Formaldehyde oxidation and methanogenesis

Formaldehyde oxidation by cell-free extracts of Methanobacterium thermoautotrophicum was shown to drive methanogenesis from CH3-S-coenzyme M or HCHO under a nonreductive atmosphere of N2. Under N2 when HCHO was the sole source of carbon and reducing equivalents in the reaction, it underwent oxidation and reduction events (disproportionation), the sum of the reactions being 3 HCHO + H2O----CH4 + 2 HCOO - + 2H+. This reaction predicts a CH4/HCHO ratio of 1/3, which is in agreement with the experimental finding of 1/2.9. In extracts of the mesophilic methanogen Methanococcus voltae and the extreme thermophile Methanococcus jannaschii , which exhibited formate dehydrogenase activity, the CH4/HCHO ratio was 1/2. NADPH stimulated methane formation from HCHO under N2. An unidentified, oxygen-labile cofactor, the formaldehyde activation factor, present in boiled-cell extract was discovered. Methanopterin , an oxygen-stable molecule, also substituted for boiled-cell extract.     

Specific degradation of H. pylori urease by a catalytic antibody light chain

Catalytic antibodies capable of digesting crucial proteins of pathogenic bacteria have long been sought for potential therapeutic use. Helicobacter pylori urease plays a crucial role for the survival of this bacterium in the highly acidic conditions of human stomach. The HpU-9 monoclonal antibody (mAb) raised against H. pylori urease recognized the alpha-subunit of the urease, but only slightly recognized the beta-subunit. However, when isolated both the light and the heavy chains of this antibody were mostly bound to the beta-subunit. The cleavage reaction catalyzed by HpU-9 light chain (HpU-9-L) followed the Michaelis-Menten equation with a K(m) of 1.6 x 10(-5) m and a k(cat) of 0.11 min(-1), suggesting that the cleavage reaction was enzymatic. In a cleavage test using H. pylori urease, HpU-9-L efficiently cleaved the beta-subunit but not the alpha-subunit, indicating that the degradation by HpU-9-L had a specificity. The cleaved peptide bonds in the beta-subunit were L121-A122, E124-G125, S229-A230, Y241-D242, and M262-A263. BSA was hardly cleaved by HpU-9-L, again indicating the digestion by HpU-9-L was specific. In summary, we succeeded in the preparation of a catalytic antibody light chain capable of specifically digesting the beta-subunit of H. pylori urease.     

Photosensitized and catalytic oxidation of DNA by metallophthalocyanine-oligonucleotide conjugates

The synthesis of new metallophthalocyanine-oligonucleotide conjugates is reported. These conjugates can cause sequence-specific photosensitized or catalytic oxidation of DNA by molecular oxygen.     

Ferritin oxidation and proteasomal degradation: protection by antioxidants

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H₂O₂ induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation.

Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol^® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin.     

Ferritin oxidation and proteasomal degradation: Protection by antioxidants

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H₂O₂ induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation.

Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol^® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin.     

One-electron oxidation in the degradation of creosote polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium.

The abilities of whole cultures of Phanerochaete chrysosporium and P. chrysosporium manganese peroxidase-mediated lipid peroxidation reactions to degrade the polycyclic aromatic hydrocarbons (PAHs) found in creosote were studied. The disappearance of 12 three- to six-ring PAHs occurred in both systems. Both in vivo and in vitro, the disappearance of all PAHs was found to be very strongly correlated with ionization potential. This was true even for compounds beyond the ionization potential thresholds of lignin peroxidase and Mn3+. Deviations from this correlation were seen in the cases of PAHs which are susceptible to radical addition reactions. These results thus begin to clarify the mechanisms of non-lignin peroxidase-labile PAH degradation in the manganese peroxidase-lipid peroxidation system and provide further evidence for the ability of this system to explain the in vivo oxidation of these compounds.     

Pyrene degradation by a Mycobacterium sp.: identification of ring oxidation and ring fission products.

