Distal Oxygen Research Articles

Heme oxygenase (HO-1). Evidence for electrophilic oxygen addition to the porphyrin ring in the formation of alpha-meso-hydroxyheme.

Previous studies have established that reaction of the rat heme-heme oxygenase complex with H2O2 proceeds normally to give verdoheme, whereas reaction of the complex with meta-chloroperbenzoic acid yields a ferryl (FeIV = O) species and a protein radical but no verdoheme. The heme-heme oxygenase complex is shown here to react regiospecifically with ethyl hydroperoxide to give alpha-meso-ethoxyheme. Formation of this product exactly parallels the formation of alpha-meso-hydroxyheme in the normal reaction supported by cytochrome P450 reductase/NADPH or H2O2. These results rule out a nucleophilic mechanism for the alpha-meso-hydroxylation catalyzed by heme oxygenase and indicate that it involves electrophilic (or possibly radical) addition of the distal oxygen of iron-bound peroxide (FeIII-OOH) to the porphyrin ring.

Intramolecular and intermolecular hydrogen bonding in triphenylphosphine derivatives of copper(I) carboxylates, (Ph3P)2CuO2C(CH2)nCOOH. Role of copper(I) in the decarboxylation of malonic acid and its derivatives

The air-sensitive copper(I) hydrogen dicarboxylate complexes (Ph 3 P) 2 CuO 2 C(CH 2 ) n COOH (n=1, 2) and derivatives thereof have been synthesized from 1 equiv of the corresponding dicarboxylic acid and cuprous butyrate. The solid-state structures of these derivatives exbibit quite different hydrogen bonding motifs. That is, the malonate derivative (1) contains a three-coordinate copper(1) center composed of two phosphine ligands and a monodentate carboxylate ligand. The appended carboxylic acid forms a strong intramolecular hydrogen bond with the distal oxygen atom of the carboxylate group bound to copper

Partial Left Heart Bypass for Thoracic Aorta Repair

Emergency repair of the torn descending thoracic aorta has been associated with an almost 15% incidence of paraplegia. The literature to date suggests that the incidence of paraplegia is not influenced by mechanical adjuncts to enhance distal aortic perfusion during cross-clamping and therefore, "clamp and sew" has been considered an acceptable technique. The purpose of the present study was to review our experience with repair of descending thoracic aortas using partial left heart bypass and to compare this favorable initial experience with the available data on the use of the heparinless centrifugal pump. A retrospective review of the routine use of partial left heart bypass in 16 consecutive patients with descending thoracic aorta disruptions. Results were compared with similar reports in the recent literature on trauma. A level 1 trauma facility in the Denver, Colo, metropolitan area. All patients with multisystem blunt trauma with a mean injury severity score of 36. Repair of the descending thoracic aorta disruption using partial left heart bypass with a heparinless centrifugal pump. Primary outcome measures were survival and paraplegia; other monitored variables included proximal and distal aortic pressure, flow rates, and oxygen transport. Among the 14 survivors (88%) there were no cases of paraplegia, and intraoperative hemodynamics and oxygen transport were well maintained with partial left heart bypass. This experience is added to the available reported data on the use of the centrifugal pump. In these additional 42 patients, the mortality rate was 7%, with no incidence of paraplegia. This collective experience failed to disclose a single case of paraplegia when partial left heart bypass was employed for repair of descending thoracic aorta. Moreover, the use of partial left heart bypass in this cohort of critically injured patients is associated with survival and perioperative morbidity rates comparable with the best recent reports of emergency thoracic aortic repair.

Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the X-ray structure.

Thiocamphor is an unusual substrate for P450cam in that in the X-ray structure it binds in the active site pocket in two distinct orientations and neither of these orientations are consistent with the 5-alcohol being the primary product. Other camphor analogs such as norcamphor or camphane bind in a single orientation consistent with the 5-alcohol being a major product. We present an analysis of four 175 ps molecular dynamics trajectories of thiocamphor-bound cytochrome P450cam. The first two trajectories were calculated for cytochrome P450cam with thiocamphor bound in both its major and minor crystallographic orientations. In the second set of simulations, a single oxygen atom was added as a distal ligand to the heme group in order to model the putative ferryl oxygen reaction intermediate. Trajectories were again calculated starting with thiocamphor in its major and minor orientations. While the protein dynamics were quite similar in all four trajectories, the substrate showed distinctly different motions in each of the trajectories. In particular, the preferred substrate orientations were very different in the presence of the ferryl oxygen than in the absence of that oxygen. The preferred orientations in the absence of the distal oxygen were consistent with the 3-alcohol being the major product, while the preferred orientations in the presence of the distal oxygen were consistent with the 5-alcohol being a major product. These simulations offer an explanation for the inconsistency between the X-ray data and the product profile.

