Fe–O versus O–O bond cleavage in reactive iron peroxide intermediates of superoxide reductase

It is generally accepted that the catalytic cycles of superoxide reductases (SORs) and cytochromes P450 involve a ferric hydroperoxo intermediate at a mononuclear iron center with a coordination sphere consisting of four equatorial nitrogen ligands and one axial cysteine thiolate trans to the hydroperoxide. However, although SORs and P450s have similar intermediates, SORs selectively cleave the Fe–O bond and liberate peroxide, whereas P450s cleave the O–O bond to yield a high-valent iron center. This difference has attracted the interest of researchers, and is further explored here. Meta hybrid DFT (M06-2X) results for the reactivity of the putative peroxo/hydroperoxo reaction intermediates in the catalytic cycle of SORs were found to indicate a high-spin preference in all cases. An exploration of the energy profiles for Fe–O and O–O bond cleavage in all spin states in both ferric and ferrous models revealed that Fe–O bond cleavage always occurs more easily than O–O bond cleavage. While O–O bond cleavage appears to be thermodynamically and kinetically unfeasible in ferric hydrogen peroxide complexes, it could occur as a minor (significantly disfavored) side reaction in the interaction of ferrous SOR with hydrogen peroxide.     

The Synthesis of Dimethyl Esters of dl-O,O′-Dimethylfukiic Acid and dl-O,O′-Dimethylepifukiic Acid

The synthesis of dimethyl esters of dl-O,O′-dimethylfukiic acid (11) and dl-O,O′-dimethylepifukiic acid (12) are described.     

The possession of coli surface antigen CS6 by enterotoxigenicEscherichia coli of serogroups O25, O27, O148, and O159: a possible colonization factor?

A total of 134 enterotoxigenicEscherichia coli (ETEC) of serogroups O25, O27, O148, and O159 were tested in the enzyme-linked immunosorbent assays for the colonization factor antigens I (CFA/I), CFA/II (coli surface antigens CS1, 2 and 3) and putative colonization factor (PCF) 8775 (CS4, 5 and 6). CS6 was detected without CS4 or CS5 in 94% of the strains of serogroup O25, 86% of strains of serogroup O27, 87% of strains of serogroup O148, and 29% of strains of serogroup, O159. The frequency with which CS6 occurs in ETEC of common serotypes without the antigens CS4 or CS5 suggests that it might be a colonization factor.     

Crystal structure of the O intermediate of the Leu93→Ala mutant of bacteriorhodopsin

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ～250 in the Leu93‐to‐Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue‐shifted λ_max (540 nm) of the mutant. A long‐lived substate of O with a red‐shifted λ_max (～565 nm) was trapped when the crystal of BR_L93A was flash‐cooled after illumination with green light. This substate (O_slow) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O_slow, however, the main chain of Lys216 is less distorted and retinal takes on the 13‐cis/15‐syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK_a value of which is suggested to be much lower in O_slow than in M. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

N,O versus O,O coordination in β-imino diketonato complexes: Role of the metal center and of the imino substituent

β-Imino carbonyl enolato metal(II) complexes of general formula [M((RCO)(R′CO)CC(R″)NH)₂] (M = Mn, Fe, Co, Ni, Cu, Zn, Pd, R = R′ = Me, R″ = CCl₃; M = Cu, Pd, R = R′ = Me, R″ = PhCO; R = Me, R′ = Ph, R″ = PhCO; R = R′ = Ph, R″ = PhCO) are easily synthesized by the reaction of metal(II) acetates with the proper β-enaminodiones in 1/2 molar ratio in ethanol at room temperature. In all the cases the trifunctional NOO β-imino carbonyl enolate ligand acts as bidentate to give ML₂ complexes, whose structure depends on the metal center and on the nature of the substituent R″ at the imino carbon. With R″ = CCl₃ an O,O coordination is observed for all the metal centers but one, in fact palladium(II) exhibits an N,O coordination through the imino nitrogen and one keto oxygen. By contrast with R″ = PhCO the ligand is always coordinated through the imino nitrogen and one keto oxygen atom.     

