4 to 16 microM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn2+, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn3+-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Protein phosphatase inhibitors induce the release of serotonin from rat basophilic leukemia cells (RBL-2H3)

Oxidation capacities of laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) from Phlebia radiata were compared using non-phenolic (veratryl alcohol and ABTS) and phenolic (syringaldazine, vanillalacetone and Phenol red) compounds as reducing substrates. The effect of Mn(II) on enzyme reactions was also studied. Highest specific activities were recorded with laccase in the oxidation of phenolic compounds or ABTS and irrespective of Mn(II) concentration. LiP and MnP oxidized all these substrates but only the catalysis of MnP was dependent upon Mn(II). Only LiP clearly oxidized veratryl alcohol. However, Mn(II) interfered with this reaction by repressing veratraldehyde formation. These results point to multiple participation of manganese ions, either as a reducing (Mn(II)) or oxidizing (Mn(III)) agent in the enzymatic reactions.     

Characterization of a novel manganese peroxidase-lignin peroxidase hybrid isozyme produced by Bjerkandera species strain BOS55 in the absence of manganese

A novel manganese-dependent peroxidase (MnP) isozyme produced in manganese-free cultures of Bjerkandera sp. strain BOS55 was purified and characterized. The production of the enzyme was greatly stimulated by the exogenous addition of various physiological organic acids such as glycolate, glyoxylate, and oxalate. The physical properties of the enzyme are similar to those of MnP isozymes from different white rot fungi (Mr = 43,000, pI 3.88, and epsilon407 nm = 123 mM-1 cm-1). The Bjerkandera MnP was efficient in the oxidation of Mn(II), as indicated by the kinetic constants (low Km of 51 microM and turnover number of 59 s-1). Furthermore, the isozyme was able to oxidize various substrates in the absence of manganese, such as 2,6-dimethoxyphenol, guaiacol, ABTS, 3-hydroxyanthranilic acid, and o- and p-anisidine. An interesting characteristic of the isozyme was its ability to oxidize nonphenolic substrates, veratryl alcohol and 1,4-dimethoxybenzene, without manganese addition. The affinity for veratryl alcohol (Km = 116 microM) and its turnover number (2.8 s-1) are comparable to those of lignin peroxidase (LiP) isozymes from other white rot fungi. Manganese at concentrations greater than 0.1 mM severely inhibited the oxidation of veratryl alcohol. The results suggest that this single isozyme is a hybrid between MnP and LiP found in other white rot fungi. The N-terminal amino acid sequence showed a very high homology to those of both MnP and LiP isozymes from Trametes versicolor.     

pH-linked binding of Mn(II) to manganese peroxidase

The stability of Mn(II) binding to manganese peroxidase (MnP) has been studied as a function of pH by spectrophotometric and potentiometric titrations. The sensitivity of the potentiometric titrations allows collection of data that are consistent with a high-affinity and a low-affinity Mn(II) binding site on the peroxidase. The two sites differ in affinity by 4 to 900-fold between pH 4 and 6.5. The stability of Mn(II) binding to the high-affinity site increases with increasing pH, while the stability of Mn(II) binding to the low-affinity site decreases with increasing pH. Interestingly, at pH values above 5.0, the high-affinity site appears to be partially unavailable for binding Mn(II). A pH-dependent structural change in the Mn(II) binding site is proposed to account for this partial inactivation at elevated pH.     

Altering substrate specificity at the heme edge of cytochrome c peroxidase

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.     

Structural properties of peroxidases

Peroxidases are heme proteins which are able to catalyze the oxidation of a large variety of substrates through the reaction with hydrogen peroxide. The specific biological function, the reduction potential of the iron and the nature of the substrates which can be oxidized, are strongly determined by the structural features of the protein matrix around the prosthetic group. In particular, two main features are considered to be responsible of the specificity of the biological function: the strong anionic character of the fifth, proximal ligand to the iron, which is able to stabilize high oxidation states, and the hydrophilic nature of the residues in the distal pocket. Beside the correct reduction potential for the oxidation reaction, the specificity towards different substrates also depends on the protein structural arrangement which can determine specific binding sites for substrates and mediators. Particularly, in the case of MnP,the Mn2+ binding site has been individuated in the X-ray structure. NMR studies were previously reported which provided an iron-manganese distance consistent with that from the X-ray structure. This information can help in defining the possible pathway for the electron transfer from the Mn2+ ion to the iron. On the contrary, in the case of LiP no information is available on the possible binding site of veratryl alcohol as well as of other aromatic substrates. This article reviews these structural properties of peroxidases with particular emphasis to their implications in the catalytic process. Finally, the calcium ions have been located in the structure of LiP and the MnP: their structural relevance will be discussed on the light of the possible role in determining the optimal arrangement of residues in the distal cavity for the enzymatic reaction.     