The degradation of pyrene, a polycyclic aromatic hydrocarbon containing four aromatic rings, by pure cultures of a Mycobacterium sp. was studied. Over 60% of [14C]pyrene was mineralized to CO2 after 96 h of incubation at 24 degrees C. High-pressure liquid chromatography analyses showed the presence of one major and at least six other metabolites that accounted for 95% of the total organic-extractable 14C-labeled residues. Analyses by UV, infrared, mass, and nuclear magnetic resonance spectrometry and gas chromatography identified both pyrene cis- and trans-4,5-dihydrodiols and pyrenol as initial microbial ring-oxidation products of pyrene. The major metabolite, 4-phenanthroic acid, and 4-hydroxyperinaphthenone and cinnamic and phthalic acids were identified as ring fission products. 18O2 studies showed that the formation of cis- and trans-4,5-dihydrodiols were catalyzed by dioxygenase and monooxygenase enzymes, respectively. This is the first report of the chemical pathway for the microbial catabolism of pyrene.     

Probing the topography of proteins in solution by photosensitized oxidation. The catalytic region of papain

The thiol function of the single cysteinyl residue at the active site of papain was selectively conjugated with either the dinitrophenyl or the fluorescein thiocarbamyl group. Absorption, fluorescence and circular dichroism studies showed that, in both cases, the introduction of the chromophoric moiety caused no significant alterations of the spatial geometry characteristic of native papain. Irradiation of the dinitrophenyl derivative by visible light resulted in the specific photo-oxidation of histidine-159 and trytophan-177, which appeared to be the only potentially photo-oxidizable amino acids adjacent to the labelling group; their distance from the thiol function of cysteine-25 was evaluated to be about 5 Å. These two residues do not appear to make an essential contribution to the structural stability of papain, since the oxidative modification of their side chains induced only limited modifications of the over-all conformation of the protein. On irradiation of the fluorescein-papain complex, the preferential photooxidation of tryptophan-177, histidine-159 and tryptophan-26 took place; in parallel, there was a drastic collapse of the tertiary structure of the protein molecule. It is concluded that tryptophan-26 is oriented in a direction different from that of tryptophan-177 and histidine-159; hence, this residue is probably not directly involved in the catalytic function of the enzyme. However, the intactness of its side chain is critical for maintaining the native three-dimensional structure of papain.     

First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models.

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri-tert-butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.     

Pyrene degradation by a Mycobacterium sp.: identification of ring oxidation and ring fission products

The degradation of pyrene, a polycyclic aromatic hydrocarbon containing four aromatic rings, by pure cultures of a Mycobacterium sp. was studied. Over 60% of [14C]pyrene was mineralized to CO2 after 96 h of incubation at 24 degrees C. High-pressure liquid chromatography analyses showed the presence of one major and at least six other metabolites that accounted for 95% of the total organic-extractable 14C-labeled residues. Analyses by UV, infrared, mass, and nuclear magnetic resonance spectrometry and gas chromatography identified both pyrene cis- and trans-4,5-dihydrodiols and pyrenol as initial microbial ring-oxidation products of pyrene. The major metabolite, 4-phenanthroic acid, and 4-hydroxyperinaphthenone and cinnamic and phthalic acids were identified as ring fission products. 18O2 studies showed that the formation of cis- and trans-4,5-dihydrodiols were catalyzed by dioxygenase and monooxygenase enzymes, respectively. This is the first report of the chemical pathway for the microbial catabolism of pyrene.     

The role of peroxide in haem degradation. A study of the oxidation of ferrihaems by hydrogen peroxide.

The oxidation of ferrihaems by H2O2 was studied as a model for haem catabolism. Rates of ferrihaem oxidation were evaluated by using a new computer-based method that measures the loss in catalytic activity of the ferrihaem during oxidation. For protoferrihaem, deuteroferrihaem, coproferrihaem and mesoferrihaem, oxidation proceeded via the monomeric species and no dimer contribution was detectable. The pH-dependence of oxidation was studied in the range 6.5--11. Within experimental error, the data were compatible with an inverse linear dependence on [H+]. This was interpreted in terms of attack by HO2- on monomeric ferrihaem. The specific second-order rate constants for oxidation of monomeric species by HO2- were of the same order of magnitude for all the ferrihaems, and were in the sequence coproferrihaem greater than protoferrihaem greater than mesoferrihaem congruent to deuteroferrihaem. A model is suggested involving formation of a ferrihaem monomerperoxide complex, which may either dissociate with the formation of a peroxidatic intermediate or be involved in an intramolecular oxidation of the ferrihaem. Haem catabolism may occur via the same or a similar intermediate.     