Influence of C-H...O interactions on the structural stability of β-lactamases

β-Lactamases produced by pathogenic bacteria cleave β-lactam antibiotics and render them ineffective. Understanding the principles that govern the structural stability of β-lactamases requires elucidation of the nature of the interactions that are involved in stabilization. In the present study, we systematically analyze the influence of CH...O interactions on determining the specificity and stability of β-lactamases in relation to environmental preferences. It is interesting to note that all the residues located in the active site of β-lactamases are involved in CH...O interactions. A significant percentage of CH...O interactions have a higher conservation score and short-range interactions are the predominant type of interactions in β-lactamases. These results will be useful in understanding the stability patterns of β-lactamases.     

Climatic potential of δ18O of Abies spectabilis from the Nepal Himalaya

A 50-year tree-ring δ¹⁸O chronology of Abies spectabilis growing close to the tree line (3850 m asl) in the Nepal Himalaya is established to explore its dendroclimatic potential. Response function analysis with ambient climatic records revealed that tree-ring δ¹⁸O is primarily governed by rainfall during the monsoon season (June–September), and the regression model accounts for 35% of the variance in rainfall. Extreme dry years identified in instrumental weather data are detected in the δ¹⁸O chronology. Further, tree-ring δ¹⁸O is much more sensitive to rainfall fluctuations than other tree-ring parameters such as width and density typically used in dendroclimatology. Correlation analyses with Niño 3.4 SST reveal time-dependent behavior of ENSO–monsoon relationships.     

Effects of H₂O₂ on electrical membrane properties of skeletal myotubes

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H₂O₂) to skeletal myotubes. H₂O₂ facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H₂O₂ was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H₂O₂. However, the fast inward rectifier K⁺ (K_IR) currents, activated by hyperpolarization, were reduced by H₂O₂, similar to the action of the potassium channel blockers Ba²⁺ and Cs⁺. The block of the outward tail current contributing to K_IR deactivation can explain the shorter latency for spike initiation. We propose that the K_IR current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells.     

RFLP Analysis of Progeny from Hybrid of Oryza alta×O.sativa

Oryza alta Swallen is a species of allotetraploid wild rice (CCDD, 2n = 48) with high biomass production and resistant to several insects and diseases. The high polymorphism (RFLP) between O. alta and O. sativa L. (average 90%) reveals the long phylogenical distance between these two species which may impede the crossing. The chromosome constitutions of several progeny from O. alta × O. sativa were analyzed with 22 mapped rice RFLP markers. The results showed that almost all triploid sterile plants which had 36 chromosomes were, as expected, composed of the chromosomes from two parents. But the two partial fertile regenerated plants from 02428 (O. sativa) × WHS601-2 (O. alta) which had only 24 and 25 chromosomes respectively, in most cases, had RFLP patterns similiar to their O. sativa parent. Therefore the chromosomes constituted their genomes were mainly from O. sativa or have high homology with the chromosomes of O. sativa. It seems evident that chromosome elimination had occurred during the wide hybridization as O. alta RFLP patterns could be found in them. Furthermore, that the introgression of O. alta chromosome fragment into that of O. sativa in an F2 plant was indicative that some kind of recombination also occurred. Regarding the rare chromosome pairing in PMCs, the probability of the existence of a nonconventional mechanism for the wild rice chromosomes (chromosome fragments ) to introgressing into cultivated rice genomes in a relatively short time was discussed.     

Does copper-d-penicillamine catalyze the dismutation of O2−?