Crystal structures of substrate binding site mutants of manganese peroxidase

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus, Phanerochaete chrysosporium, catalyzes the oxidation of MnII to MnIII. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed MnII binding site of MnP consists of a heme propionate, three acidic ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of MnII bound to the protein. Crystals grown in the absence of MnII, or in the presence of EDTA, exhibited diminished electron density at this site. Crystals grown in excess MnII exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major MnII binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined. The mutant structures lack a cation at the MnII binding site. The structure of the MnII binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.     

Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-amino-acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58-60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (C alpha-distance 1.2 A). However, this peroxidase includes a Mn2+ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn2+. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.     

Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes

The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.     

DNA damage checkpoints update: getting molecular

A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I-P-X-P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5kb contained the complete sequence of the Cs-mnp1 gene, including 162bp and 770bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT-AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9kb was amplified using inverse PCR. A putative TATAA element was identified 5' of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.     

NMR investigation of isotopically labeled cyanide derivatives of lignin peroxidase and manganese peroxidase

The 1H NMR spectroscopy was used to study lignin peroxidase (LiP) and manganese peroxidase (MnP) containing deuterated histidines. LiP and MnP were obtained from a histidine auxotroph of the fungus Phanerochaete chrysosporium grown in the presence of deuterated histidines. The derivatives with deuterated histidines have allowed a firm assignment of the protons of the distal and proximal histidines. We have also found that the LiP from this strain exhibits different orientations of the 2-vinyl group compared to the LiP from the strain previously studied. Mobility of the group has also been detected, thus explaining the apparent inconsistency between X-ray solid-state and NMR solution data. The 15N shift values of 15N-enriched CN- in the cyanide derivatives of LiP and MnP have also been measured. The shift patterns, both for 15N and for the proximal histidine protons of several peroxidases, are consistent with predominant contact shift contributions which reflect the bond strength of the metal-axial ligand. Finally, our results confirm a correlation between shift values of 15N and those of proximal histidine protons and the Fe3+/Fe2+ redox potentials.     

Altering the binuclear manganese cluster of arginase diminishes thermostability and catalytic function

Arginase is a thermostable (Tm = 75 degrees C) binuclear manganese metalloenzyme which hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of native metal-depleted arginase, metal-loaded H101N arginase, and metal-depleted H101N arginase have been determined by X-ray crystallographic methods to probe the roles of the manganese ion in site A (Mn2+A) and its ligand H101 in catalysis and thermostability. We correlate these structures with thermal stability and catalytic activity measurements reported here and elsewhere [Cavalli, R. C., Burke, C. J., Kawamoto, S., Soprano, D. R., and Ash, D. E. (1994) Biochemistry 33, 10652-10657]. We conclude that the substitution of a wild-type histidine ligand to Mn2+A compromises metal binding, which in turn compromises protein thermostability and catalytic function. Therefore, a fully occupied binuclear manganese metal cluster is required for optimal catalysis and thermostability.     