Calixarenes. 27. Synthesis, characterization, and complexation studies of double-cavity calix[4]arenes

The ease with which calix[4]arenes can be selectively substituted at the distal phenolic oxygens is employed to advantage to build a second cavity and create two classes of «double-cavity calixarenes». Through the use of 3,5-dinitrobenzoyl chloride, 3-nitro-5-carbomethoxybenzoyl chloride, 3,5-dinitrobenzyl chloride, or 3-nitro-5-carbomethoxybenzyl chloride the diesters 2 and 16, diethers 3 and 17, and ether-ester 10 have been prepared. The second cavity is built by reduction of the nitro groups to amino groups to give compounds 4, 5, 13, 18, and 19 followed by treatment with a diacyl chloride

Oxylipin metabolism in the red alga Gracilariopsis lemaneiformis: Mechanism of formation of vicinal dihydroxy fatty acids

Conversion of arachidonic acid into the vicinal diol fatty acid 12 R,13 S-dihydroxy- 5 Z,8 Z,10 E,14 Z-eicosatetraenoic acid using an acetone powder of the marine red alga, Gracilariopsis lemaneiformis, occurred via intermediate formation of 12 S-hydroperoxy-5 Z,8 Z, 10 E,14 Z-eicosatetraenoic acid. Incubations of the linoleic acid-derived 13 S- and 13 R-hydroperoxy-9 Z,11 E-octadecadienoic acids led to the formation of 13 R,14 S-dihydroxy-9 Z, 11 E-octadecadienoic acid and 13 S,14 S-dihydroxy-9 Z,11 E-octadecadienoic acid, respectively, whereas incubation of 9 S-hydroperoxy-10 E,12 Z-octadecadienoic acid resulted in the formation of 8 S,9 R-dihydroxy-10 E, 12 Z-octadecadienoic acid. Experiments with 18O 2-labeled 13 S-hydroperoxyoctadecadienoic acid demonstrated that the oxygens of the two hydroxyl groups of 13 R,14 S-dihydroxy-9 Z,11 E-octadecadienoic acid originated in the hydroperoxy group of the substrate. Furthermore, experiments with mixtures of unlabeled and 18O 2-labeled 13 S-hydroperoxyoctadecadienoic acid showed that conversion into 13 R,14 S-dihydroxyoctadecadienoic acid occurred by a reaction involving an intramolecular hydroxylation at C-14 by the distal hydroperoxide oxygen. The existence of a hydroperoxide isomerase in G. lemaneiformis which catalyzes the conversion of fatty acid hydroperoxides into vicinal diol fatty acids is postulated.

Shortcomings of Using Two Jet Nebulizers in Tandem with an Aerosol Face Mask for Optimal Oxygen Therapy

Herein, a laboratory model which allows measurement of simulated distal airway oxygen percentage at different breathing patterns is described to illustrate the shortcomings of conventional O2 devices and, in particular, the aerosol face mask with two jet nebulizers (AFM-DF) in tandem. A table showing the degree of dilution which occurred during simulation of various breathing patterns while using the AFM-DF is also presented. Data revealed that when 60 percent was desired, 13 of 27 measurements were less than 55 percent. The worst-case scenario for 60 percent desired was 48 percent measured. When 80 percent was desired, less than 70 percent was delivered in 24 of the 27 breathing patterns simulated. Less than 60 percent was measured on 12 occasions, with 51 percent being the lowest measurement. When 100 percent O2 was desired, less than 80 percent was measured in 25 of 27 breathing patterns. Less than 60 percent was measured in ten of those. Fifty percent was the lowest analyzed value for the 100 percent setting. The inadequacy of AFM-DF is described in three case studies. A high-flow nonrebreathing face mask (HFM) to address the subset of patients is also discussed. A peak inspiratory flow prediction chart is also documented and may be useful in setting optimal flows when using high-flow systems. The patients in whom intubation and mechanical ventilation (or use of continuous mask CPAP) are indicated can be more clearly identified with a trial of high-flow O2 therapy (with a system that assures adequate flow to meet the patient's peak inspiratory flow demands). In the remainder of patients, those higher-risk modalities will be precluded.

