The Deuterium Isotope Effect as a Tool to Investigate Enzyme Catalysis: Proton-Transfer Control Mechanisms in Cytochrome c Oxidase

Cytochrome c oxidase is a membrane-bound redox-driven proton pump. The coupling of the exergonic electron-transfer reactions from cytochrome c to oxygen to proton translocation across the membrane requires control of internal electron- and proton-transfer reactions. In this work, we focus on the kinetics of electron and proton transfer during those reaction steps that are coupled to proton pumping in cytochrome c oxidase. The results show that during the first pumping step (peroxy → oxoferryl transition), proton transfer regulates intramolecular electron transfer. The proton transfer takes place in two steps: (1) Internal proton transfer from a protonatable group, proposed to be Glu(I-286), in the so-called D-pathway, to an oxygen intermediate at the binuclear center (τ ≅ 100 μs); (2) Rapid re-protonation of Glu(I-286) from the bulk solution (τ < 100 μs). Only after proton uptake from solution the last (fourth) electron is transferred “one step closer” towards the binuclear center (from Cu_A to heme a). During the second proton-pumping step (oxoferryl → oxidized), this electron is transferred to the binuclear center, linked to the uptake of a proton through the D-pathway. The electron-transfer rate displays a kinetic-isotope effect (k_H/k_D) of 6 ± 1 (in a pH range in which the pH dependence of the rate is small), which indicates that the electron transfer is rate-limited by the proton transfer. The entry into the D-pathway (around Asp(I-132)) is composed of a cluster of negatively-charged amino acid residues together with a number of histidines, forming a so-called proton-collecting antenna designed to allow rapid protonation of groups within the proton-transfer pathway.     

Examination of Sugars, Sugar Alcohols, and Artificial Sweeteners as Substitutes for Sucrose in Strawberry Jam. Product Development

Preparation of acceptable low sugar jams and replacement of sucrose by other sweeteners in jam were studied. Strawberry jam was sweetened with sucrose, fructose, high fructose syrup (HFS), xylitol, sorbitol, lactose, saccharin, cyclamate, or with combinations of these. It was technologically possible to prepare jams with lower amounts of sucrose than currently used and still attain an acceptable product. In addition, sucrose can be replaced in strawberry jam by other sweeteners or by combinations of sweeteners. The attainment of a suitable texture may be more difficult in xylitol and sorbitol jams than in jams with other sweeteners. The use of maltodextrin as bulking agent in jam is limited by the abnormal appearance and taste it gives to the product.     

On correction of translational misalignments between section planes in 3D EBSD

Three‐dimensional electron backscatter diffraction allows obtaining the 3D image of a material from the stack of 2D sections. This is achieved by repeated application of two different beams; electron beam for electron backscatter diffraction mapping of the surface and focused ion beam for removing a thin layer of material from the surface. In most of these systems with two beams, the experiment requires stage movements for correct positioning of the sample to the respective beams. However, imperfections in this positioning are difficult to avoid, which yield small translational misalignments between the sections in the output data. In this work, we deal with an important task of correcting these misalignments between the sections such that the 3D image is recovered properly. On a simple example, we demonstrate that commonly used methods fail in case there is a structural anisotropy in the material under consideration. We propose an improved alignment algorithm which can neglect this behaviour with the use of external support information on a systematic trend in the translational misalignments. Efficiency of the algorithm is proven on a number of simulated data with different kinds of anisotropy. Application to a real data sample of a fine grained aluminium alloy is also given. The algorithm is available in an open‐source library.     

Properties of magnetron-sputtered electrically insulating Al2O3 coatings on copper

Amorphous Al₂O₃ coatings were deposited on Cu substrates with metallic bond layers by different magnetron-sputtering processes. Such sputtering conditions as the type of discharge, target material, total pressure, sputtering gas composition and substrate temperature were varied to study the process effects on the structure and properties of the coatings deposited. The structure and general properties were found to be strongly dependent on the type of process and parameters. The breakdown voltages did not show any significant lowering as the temperature was increased from 20 to 400–500 C. The maximum temperature without formation of cracks in the Al₂O₃ coating on Cu was about 700 C. The d.c. electrical conductivities of the Al₂O₃ were similar to that of bulk Al₂O₃ at different temperatures. The results reveal the potential use of magnetron-sputtered Al₂O₃ coatings on Cu for electrical insulation and oxidation protection in different high-temperature applications, e.g. on metallic magnetohydrodynamic electrode and insulator modules.     

Experiences of Biological Iron and Manganese Removal in Finland

Even though groundwater resources in Finland are adequate, and theoretically the total water consumption could be supplied from this source, in practice only about 50% of the total consumption is from groundwater. There are thousands of unconnected small aquifers dispersed all over the country, and the use of these as water supplies is not economically possible.
Biological iron and manganese removal systems appear to be a promising alternative to chemical treatment. In Finland the following biological treatment systems are in use: (i) VYREDOX method; (ii) overland flow method; (iii) slow sand filtration; (iv) re-infiltration; and (v) biological reactors.
Active iron and manganese bacteria play the most essential role in all these applications, and different methods of removal are discussed in detail in the paper.     

Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy

Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico‐ to nano‐molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation spectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non‐ideal confocal volume can be experimentally determined and validated.