Metal ion activation of galactosyltransferase

Galactosyltransferase, which functions as the catalytic component of lactose synthase and in the glycosylation of glycoproteins, has been previously reported to have an absolute dependence on Mn2+ for activity, with a Kd for Mn2+ (10(-3) M) 2 to 3 orders of magnitude greater than the physiological range of Mn2+ concentrations (v 10(-6) M). Reinvestigation of the metal ion dependence of this enzyme has shown that Zn2+, Cd2+, Fe2+, Co2+, and Pr3+ also produce activation, although with lower activities at saturation than that attained with Mn2+. Velocity against metal ion concentration curves for all metals, including Mn2+, are sigmoid, suggesting the presence of two or more activating metal binding sites on the enzyme. The presence of two sites is confirmed by studies using both Mn2+ and Ca2+. While galactosyltransferase is inactive in the presence of Ca2+ alone, at low concentrations of Mn2+ (10(-5) M), enzyme activity is stimulated by Ca2+. A more detailed investigation by steady state kinetics has revealed that there is a tight binding site for Mn2+ (site I: Kd of 2 X 10(-6) M) from which Ca2+ is excluded, and a site at which Ca2+ can replace Mn2+ (site II: Kd for Ca2+ of 1.76 X 10(-3) M), to which metal binding has a specific synergistic effect on UDP-galactose binding, possibly as a result of the formation of an enzyme-Ca2+-UDP-galactose bridge complex. The site I Mn2+, site II Ca2+-activated enzyme has a maximum velocity similar to that of the Mn2+-activated enzyme, and is the enzyme form that must act in lactose synthesis in vivo. A trypsin-degraded form of galactose transferase (galactosyltransferase-T) (Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228) appears to lack site I and is activated by Ca2+ in the absence of Mn2+.     

Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism

Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase [CCP(MI)] and horseradish peroxidase (HRP) in their ferric forms. CCP(MI) unfolds in urea and in guanidine hydrochloride (GdHCl) at pH 7.0, while HRP loses its secondary structure only in the presence of GdHCl. CCP(MI) unfolds in urea by two distinct steps as monitored by fluorescence, but the loss of its secondary structure as monitored by UV/CD occurs in a single step between 3.4 and 5 M urea and 1.5 and 2.5 M GdHCl. The localized changes detected by fluorescence involve the CCP(MI) heme cavity since the Soret maximum red-shifts from 408 to 416 nm, and the heme CD changes examined in urea are biphasic. The polypeptide of HRP also loses secondary structure in a single step between 1.2 and 2.7 M GdHCl as monitored by UV/CD, and a fluorescence-monitored transition involving conformational change in the Trp117-containing loop occurs above 4 M GdHCl. Free energies of denaturation extrapolated to 0 M denaturant (delta Gd,aq) of approximately 6 and approximately 4 kcal/mol were calculated for CCP(MI) and HRP, respectively, from the UV/CD data. The refolding mechanisms of the two peroxidases differ since heme capture in CCP(MI) is synchronous with refolding while apoHRP captures heme after refolding. Thus, the denatured form of apoHRP does not recognize heme and has to correctly refold prior to heme capture. The half-life for unfolding of native HRP in 6 M GdHCl is slow (519 s) compared to that for CCP(MI) (14.3 s), indicating that HRP is kinetically much more stable than CCP(MI). Treatment with EDTA and DTT greatly destabilizes HRP, and unfolding in 4 M GdHCl occurs with t1/2 = 0.42 s.     

Interaction of peroxynitrite with mitochondrial cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of enzyme activity

Purified mitochondrial cytochrome c oxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear heme a3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a3 nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreased Vmax and a raised Km for oxygen. Under these conditions, the redox state of cytochrome a is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 microM peroxynitrite to 10 microM fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochrome a3, partial reduction of cytochrome a, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 microM) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a and a3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase.     

Modeling Manganese Oxidation with KMnO4 for Drinking Water Treatment

Manganese (Mn) in a drinking water distribution system can cause multiple aesthetic problems including discolored water and fouling or scaling of fixtures. Oxidation and solid-liquid separation processes are typically employed at a treatment plant to limit the concentration of Mn entering the distribution system. Potassium permanganate (KMnO4) is commonly used to oxidize the manganous ion (Mn+2) to manganese oxide (MnO2). In this study, a mechanistic model is applied to the oxidation of manganese at a treatment plant. Literature kinetic constants (determined with artificial water) are compared with the values obtained for the plant's natural water. The solution and surface phase oxidation rate constants determined with the natural water are two to six orders of magnitude less than those determined with the artificial water. The reduced oxidation rate in the natural water is attributed to the presence of dissolved organic matter, which can exert a competitive demand on the oxidant and interfere with the oxidation by complexing Mn+2. The development of an additional rate constant for the oxidation of dissolved organic matter improves the modeling results for KMnO4 concentration versus time, but only marginally explains the Mn+2 oxidation rate differences.     

