Lipid membrane domain formation and alamethicin aggregation studied by calorimetry, sound velocity measurements, and atomic force microscopy

An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems.     

Strong excitonic interactions in the oxygen-reducing site of bd-type oxidase: the Fe-to-Fe distance between hemes d and b595 is 10 A

Absorption and circular dichroism (CD) spectra of cytochrome bd from Escherichia coli have been compared for the wild type enzyme and an inactive mutant in which a highly conserved E445 in subunit I has been replaced by alanine [Zhang, J., Hellwig, P., Osborne, J. P., Huang, H. W., Moenne-Loccoz, P., Konstantinov, A. A., and Gennis, R. B. (2001) Biochemistry 40, 8548-8556]. The absorption bands of ferrous heme b595 are absent from the spectrum of the dithionite-reduced E445A form of cytochrome bd. The difference between the spectra of the dithionite-reduced WT and E445A enzymes indicates that in the mutant, heme b595 is present but is not reducible by dithionite. Cytochrome bd reveals intense CD signals dominated by heme d, with almost no contribution from heme b595 or heme b558. The CD spectrum of the reduced wild type enzyme in the Soret band indicates strong excitonic interactions between ferrous heme d and ferrous heme b595, and these interactions are not observed in dithionite-reduced E445A mutant, in which heme b595 remains in the ferric state. Modeling the excitonic interactions in both absorption and CD spectra has been carried out, yielding an estimate of the Fe-to-Fe distance between heme d and heme b595 of about 10 A. The physical proximity supports the hypothesis that heme d and heme b595 can form a di-heme oxygen reducing site, a unique structure for respiratory oxidases.     

Structural organization of the bacterial nucleoid using endonucleolysis

The fragmentation of bacterial deoxyribonucleoprotein (bDNP) in the spheroplasts of Escherichia coli, Serratia marcescens, Pseudomonas fluorescens and Micrococcus luteus by bacterial intracellular Ca2+ or Ca2+, Mg2+-dependent endonucleases in situ was studied. An electrophoresis of the extracted nuclease-split bDNP revealed the presence of high molecular weight (nuclease-resistant) and low molecular weight multiple fragments (100--120 nucleotide pairs). The electrophoretic mobility of the smallest nuclease-split DNA fragments in all bacterial species under study was similar, indicating the orderly structure of bDNP. Two total fractions whose electrophoretic mobility corresponded to that of the histones H2a and H2b from calf thymus were prevalent in the spectrum of acid-soluble bDNP proteins of gram-negative species. The heterogeneity of DNP with respect to its sensitivity to nucleases, is interaction with membranes and protein distribution pattern were revealed by treatment of the bacterial nucleoid with endogenous endonucleases, which probably reflects differences in the functional state of individual sites of the genome.     

Cytochrome bd from Azotobacter vinelandii: evidence for high-affinity oxygen binding

Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements of O2 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give Kd(O2) = 0.5 microM, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 microM); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with O2 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate of O2 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.     

Oxylipins and oxylipin synthesis pathways in fungi

Oxylipins are a family of oxygenated fatty acids that are very diverse with regard to origin, structure, and functions. These compounds are found in almost all living beings and serve both as autoregulators of the development of organisms and as communication molecules. The autoregulatory role of oxylipins in fungi is to control the development, reproduction, synthesis of secondary metabolites (including mycotoxins), and adaptive responses. The role of oxylipins in the regulation of pathogenesis accounts for an important aspect of research on the biological activity of these compounds. The synthetic pathways and functions of oxylipins of fungi, the differences between fungal oxylipins and oxylipins from bacteria, higher plants, and mammals, and the role of oxylipins in the interaction of fungi with other organisms are considered in the present review.     

Heterometallic complex formation on p-sulfonatothiacalix[4]arene platform resulting in pH- and redox-modification of [Ru(bpy)3] luminescence

The pH-dependent heterometallic complex formation with p-sulfonatothiacalix[4]arene (TCAS) as bridging ligand in aqueous solutions was revealed by the use of spectrophotometry, nuclear magnetic relaxation and fluorimetry methods. The novelty of the structural motif presented is that the appendance of emission metal center ([Ru(bpy)₃]²⁺) is achieved through the cooperative non-covalent interactions with the upper rim of TCAS. The second metal block (Fe(III), Fe(II) and Mn(II)), bound with the lower rim of TCAS in the inner sphere coordination mode is serving as quencher of [Ru(bpy)₃]²⁺ emission. The difference between the complex ability of Fe(III) and Fe(II) ions provides pH conditions for redox-dependent emission of [Ru(bpy)₃]²⁺.