Mechanistic insight into the catalytic inhibition by nitroxides of tyrosine oxidation and nitration

BackgroundNitroxide antioxidants (RNO^•) protect from injuries associated with oxidative stress. Tyrosine residues in proteins are major targets for oxidizing species giving rise to irreversible cross-linking and protein nitration, but the mechanisms underlying the protective activity of RNO^• on these processes are not sufficiently clear.MethodsTyrosine oxidation by the oxoammonium cation (RN⁺=O) was studied by following the kinetics of RNO^• formation using EPR spectroscopy. Tyrosine oxidation and nitration were investigated using the peroxidase/H₂O₂ system without and with nitrite. The inhibitory effect of RNO^• on these processes was studied by following the kinetics of the evolved O₂ and accumulation of tyrosine oxidation and nitration products.ResultsTyrosine ion is readily oxidized by RN⁺=O, and the equilibrium constant of this reaction depends on RNO^• structure and reduction potential. RNO^• catalytically inhibits tyrosine oxidation and nitration since it scavenges both tyrosyl and ^•NO₂ radicals while recycling through RN⁺=O reduction by H₂O₂, tyrosine and nitrite. The inhibitory effect of nitroxide on tyrosine oxidation and nitration increases as its reduction potential decreases where the 6-membered ring nitroxides are better catalysts than the 5-membered ones.ConclusionsNitroxides catalytically inhibit tyrosine oxidation and nitration. The proposed reaction mechanism adequately fits the results explaining the dependence of the nitroxide inhibitory effect on its reduction potential and on the concentrations of the reducing species present in the system.General significanceNitroxides protect against both oxidative and nitrative damage. The proposed reaction mechanism further emphasizes the role of the reducing environment to the efficacy of these catalysts.     

Protein oxidation and degradation during postmitotic senescence

Oxidized and cross-linked proteinacious materials (lipofuscin, age pigments, ceroid, etc.) have long been known to accumulate in aging and in age-related diseases, and some studies have suggested that age-dependent inhibition of the proteasome and/or lysosomal proteases may contribute to this phenomenon. Cell culture studies trying to model these aging effects have almost all been performed with proliferating (divisionally competent) cell lines. There is little information on nondividing (postmitotic) cells; yet age-related accumulation of oxidized and cross-linked protein aggregates is most marked in postmitotic tissues such as brain, heart, and skeletal muscles. The present investigation was undertaken to test whether oxidized and cross-linked proteins generally accumulate in nondividing, IMR-90 and MRC-5, human cell lines, and whether such accumulation is associated with diminished proteolytic capacities. Since both protein oxidation and declining proteolytic activities might play major roles in the age-associated accumulation of intracellular oxidized materials, we tested for protein carbonyl formation, proteasomal activities, and lysosomal cathepsin activities. For these studies, confluent, postmitotic IMR-90 and MRC-5 fibroblasts (at various population doubling levels) were cultured under hyperoxic conditions to facilitate age-related oxidative senescence. Our results reveal marked decreases in the activity of both the proteasomal system and the lysosomal proteases during senescence of nondividing fibroblasts, but the peptidyl-glutamyl-hydrolyzing activity of the proteasome was particularly inhibited. This decline in proteolytic capacity was accompanied by an increased accumulation of oxidized proteins.     

Rate of catalytic oxidation and decomposition of NADH by graphite in aqueous solutions

Graphites of different manufacture and origin exert positive but different accelerations on the rate of oxidation of NADH to NAD⁺ in aqueous solution; different proportions of the oxidized form of NAD retain enzymatic activity depending on the nature of the graphite. Oxidative pretreatment of the graphite surfaces increases the rates of NADH oxidation, but subsequent silanization of the surfaces to attach alkylamine groups causes the rates to decrease. The experimental results suggest the presence of at least two types of sites on graphite surfaces: One very reactive site which produces a high percentage of an enzymatically inactive reaction product of NADH and is itself deactivated during the course of reaction, and another type of site which promotes the oxidation of NADH to enzymatically active NAD⁺ in high yields.     

Iron oxidation by casein.

Casein accelerates the oxidation of Fe(II) to Fe(III) and the resulting Fe(III) remains strongly bound to the casein. Removal of phosphate from the casein abolishes the oxidative process. The oxidation rate is proportional to the casein concentration, and with high casein concentrations the rate is pseudo-first-order with respect to Fe(II) with a half-life of approximately 2 minutes. The oxidized iron is stoichiometrically bound to the casein, each mg of casein binding approximately 10 micrograms of iron. The physiological significance is discussed.