Calcium induces binding and formation of a spin-coupled dimanganese(II,II) center in the apo-water oxidation complex of photosystem II as precursor to the functional tetra-Mn/Ca cluster

Two new intermediates are described which form in the dark as precursors to the light-induced assembly of the photosynthetic water oxidation complex (WOC) from the inorganic components. Mn2+ binds to the apo-WOC-PSII protein in the absence of calcium at a high-affinity site. By using a hydrophobic chelator to remove Mn2+ and Ca2+ from the WOC and nonspecific Fe3+, a new EPR signal becomes visible upon binding of Mn2+ to this site, characterized by six-line 55Mn hyperfine structure (DeltaHpp = 96 +/- 1 G) and effective g = 8.3. These features indicate a high-spin electronic ground state (S = 5/2) for Mn2+ and a strong ligand field with large anisotropy. This signal is eliminated if excess Ca2+ or Mg2+ is present. A second Mn2+ EPR signal forms in place of this signal upon addition of Ca2+ in the dark. The yield of this Ca-induced Mn signal is optimum at a ratio of 2 Mn/PSII, and saturates with increasing [Ca2+] >/= 8 mM, exhibiting a calcium dissociation constant of KD = 1.4 mM. The EPR signal of the Ca-induced Mn center at 25 K is asymmetric with major g value of approximately 2.04 (DeltaHpp = 380 G) and a shoulder near g approximately 3.1. It also exhibits resolved 55Mn hyperfine splitting with separation DeltaHpp = 42-45 G. These spectral features are diagnostic of a variety of weakly interacting Mn2(II, II) pairs with electronic spins that are magnetic dipolar coupled in the range of intermanganese separations 4.1 +/- 0.4 A, and commonly associated with one or two carboxylate bridges. The calcium requirement for induction of the Mn2(II,II) signal matches the value observed for steady-state O2 evolution (Michaelis constant, KM approximately 1.4 mM), and for light-induced assembly of the WOC by photoactivation. The Ca-induced Mn2(II,II) center is a more efficient electron donor to the photooxidized tyrosine radical, TyrZ+, than is the mononuclear Mn center present in the absence of Ca2+. The Ca-induced Mn2(II,II) signal serves as a precursor for photoactivation of the functional WOC and is abolished by the presence of Mg2+. Formation of the Mn2(II,II) EPR signal by addition of Ca2+ correlates with reduction of flash-induced catalase activity, indicating that calcium modulates the accessibility or reactivity of the Mn2(II,II) core with H2O2. We propose that calcium organizes the binding site for Mn ions in the apo-WOC protein and may even interact directly with the Mn2(II,II) pair via solvent or protein-derived bridging ligands.     

Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli

The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.     

590 MPa含锰钢带罩式炉退火氧化色分析与控制

针对罩式炉工业化生产590 MPa含锰低合金冷轧钢带表面氧化色缺陷,分析了表面氧化色的主要组成。实验室采用马弗炉模拟了罩式炉退火工艺,验证了金属锰薄片对钢带表面氧化色的抑制作用。结果表明,金属锰薄片可以通过消耗炉内氧化性气氛,保护试样表面在退火过程中不被氧化。罩式炉工业化退火采用冷点温度620 ℃、热点温度630 ℃、保温时间25 h及全过程40 m3/h氢气流量吹扫制度,同时退火过程中在每个对流板中心处装入125 kg纯金属锰薄片,可避免工业化生产590 MPa含锰低合金冷轧钢带边部出现氧化色缺陷,同时力学性能满足要求。     

纳米锰方硼石的合成与结构性能表征

通过溶胶?凝胶（Sol?Gel）法成功合成了纳米锰方硼石并对其进行了稀土Eu3+掺杂。使用X射线衍射、透射电子显微镜和高分辨透射电子显微镜等表征了锰方硼石晶体结构,并通过荧光光谱测试对其发光性能进行了研究。结果表明:合成纳米锰方硼石为粒径小于50 nm的球状颗粒,与天然锰方硼石的物相结构相同,属于斜方晶系,与尖晶石类似,（010）晶面的晶面间距为0.8565 nm。在490 nm激发光激发下,天然锰方硼石、合成锰方硼石和稀土Eu3+掺杂锰方硼石晶体中的Mn2+发光,其中发绿光的Mn2+在晶体中占据四面体格位中心,发红光的Mn2+在晶体占据八面体格位中心。合成的锰方硼石随激发波长变长,产生发射光谱的红移现象,有利于实现冷暖发光转换;在稀土Eu3+掺杂的纳米锰方硼石光谱的发光强度得到了提升。