It has been reported (M. Younes and U. Weser, 1977, Biochem. Biophys. Res. Commun.78, 1247–1253; E. Lengfelder and E. F. Elstner, 1978, Hoppe-Seyler's Z. Physiol. Chem.359, 751–757) that the complex [Cu(I)₈Cu(II)₆(D-penicillamine)₁₂Cl]^5?-efficiently catalyzes the dismutation of O₂^? and that this activity is resistant to both EDTA and CN^?. However, careful study has demonstrated that this complex is unable to catalyze the dismutation of O₂^?, but that it slowly decomposes to simpler copper complexes which are active. Moreover, the activity which is observed is suppressed by EDTA or by Chelex 100 treatment.     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Controlling conformational flexibility of an O₂-binding H-NOX domain

Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O(2) association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 ? crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O(2) as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.     

Evaluation of maximal O₂ uptake with undergraduate students at the University of La Reunion

The maximal rate of O? consumption (VO? max) constitutes one of the oldest fitness indexes established for the measure of cardiorespiratory fitness and aerobic performance. Procedures have been developed in which VO? max is estimated from physiological responses during submaximal exercise. Generally, VO? max is estimated using the classical renowned Astrand-Ryhming test. In young adults, poor fitness and low aerobic performance are often associated with a sedentary lifestyle, which is a well-described factor for the development of obesity and its related disorders such as cardiovascular diseases and type 2 diabetes. In the Indian Ocean, the inhabitants of La Reunion Island, a French overseas department, exhibit an increasing prevalence of obesity and type 2 diabetes. At the University of La Reunion, a new laboratory course involving students was designed to teach the indirect evaluation of their VO? max from the classical Astrand-Ryhming test and using a cycle ergometer as the exercise mode. Inverse and significant correlations were established between the students' fat mass percentages and their VO? max and between their waist-to-hip ratio and VO? max as well. Results from the international physical activity questionnaire showed that most participants in this laboratory were sedentary students. Therefore, this laboratory makes the students practice and understand the use of a classical test to estimate their VO? max. It also alerts them to the correlation between a sedentary lifestyle and higher body fat content. This exercise allowed students to use a scientific method to engage the problem of sedentary lifestyle, which is a real world issue.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O1 (Lányi), O3 (Habs), O13 and O14 (Wokatsch), and the serologically related strain NCTC 8505

Lipopolysaccharides from Pseudomonas aeruginosa O1 (Lányi classification), O3 (Habs classification), O13 and O14 (Wokatsch classification), and strain NCTC 8505, which is also related to serogroup O3 (Habs), have structurally similar O-specific polysaccharide chains built up of tetrasaccharide repeating units involving L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and a di-N-acyl derivative of bacillosamine (BacN): 2,4-diacetamido-2,4,6-trideoxy-D-glucose or 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose. The latter derivative was obtained free by solvolysis with hydrogen fluoride of carboxyl-reduced Habs O3 polysaccharide, and was identified by 1H-nuclear magnetic resonance spectroscopy and by mass spectrometry of the corresponding methylated alditol. Habs O3, Lányi O1, and Wokatsch O14 polysaccharides contained O-acetyl groups. Solvolysis with hydrogen fluoride of the native Habs O3 polysaccharide resulted in selective cleavage of the glycosidic linkages of 6-deoxy sugars to give the trisaccharide fragment involving all three N-acylated amino sugars. Similar solvolysis of NCTC 8505 polysaccharide afforded a mixture of disaccharide and trisaccharide with N,N'-diacetylbacillosamine at the reducing end. Smith degradation of Habs O3 polysaccharide resulted in selective oxidation of rhamnose to give a glycoside of a trisaccharide with glyceraldehyde as the aglycone. Smith degradation of NCTC 8505 polysaccharide was complicated by the formation of the glycoside of a trisaccharide with an aglycone of unknown structure. A trisaccharide with rhamnose at the reducing end was also isolated after Smith degradation of the latter polysaccharide. Analysis of the composition and structure of all oligosaccharides obtained, and detailed examination of the 13C-nuclear magnetic resonance spectra of these oligosaccharides, and of both intact and modified polysaccharides, revealed the following structures of the repeating units. The structure for the NCTC 8505 polysaccharide differs from that proposed previously [Tahara, Y. and Wilkinson, S.G. (1983) Eur. J. Biochem. 134, 299-304] in the configurations assigned to the glycosidic linkages of rhamnose and bacillosamine. The results obtained show the P. aeruginosa strains studied to represent three different O-serotypes in a single O-serogroup (Formula: see text).     