Hemoglobins of the Lucina pectinata/bacteria symbiosis. II. An electron paramagnetic resonance and optical spectral study of the ferric proteins.

We report an optical and EPR spectral study of three hemoglobins, Hb I, II, and III, from the gill of the clam Lucina pectinata. Hemoglobin I reacts much more avidly with hydrogen sulfide than do Hbs II and III. The proximal ligand to the heme iron of each hemoglobin is histidyl imidazole. The acid/alkaline transition of ferric Hb I occurs with pK 9.6; those of ferric Hbs II and III with pK 6.6 and 5.9, respectively. At their acid limits each ferric hemoglobin exists as aquoferric hemoglobin. Broadening of the g = 6 resonance suggests that the bound water enjoys great positional freedom. Ferric Hb I, at the alkaline limit (pH 11), exists as ferric hemoglobin hydroxide. Ferric Hbs II and III, at their alkaline limit (pH 7.5), each exist as equal mixtures of two species. The low spin species with optical maxima near 541 and 576 nm and g values of 2.61, 2.20, and 1.82, are identified as ferric hemoglobin hydroxide. The high spin species, with optical maxima near 486 and 603 nm and g values of 6.71, 5.87, and 5.06, resemble Dicrocoelium hemoglobin and hemoglobin MSaskatoon. Here we show that Hbs II and III resemble hemoglobin MSaskatoon in which a distal tyrosinate oxygen ligated to the ferric heme iron at alkaline pH is displaced by water at acid pH. The H2S product of ferric Hb I is identified as ferric hemoglobin sulfide.

Analysis of the active site of the flavoprotein p-hydroxybenzoate hydroxylase and some ideas with respect to its reaction mechanism

The flavoprotein p-hydroxybenzoate hydroxylase has been studied extensively by biochemical techniques by others and in our laboratory by X-ray crystallography. As a result of the latter investigations, well-refined crystal structures are known of the enzyme complexed (i) with its substrate p-hydroxybenzoate and (ii) with its reaction product 3,4-dihydroxybenzoate and (iii) the enzyme with reduced FAD. Knowledge of these structures and the availability of the three-dimensional structure of a model compound for the reactive flavin 4a-hydroperoxide intermediate has allowed a detailed analysis of the reaction with oxygen. In the model of this reaction intermediate, fitted to the active site of p-hydroxybenzoate hydroxylase, all possible positions of the distal oxygen were surveyed by rotating this oxygen about the single bond between the C4a and the proximal oxygen. It was found that the distal oxygen is free to sweep an arc of about 180 degrees in the active site. The flavin 4a-peroxide anion, which is formed after reaction of molecular oxygen with reduced FAD, might accept a proton from an active-site water molecule or from the hydroxyl group of the substrate. The position of the oxygen to be transferred with respect to the substrate appears to be almost ideal for nucleophilic attack of the substrate onto this oxygen. The oxygen is situated above the 3-position of the substrate where the substitution takes place, at an angle of about 60 degrees with the aromatic plane, allowing strong interactions with the pi electrons of the substrate. Polarization of the peroxide oxygen-oxygen bond by the enzyme may enhance the reactivity of flavin 4a-peroxide.

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 Å resolution : Analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 Å. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 Å. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 Å structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 Å structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 Å and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14∘ compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10∘ for the enzyme-substrate complex and 19∘ for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme—product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring. This arrangement may stabilize negatively charged forms of the isoalloxazine ring that occur during catalysis. Two solvent molecules in the active site are in ban der Waals' contact with the flavin ring. Of particular interest is the position of a solvent molecule near the N terminus of helix H10. This solvent molecule is within hydrogen-bonding distance of two peptide nitrogen atoms and could be replaced by the distal oxygen atom of the flavin 4a-peroxide anion after reaction with oxygen.

Structure of the dysprosium-glycocholate complex in submicellar aqueous solution: paramagnetic mapping by proton nuclear magnetic resonance spectroscopy. An approximation for the intrinsic "bound" relaxation rates in the case of nondilute paramagnetic systems.

