Single-Step Reconstitution of Apo-Hemoproteins at the Disruption Stage of Escherichia coli Cells

Hemoproteins on their metal: We report a novel strategy for the reconstitution of hemoproteins with non-natural metal complexes; simple addition of manganese and ruthenium porphyrin to E. coli cells immediately prior to homogenization yields the reconstituted proteins. We believe that this simple approach could become a standard reconstitution method for hemoproteins.     

Peptide‐Based Scaffold for Nitric Oxide Induced Differentiation of Neuroblastoma Cells

Nitric oxide is a gaseous messenger involved in neuronal differentiation, development and synaptogenesis, in addition to many other physiological functions. Therefore, it is imperative to maintain an optimal nitric oxide concentration to ensure its biochemical function. A sustained nitric oxide releasing scaffold, which supports neuronal cell differentiation, as determined by morphometric analysis of neurite outgrowth, is described. Moreover, the effect of nitric oxide on the neuroblastoma cell line was also confirmed by immunofluorescent analysis of neuronal nuclear protein (NeuN), specific neuronal marker and neurofilament (NF) protein, which revealed a significant increase in their expression levels, in comparison with undifferentiated cells.     

自养型生物过滤器硝化氧化一氧化氮

Carbon foam-a, kind of new engineering material as packing material was adopted in three biofilters with different pore dimensions and adapted autotrophic nitrite nitrobacteria to investigate the purification of nitric oxide (NO) in a gas stream. The biofilm was developed on the surface of carbon foams using nitrite as its only nitric source. The moisture in the filter was maintained by ultrasonic aerosol equipment which can minimize the thickness of the liquid film. The liquid phase nitrification test was conducted to determine the variability and the potential of performance among the three carbon foam biofilters. The investigation showed that during the NO2--N inlet concentration of 200g·L-1-min-1 to 800g·L-1-min-1, the 24PPC (pores per centimeter) carbon foam biofilter had the greatest potential, achieving the NO--N removal efficiency of 94% to 98%. The 8PPC and 18PPC carbon foam biofilters achieved the NO2--N removal efficiency of 15% to 21% and of 30% to 40%, respectively. The potential for this     

Enhancement of Peroxidase Activity in Artificial Mimochrome VI Catalysts through Rational Design

Rational design provides an attractive strategy to tune and control the reactivity of bioinspired catalysts. Although there has been considerable progress in the design of heme oxidase mimetics with active‐site environments of ever‐growing complexity and catalytic efficiency, their stability during turnover is still an open challenge. Herein, we show that the simple incorporation of two 2‐aminoisobutyric acids into an artificial peptide‐based peroxidase results in a new catalyst (Fe^III‐MC6*a) with higher resistance against oxidative damage and higher catalytic efficiency. The turnover number of this catalyst is twice as high as that of its predecessor. These results point out the protective role exerted by the peptide matrix and pave the way to the synthesis of robust bioinspired catalysts.     

加压条件下稀硝酸吸收氮氧化物的实验研究

为治理工业过程中产生的高浓度氮氧化物(NOx)废气并从中回收硝酸,对加压条件下用稀硝酸吸收法处理高浓度NOx模拟废气进行了研究.分别在0.4、0.6和0.8 MPa下,用质量分数为35％～50％的硝酸作吸收剂进行实验,研究了操作条件对吸收效率的影响.结果表明,吸收效率随着NOx的进口浓度和系统压强的增大而升高;随着硝酸浓度的增大吸收效率下降,但可以达到硝酸回收率50％的目的;液气比增大吸收效率升高,但是液气比大于6 L/m2后,吸收效率的增幅趋于平缓;温度升高不利于NOx的吸收.     

Using Biosynthetic Models of Heme-Copper Oxidase and Nitric Oxide Reductase in Myoglobin to Elucidate Structural Features Responsible for Enzymatic Activities

In biology, a heme-Cu center in heme-copper oxidases (HCOs) is used to catalyze the four-electron reduction of oxygen to water, while a heme-nonheme diiron center in nitric oxide reductases (NORs) is employed to catalyze the two-electron reduction of nitric oxide to nitrous oxide. Although much progress has been made in biochemical and biophysical studies of HCOs and NORs, structural features responsible for similarities and differences within the two enzymatic systems remain to be understood. Here, we discuss the progress made in the design and characterization of myoglobin-based enzyme models of HCOs and NORs. In particular, we focus on use of these models to understand the structure-function relations between HCOs and NORs, including the role of nonheme metals, conserved amino acids in the active site, heme types, and hydrogen-bonding networks, in tuning enzymatic activities and total turnovers. Insights gained from these studies are summarized and future directions are proposed.     

