The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.     

Quantification of cuticular permeability in genetically modified plants

More and more studies on genetically modified plants are identifying parts of the genetic code with putative involvement in creating the cuticular barrier. Unfortunately, many of these studies suffer from the inadequacy of the chosen methods to quantify, in a reasonably unambiguous way, if and how the efficacy of the cuticular barrier is affected by the genetic change. A short overview of relevant findings is given and a more stringent experimental approach to quantifying effects on cuticular permeability in genetically modified plants proposed.     

Survival strategies of plants during secondary growth: barrier properties of phellems and lenticels towards water, oxygen, and carbon dioxide

Ever since plants began to conquer the terrestrial environment, a simple but effective evolutionary strategy has been employed to cope with the combined necessities of preventing an excessive loss of water via the aerial surface while also supporting the vital exchange of CO(2) and O(2) for photosynthesis and respiration. Large areas of the primary above-ground surface of plants are covered by a hydrophobic, non-cellular cuticle which effectively minimizes evaporation and very strongly reduces exchange of CO(2) and O(2). Hence, gas exchange is controlled by regulating stomatal apertures. Upon wounding or entering into secondary growth, however, the epidermis, cuticle, and stomata are replaced by a phellem (cork), which is produced by a lateral cambium, the phellogen. Former stomata are replaced by lenticels, which are multicellular structures and functionally analogous to stomata. In the secondary plant body, phellems effectively prevent the loss of water from the cortex of the stem while lenticels support the exchange of vital gases such as CO(2), O(2), and water vapour. The permeance of these gases via the lenticels reaches a maximum during July and is minimal during autumn and winter. In contrast to stomatal control, gas exchange through phellems is regulated by long-term structural changes. The permeances of cuticles, phellems, and lenticels are compared and discussed.     

Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1

Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.     

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and M?ssbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> induces aponecrosis in human aortic smooth muscle cells

Background  

The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis.     

Electronic structure of Q-A in reaction centers from Rhodobacter sphaeroides. I. Electron paramagnetic resonance in single crystals.

The magnitude and orientation of the electronic g-tensor of the primary electron acceptor quinone radical anion, Q-A, has been determined in single crystals of zinc-substituted reaction centers of Rhodobacter sphaeroides R-26 at 275 K and at 80 K. To obtain high spectral resolution, EPR experiments were performed at 35 GHz and the native ubiquinone-10 (UQ10) in the reaction center was replaced by fully deuterated UQ10. The principal values and the direction cosines of the g-tensor axes with respect to the crystal axes a, b, c were determined. Freezing of the single crystals resulted in only minor changes in magnitude and orientation of the g-tensor. The orientation of Q-A as determined by the g-tensor axes deviates only by a few degrees (< or = 8 degrees) from the orientation of the neutral QA obtained from an average of four different x-ray structures of Rb. sphaeroides reaction centers. This deviation lies within the accuracy of the x-ray structure determinations. The g-tensor values measured in single crystals agree well with those in frozen solutions. Variations in g-values between Q-A, Q-B, and UQ10 radical ion in frozen solutions were observed and attributed to different environments.     

NADPH/NADP ratios in photosynthesizing reconstituted chloroplasts

Levels of reduced and oxidized triphosphopyridine nucleotides have been determined in reconstituted spinach chloroplasts and compared with levels in whole isolated chloroplasts during photosynthesis and darkness. The ratio of NADPH/NADP⁺ reaches values slightly above 1.0 at the beginning of photosynthesis, less than half the ratio attained with whole chloroplasts. Nonetheless these lower ratios are sufficient to maintain high rates of photosynthetic carbon dioxide fixation and reduction, which are comparable in the reconstituted chloroplasts to the rates found with whole chloroplasts. As with whole chloroplasts there is a decline in the ratio of NADPH/NADP⁺ as a function of time of photosynthesis. The effect of addition of bicarbonate (6 mM) in causing a transient drop in the ratio of NADPH/NADP⁺ is described and discussed in terms of the reversibility of the reduction of 3-phosphoglycerate to triose phosphate. The ratio NADPH/NADP⁺ can be improved by the addition of more lamellae either before or during the course of photosynthesis, and this improvement in ratio is accompanied by an improved rate of CO₂ fixation or a more sustained rate of CO₂ fixation with time of photosynthesis. The importance of NADPH/NADP⁺ ratio not only to the reduction of 3-phosphoglycerate to triose phosphate but also to the activation of the ribulose-1,5-diphosphate carboxylasemediated step is discussed.