The effect of air containing O2 −,O2 +, CO2 − and CO2 + on the growth of seedlings ofHordeum Vulgaris

Equipment was devised which permitted the addition of specific gaseous ions to the atmosphere of plastic chambers in which seedlings of HORDEUM VULGARIS were grown in sand culture supplied with chemically defined nutrient solutions. Moderate densities of O₂ ^– or O₂ ⁺ ions (1.8×10⁴/cm³)in air containing an added 8% of O₂ accelerated the growth rate. A like number of CO₂ ^– or CO₂ ⁺ ions in air containing 8% of CO₂ inhibited growth, impeded the production of chlorophyll and devitalized the young seedlings. Evidence is presented that O₂ ^– and O₂ ⁺ stimulate the production of cytochromes and other Fe-containing enzymes through their action on the plant regulatory system responsible for the control of Fe metabolism. The toxic effect of CO₂ ^– and CO₂ ⁺ cannot be explained as yet.

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Zusammenfassung Eine Apparatur wurde entwickelt, die die Zufuhr von ionisiertem Gas der AtmosphÄre in Kammern gestattet. Darin wurden Keimlinge von HORDEUM VULGARIS in Sand mit chemisch definierten NÄhrlösungen gezüchtet. Konzentrationen von 1,8×10⁴/cm³ O₂ ^– und O₂ ⁺ in Luft mit zusÄtzlich 8% O₂ beschleunigten die Wachstumsrate. Die gleiche Menge CO₂ ^– und CO₂ ⁺ in Luft mit zusÄtzlich 8% CO₂ hemmte die Wachstumsrate, die Bildung von Chlorophyll und entkrÄftigte die Keimlinge. Es wird gezeigt,dass O₂ ^– und O₂ ⁺ die Bildung von Cytochrom und anderen eisenhaltigen Enzymen anregen durcn ihre Wirkung auf das den Fe-Stoffwechsel regulierende System der Pflanze. Die toxische Wirkung von CO₂ ^– und CO₂ ⁺ lÄsst sich noch nicht erklÄren.

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Resume On a construit un appareil permettant d'introduire dans 1'atmosphères des ions de gaz déterminés. On a alors effectué de telles adjonctions à l'air contenu dans des cellules de plastique dans lesquelles on cultivait HORDEUM VULGARIS sur du sable et dans une solution nutritive chimiquement définie. Des densités modérées d'ions O₂ ^– ou O₂ ⁺ (1,8×10⁴/cm³) dans de l'air additionné de 8% d'O₂ accélèrent la croissance. La meme concentration de CO₂ ^– et CO₂ ⁺ additionnée de 8% de CO₂ a ralenti la croissance et la formation de chlorophylle et a diminué la vitalite des plantes nouvellement germées. On démontre que O₂ ^– et O₂ ⁺ active la formation de cytochrome et d'autres enzymes ferreuses par suite de l'action de ces ions sur le système régularisant le métabolisme du fer dans la plante. L'effet toxique du CO₂ ^– et CO₂ ⁺ reste encore inexpliqué.

     

Do the higher oxidation states of the photosynthetic O2-evolving system contain bound H2O?

A modified mass spectrometer was used to determine whether the higher oxidation states of the photosynthetic O2-evolving system contain substrate water that is not freely exchangeable with the external medium. Our data indicated that the higher oxidation states contain no appreciable bound, non-exchangeable H2O. This suggests that H2O oxidation takes place via a rapid, concerted, all-or-none mechanism rather than by a mechanism involving stable, partially oxidized, H2O-derived intermediates. These findings set definite constraints on possible mechanisms of O2 evolution.