How does heme axial ligand deletion affect the structure and the function of cytochrome b562?

We have recently generated a new mutant of cytochrome b₅₆₂ (cytb₅₆₂)in which Met7, one of the axial heme ligands, is replaced byAla (M7A cytb₅₆₂). The M7A cytb₅₆₂ can bind heme and the UV-visibleabsorption spectrum is of a typical high-spin ferric heme. Toinvestigate the effect of the lack of Met7 ligation on the structuralintegrity of cytb₅₆₂, thermal transition analyses of M7A cytb₅₆₂were conducted. From the thermodynamic parameters obtained,it is concluded that the folding of M7A cytb₅₆₂ is comparableto the apoprotein despite the presence of heme. On the otherhand, exogenous ligands such as cyanide and azide ions are readilybound to the heme iron, indicating that the axial coordinationsite is available for substrate binding. The peroxidase activityof this mutant is thus examined to evaluate new enzymatic functionat this site and M7A cytb₅₆₂ was found to catalyze an oxidationreaction of aromatic substrates with hydrogen peroxide. Theseobservations demonstrate that the Met7/His102 bis-ligation tothe heme iron is crucial for the stable folding of cytb₅₆₂,whereas the functional conversion of cytb₅₆₂ is successfullyachieved by the loose folding together with the open coordinationsite.     

Decomposition of a cyanine dye in binary nanosheet colloids of photocatalytically active niobate and inert clay

Binary colloids of inorganic nanosheets prepared by exfoliation of two different layered crystals form phase-separated structures with demixing of the two nanosheets. The phase-separated colloids of photocatalytic niobate and photochemically inert clay nanosheets exhibit unusual photochemical reactions based on the phase-separated structure. The present paper reports photocatalytic decomposition of a cyanine dye in this binary colloid, where the dye is selectively adsorbed on the clay nanosheets to be spatially separated from the photocatalytic nanosheets. Upon UV irradiation, the dye is photocatalytically decomposed in the colloids containing the niobate nanosheets, but self-photolysis of the dye is observed in the colloid lacking the photocatalytic nanosheets. Faster decomposition in nitrogen than in air suggests contribution of the conduction-band electrons generated in the niobate nanosheets to the photocatalytic reaction. In the binary colloid, the degradation is retarded compared with the single-component niobate colloid. Larger clay content more stabilized the dye against the decomposition. In contrast, irradiation of the colloids with visible light causes self-photolysis of the dye even in the presence of the niobate nanosheets, indicating the absence of electron transfer from the photoexcited dye to the photocatalytic nanosheets.     

Biotransformation of arsenic species by activated sludge and removal of bio-oxidised arsenate from wastewater by coagulation with ferric chloride

The potential of activated sludge to catalyse bio-oxidation of arsenite [As(III)] to arsenate [As(V)] and bio-reduction of As(V) to As(III) was investigated. In batch experiments (pH 7, 25 degrees C) using activated sludge taken from a treatment plant receiving municipal wastewater non-contaminated with As, As(III) and As(V) were rapidly biotransformed to As(V) under aerobic condition and As(III) under anaerobic one without acclimatisation, respectively. Sub-culture of the activated sludge using a minimal liquid medium containing 100mg As(III)/L and no organic carbon source showed that aerobic arsenic-resistant bacteria were present in the activated sludge and one of the isolated bacteria was able to chemoautotrophically oxidise As(III) to As(V). Analysis of arsenic species in a full-scale oxidation ditch plant receiving As-contaminated wastewater revealed that both As(III) and As(V) were present in the influent, As(III) was almost completely oxidised to As(V) after supply of oxygen by the aerator in the oxidation ditch, As(V) oxidised was reduced to As(III) in the anaerobic zone in the ditch and in the return sludge pipe, and As(V) was the dominant species in the effluent. Furthermore, co-precipitation of As(V) bio-oxidised by activated sludge in the plant with ferric hydroxide was assessed by jar tests. It was shown that the addition of ferric chloride to mixed liquor as well as effluent achieved high removal efficiencies (>95%) of As and could decrease the residual total As concentrations in the supernatant from about 200 microg/L to less than 5 microg/L. It was concluded that a treatment process combining bio-oxidation with activated sludge and coagulation with ferric chloride could be applied as an alternative technology to treat As-contaminated wastewater.     

Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain

Structure-based design of antibody/cytokine receptor chimeras has permitted a growth signal transduction in response to non-natural ligands such as fluorescein-conjugated BSA as mimicry of cytokine-cytokine receptor systems. However, while tight on/off regulation is observed in the natural cytokine receptor systems, many chimeras constructed to date showed residual growth-promoting activity in the absence of ligands. Here we tried to reduce the basal growth signal intensity from a chimera by engineering the transmembrane domain (TM) that is thought to be involved in the interchain interaction of natural cytokine receptors. When the retroviral vectors encoding the chimeras with either the wild-type erythropoietin receptor (EpoR) TM or the one bearing two mutations in the leucine zipper motif were transduced to non-strictly interleukin-6-dependent 7TD1 cells, a tight antigen-dependent on/off regulation was attained, also demonstrating the first antigen-mediated genetically modified cell amplification of non-strictly factor-dependent cells. The results clearly indicate that the TM mutation is an effective means to improve the growth response of the antibody/receptor chimera.     

Hemoglobin(βC93A)–Albumin Cluster: Mutation of Cysteine-β93 to Alanine Allows Moderate Reduction of O2 Affinity by Inositol Hexaphosphate

Covalent wrapping of recombinant human hemoglobin (Cys-β93→Ala) variant rHb(βC93A) by human serum albumin (HSA) yielded the rHb(βC93A)-HSA₃ cluster as an artificial O₂ carrier as a red blood cell substitute. Complexation of inositol hexaphosphate to the central rHb(βC93A) core reduced the O₂ affinity moderately, in much the same way as that of naked hemoglobin. This reduction might be attributable to the inert, small Ala-β93 residue, which cannot be reacted with the bulky maleimide crosslinker.     

Applying Quantitative Microstructure Control in Advanced Functional Composites

Microstructure control in functional materials draws from a historical reserve rich in established theory and experimental observation of metallurgy. Methods such as rapid solidification, eutectoid reaction, and nucleation and growth precipitation have all proven to be effective means to produce microstructure relevant for a wide array of applications. Here, the available parameters to control structure morphology, size, and spacing are discussed using thermoelectric composites as an example. Moreover, exploiting different aspects of a material system's phase diagram enables a controlled introduction of nanostructures. While much of this discussion is pertinent to the rapidly developing field of thermal conductivity control in thermoelectric composites, these techniques can be applied to a variety of other material systems where their use may lead to novel electrical, optical, as well as thermal properties of semiconductors and insulators as it has in the past for the mechanical properties of metals.     

Preparation of a layered hexaniobate–titania nanocomposite and its photocatalytic activity on removal of phenol in water

A nanocomposite photocatalyst of two photocatalytically active semiconductor oxides was prepared by flocculation of exfoliated layered hexaniobate K₄Nb₆O₁₇ with TiO₂ fine particles in presence of acid electrolyte, and evaluated as photocatalyst by using photodegradation of phenol as a test reaction. Disappearance of (0k0) peaks which correspond to the basal spacing in the X-ray diffraction (XRD) patterns of the nanocomposite suggested that the exfoliated niobate nanosheets were randomly hybridized with TiO₂ particles without restacking to the layered structure. Scanning electron microscopy revealed that the nanocomposite was an agglomerate of closely packed niobate nanosheets and TiO₂ particles. The nanocomposite is mesoporous and has a specific surface area larger than those of the original K₄Nb₆O₁₇ and TiO₂ as indicated by N₂ adsorption–desorption isotherms. The increase in surface area is ascribed to the porous structure constructed by the exfoliated niobate nanosheets and TiO₂ particles. The photocatalytic activity of the porous nanocomposite was superior to those of K₄Nb₆O₁₇ and TiO₂ in terms of degradation of phenol.     

Artificial Protein Complexes for Biocatalysis

In nature, the macromolecular organization of multienzyme complexes has important implications for the specificity, regulation and overall efficiency of biochemical reactions. Artificial multienzyme complexes mimicking natural multienzyme organization would represent promising biocatalysts. In this review, we will give an overview of current developments and successes in the field of multienzyme complex formation using DNA scaffolds, synthetic protein scaffolds and self-assembling protein scaffolds.