A paramagnetic NMR study of the structure of the calcium-glycocholate complex in submicellar solution, utilizing dysprosium as an isomorphous lanthanide replacement of calcium, is presented. The dysprosium-induced relaxation rate (1/T1) enhancements of certain glycocholate protons have been used to estimate internuclear distances between these protons and the metal ion. An approximation to calculate the intrinsic relaxation rate (1/T1) enhancements for a nondilute paramagnetic solution is given in the Appendix. From these data, and analysis based on conformation averaging and minimum energy conformations, a molecular model of the dysprosium-glycocholate complex in submicellar aqueous solution has been constructed. In this model the metal ion has a unidentate, first-sphere interaction with the proximal oxygen atom of the glycine carboxyl. The metal ion has second-sphere interactions with the peptide bond carbonyl oxygen (3.6 A) and the distal carboxyl oxygen (4.4 A). The metal ion to hydroxyl oxygen distances (8.4-12.4 A) are not compatible with any metal ion to hydroxyl coordination. The side chain appears to exist in one predominant conformation. All six oxygen atoms of glycocholate, the peptide bond carbonyl, the carboxyl group, and the hydroxyl groups are on the alpha face of the bile salt molecule. On the basis of these features we conclude that in the submicellar state the solution structure of the dysprosium-glycocholate complex displays a metal ion enhanced segregation of polar versus nonpolar groups to the two separate faces of the molecule, which may result in a facilitated hydrophobic interaction of different complex units.

Venoarterial bypass: A technique for spinal cord protection

In the present study, we examined the effects of various levels of oxygen tension on spinal cord blood flow while using somatosensory evoked potentials to monitor spinal cord sensory function during hypoxia. In this experiment, six adult, mongrel dogs were heparinized and placed on right atrial-femoral artery bypass with an oxygenator in the bypass circuit. The aorta was cross-clamped proximal to the left subclavian artery, and bypass flow and fluid balance were adjusted so as to maintain a distal aortic perfusion pressure of greater than 80 mm Hg. Oxygen flow to the oxygenator was lowered by graded decrements to provide decreasing levels of oxygen tension, which ultimately approached pure venoarterial bypass. Each successive oxygen level was maintained for 30 minutes. Spinal cord blood flow was measured with radioactive microspheres, and latency and amplitude of somatosomatic evolved potentials were continuously monitored. The somatosensory evolved potential signal was invariably present as long as the distal aortic pressure was greater than 80 mm Hg; there were several transient hypotensive episodes (less than 5 minutes), which were accompanied by reversible loss of somatosensory evolved potentials. The spinal cord blood flow increased from 13.6 to 119.7 ml/100 gm/min as the distal oxygen tension fell to a mean value of 30 mm Hg, while latency of somatosensory evolved potentials increased 19.3% and amplitude decreased 43.3%. These results suggest the following conclusions: (1) In response to hypoxia, spinal cord blood flow dramatically increases and somatosensory evolved potentials deteriorate (increase in latency and decrease in amplitude). (2) However, during prolonged hypoxia, spinal cord sensory function can be maintained by sufficiently high flow rates and perfusion pressures. (3) Somatosensory evolved potentials can be used to monitor continuously spinal cord sensory function under these conditions.

Chemical mechanisms for cytochrome P-450 hydroxylation: evidence for acylation of heme-bound dioxygen.

Using isotopic tracer methods, we have shown that dihydrolipoic acid (2,3-thioctic acid) acylates the distal oxygen of ferrous oxygenated Pseudomonas cytochrome P-450, forming a transient acyl peroxide intermediate that facilitates oxygen-oxygen bond cleavage. Single-turnover studies with 18O2 indicate one oxygen-18 atom incorporated into the carboxylate group of lipoic acid for each oxygen-18 inserted into the substrate, camphor, forming the product, exo-5-hydroxycamphor. Such a branching ratio for label indicates that water is initially released from an unlageled position and illustrates that the general P-450 mixed-function oxidase stoichiometry generates H218O from 18O2 only after multiple-turnover equilibration with the acylating carboxylate oxygen. Formation of an acyl peroxide state is a natural intermediate in peracid, "oxene", or radical mechanisms for methylene carbone oxygenation.