Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.

An FT-IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm(-1). Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the C-H stretching vibrational population between 2,984 and 2,780 cm(-1). Carbohydrate content was determined using integration over a C-O and C-O-C stretching band area between 1,180 and 1,133 cm(-1). Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra-batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT-IR spectroscopy compared well with the results obtained from elemental analysis.

The effects of ionic strength on formation and decay of metarhodopsin I1 (MII), the active photointermediate of bovine rhodopsin, were studied in the native membrane environment by means of ultraviolet/ visible and Fourier-transform infrared (FTIR) spectroscopy. By increasing the concentration of KCI in the range from hypotonic to 4 M, the apparent pK, of the metarhodopsin I(MI)/MII equilibrium is shifted by approximately pH three, in favor of the MI1 intermediate. In addition, the apparent rate of MI1 formation is enhanced by an increase in ionic strength (about twofold in the presence of 2 M KCI). MI1 decay is independent of the salt concentration. Attenuated-total-reflectance/lTIR data show that the high-salt conditions have no effect on the rigidity of the membrane matrix and do not induce structural changes in the intermediates themselves. Different salts were tested for their ability to shift the MI/MII equilibrium; however, no clear ion dependence was observed. We interpret these results as an indication for direct involvement of the cytosolic surface charge in the regulation of the photochemical activity of bovine rhodopsi n.Keywords: bovine rhodopsin ; metarhodopsin hetarhodopsin I1 equilibrium ; pKt, ; ionic strength ; surface charge Rhodopsin is the photoreceptor protein located in the disk membranes of retinal rod photoreceptor cells, and is considered to be a model for the superfamily of guanine-nucleotide-binding regulatory protein (G protein)-coupled receptors (DeGrip et al., 1988;Hargrave and McDowell, 1992). It consists of the apoprotein opsin and the chromophore 1 1-cis-retinal, which is covalently linked through a Schiff base to Lys296 in bovine opsin. Photoexcitation of rhodopsin involves the rapid photoisomerization of the chromophore to the all-trans configuration. This primary photochemical event triggers a cascade of photointermediates involving a series of slower thermal transitions in the protein moiety. Under physiological conditions, the active metarhodopsin I1 intermediate (MII) is formed on a millisecond timescale (Emeis et al., 1982;Kibelbek et al., 1991). MI1 can bind and activate the G protein transducin, which ultimately leads to hyperpolarization of the rod photoreceptor cell and further neuronal transduction of the signal.MI1 is in equilibrium with its precursor metarhodopsin I (MI; Matthews et al., 1963). Therefore, to clarify the mechanism of visual excitation on a molecular level, understanding the factors that control the MUM11 transition is essential. The main characteristics of the MI/MII transition are considered to be (a) deprotonation of the retinal Schiff base and a net proton uptakeCorresponderzcr to

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We can induce or alter supramolecular expression of chirality in the aggregates of a series of chiral amide‐containing surfactants (1, R = C(O)C3H7, C6H5; R′ = C11H23, C17H35; X = phosphate, imidazole) by changing the pH or by addition of metal ions. Chirality at a higher level, such as in the formation of helical structures, is found to depend strongly on the head‐group organisation of the surfactant molecules.