Rational Design of Domain‐Swapping‐Based c‐Type Cytochrome Heterodimers by Using Chimeric Proteins

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three‐dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c‐type cytochrome‐based chimeric proteins have been constructed and the domains swapped. According to X‐ray crystallographic analysis, the two chimeric proteins formed a domain‐swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H₂O coordinated hemes was formed. Binding of an oxygen molecule to the His/H₂O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe?O₂ stretching band was observed at 580 cm^?1 for the reduced/oxygenated heterodimer (at 554 cm^?1 under an ¹⁸O₂ atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.     

Second-Generation CD73 Inhibitors Based on a 4,6-Biaryl-2-thiopyridine Scaffold

Various series of 4,6-biaryl-2-thiopyridine derivatives were synthesized and evaluated as potential ecto-5′-nucleotidase (CD73) inhibitors. Two synthetic routes were explored and the coupling of 4,6-disubstituted 3-cyano-2-chloro-pyridines with selected thiols allowed us to explore the structural diversity. Somehow divergent results were obtained in biological assays on CD73 inhibition using either the purified recombinant protein or cell-based assays, highlighting the difficulty to target protein-protein interface on proteins existing as soluble and membrane-bound forms. Among the 18 new derivatives obtained, three derivatives incorporating morpholino substituents on the 4,6-biaryl-2-thiopyridine core were shown to be able to reverse the adenosine-mediated immune suppression on human T cells. The higher blockade efficiency was observed for 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)-N-(isoxazol-3-yl)acetamide (with total reversion at 100 μM) and methyl 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)acetate (with partial reversion at 10 μM). Thus, this series of compounds illustrates a new chemotype of CD73 allosteric inhibitors.     

基于间接碘量法下硝酸中氮的氧化物含量的规律研究

浓硝酸强烈的氧化和硝化作用可使一些有机物如棉纱、木材等分解甚至燃烧,对人的皮肤也能严重侵蚀和烧伤。通过模拟不同温度、不同储存环境,用间接碘量法对试样中硝酸内氮的氧化物进行含量测定,掌握其变化规律,减少事故发生概率,找到该物质储存所适宜的外部环境,在可调节的范围内尽量减小氧化物的周期变化,防止硝酸中氮的氧化物出现大的变化,出现突发事故以及不可预料的情况出现。     

Communication Maps: Exploring Energy Transport through Proteins and Water

Frequency-resolved communication maps provide a coarse-grained, global mapping of energy transport channels in a protein as a function of frequency of modes that carry energy. We illustrate the approach with a study of the homodimeric hemoglobin of Scapharca inaequivalvis, which exhibits cooperativity during ligand binding. We compare energy transport between the two hemes of the unliganded and oxygenated protein, which is mediated by water as well as residues forming a hydrogen-bonding network at the interface between the globules, and lies along the pathway for allosteric transitions observed in time-resolved X-ray studies. Non-equilibrium molecular simulations on energy transport from the heme corroborate the energy transport pathways identified by the communication maps.     

Halogenated veneers: protein cross-linking and halogenation in the jaws of nereis, a marine polychaete worm

Mineralized tissues are produced by most living organisms for load and impact functions. In contrast, the jaws of the clam worm, Nereis, are hard without mineralization. However, they are peculiarly rich in halogens, which are associated with a variety of post-translationally modified amino acids, many of which are multiply halogenated by chlorine, bromine, and/or iodine. Several of these modified amino acids, namely dibromohistidine, bromoiodohistidine, chloroiodotyrosine, bromoiodotyrosine, chlorodityrosine, chlorotrityrosine, chlorobromotrityrosine, and bromoiodotrityrosine, have not been previously reported. We have found that the distributions of Cl, Br, and I differ: Cl is widespread whereas Br and I, although not colocalized, are concentrated in proximity to the external jaw surfaces. By using nanoindentation, we show that Br and I are unlikely to play a purely mechanical role, but that the local Zn and Cl concentrations and jaw microstructure are the prime determinants of local jaw hardness. Several of the post-translationally modified amino acids are akin to those found in various sclerotized structures of invertebrates, and we propose that they are part of a cross-linked protein casing.     

Determination of hemin-binding characteristics of proteins by a combinatorial peptide library approach

Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron‐ion‐containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate‐covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed. Ligands for both types of binding have been reported independently, though sometimes with different affinities for similar sequences. We applied a combinatorial approach using the library (X)₄(C/H/Y)(X)₄ to characterize peptide ligands with considerable hemin binding capacities. Some of the library‐selected peptides were comparable in terms of hemin association independently of whether or not a cysteine residue was present in the sequence. Indeed, a preference for His‐based (≈39 %) and Tyr‐based (≈40 %) sequences over Cys‐based ones (≈21 %) was detected. The binding affinities for the library‐selected peptides, as determined by UV/Vis spectroscopy, were in the nanomolar range. Moreover, selected representatives efficiently competed for hemin binding with the human BK channel hSlo1, which is known to be regulated by heme through binding to its heme‐binding domain.