Effects of melecular oxygen on lignin degradation by Phanerochaete chrysosporium

Research has demonstrated that shallow stationary cultures of white-rot wood-destroying Basidiomycetes degrade lignin (¹⁴C-lignin → ¹⁴CO₂) at much higher rates under O₂ than under air. The present study, conducted with $\text{Phanerochaete chrysosporium}$, showed that the effect on rate is a dual one: a) Immediately preceeding appearance of the lignin-degrading system the partial pressure of O₂ determines the amount of ligninolytic activity that develops in cultures; and b) after the system develops, the partial pressure of O₂ affects the rate of oxidation.     

Mechanism of the enzyme-catalyzed bioluminescent oxidation of coelenterate-type luciferin.

The use of a fully active, synthetic analogue of coelenterate-type luciferin labeled in the carbonyl position with ¹⁴C and ¹⁸O was used to probe the mechanism of the $\text{Renilla}$ luciferase catalyzed oxidative decarboxylation of this compound. In the presence of ¹⁷O₂, the CO₂ produced in this oxidation can be shown to contain approximately one ¹⁷O atom per CO₂ molecule. This result is consistent with a cyclic peroxide or dioxetanone-type mechanism. In the presence of luciferase, the oxygen in the luciferin carbonyl group is rapidly exchanged with solvent water prior to the production of CO₂. Thus, the $\text{reaction}$ CO₂ contains considerable oxygen derived from water, via exchange with the carbonyl group, and about one oxygen from O₂ via a cyclic peroxide.     

Generation of hydroxyl radical and its involvement in lignin degradation by Phanerochaete chrysosporium

Cultures of $\text{Phanerochaete chrysosporium}$ produced ethylene from methional and 2-keto-4-thiomethyl butyric acid (KTBA) only under conditions when the organism was competent to degrade [¹⁴C]-lignin to ¹⁴CO₂. The ability of several mutant strains to produce ethylene reflected their ability to degrade lignin. Hydroxyl radical scavengers including thiourea, salicylate, mannitol, 4-0-methylisoeugenol, as well as catalase, inhibited fungal lignin degradation, fungal ethylene production from methional and KTBA, as well as ethylene generation from KTBA via Fenton's reagent and γ-irradiation. In addition, methional inhibited fungal lignin degradation and lignin inhibited ethylene generation from methional. All of these results indicate that hydroxyl radical plays an important role in lignin degradation by $\text{P. chrysosporium}$.     

Mevalonate metabolism: Effect of K2Cr2O7, a renal tubular toxin

K₂Cr₂O₇ was given to one member of 10 pairs of rats 2.5 hours before injection of $\text{RS}\text{-[5-}{}^{14}\text{C]}$mevalonate. The poisoned rats expired 30.8 ± 3.5% less ¹⁴CO₂ than their unpoisoned controls in the 2.5 hours after mevalonate injection (p < .001) and incorporated 16.3 ± 7.1% less label into renal (p<.05) and 63.7 ± 12.0% more label into hepatic unsaponifiable lipids (p < .001). K₂Cr₂O₇ had no effect on the oxidation of Na[1-¹⁴C]octanoate. These changes occurred at least 14 hours before any previously demonstrated effect of K₂Cr₂O₇ on the kidney and represent an early biochemical expression of renal tubular damage.     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

Mechanism of oxidation of carbon monoxide by bacteria

Double label experiments were performed employing ¹³CO and either H₂¹⁸O or ¹⁸O₂ in the presence of a CO utilizing bacterium. CO₂ generated was trapped and $\text{m}\text{e}$ ratios $\text{47}\text{45}$ showed that the second oxygen atom in the oxidation of CO to CO₂ by this bacterium comes neither from O₂ nor H₂O.     

Kinetic studies of 18O exchange from inorganic phosphate catalyzed by Mg2+-activated unadenylylated glutamine synthetase (E. coli w).

Oxygen-18 exchange out of [¹⁸O]Pi catalyzed by Mg²⁺-activated unadenylated glutamine synthetase from $\text{E.}$$\text{coli}$ was followed by ³¹P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the ${}^{16}\text{O}{}^{18}\text{O}$ in the species P¹⁸O₄, P¹⁸O₃¹⁶O₁, P¹⁸O₂¹⁶O₂, P¹⁸O₁¹⁶O₃, P¹⁶O₄ during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of P_i. The rate constant for ${}^{16}\text{O}{}^{18}\text{O}$ exchange was 410±40 min^?1 while the rate constant for net reaction (ATP formation) was 62±4 min^?1. Thus exchange proceeds ～7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ($\text{J.}$$\text{Biol.}$$\text{Chem.}$ (1976) $\text{251}$, 5558) for the Mn²⁺-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) $\text{73}$, 476) was successfully used to fit the oxygen exchange data in this paper.     

Conversion of sterigmatocystin to aflatoxin B 1 by Aspergillus parasiticus

¹⁴C-Sterigmatocystin isolated from cultures of $\text{Aspergillus}$$\text{versicolor}$ supplemented with (1-¹⁴C)acetate was shown to be efficiently converted to aflatoxin B₁ by the resting mycelium of $\text{A. parasiticus}$. The experimental results may indicate a biosynthetic pathway leading from 5-hydroxysterigmatocystin to sterigmatocystin and then to aflatoxin B₁.     

On the role of superoxide in reactions catalyzed by rubredoxin of Pseudomonas oleovorans

In the presence of NADH, and the reductase and rubredoxin components of the ω-hydroxylation system of $\text{Pseudomonas oleovorans}$, epinephrine is oxidized to adrenochrome at pH 7.8, and the reaction is strongly inhibited by the addition of superoxide dismutase (SDM). Boiled SDM has no effect on the reaction rate. The oxidation reaction is oxygen-dependent, and approximately 1 mole of H₂O₂ is produced per mole of O₂ consumed. The stoichiometry between NADH oxidation and adrenochrome formation is approximately 2:1. Epoxidation and epinephrine oxidation are mutually competitive reactions, despite the fact that the epoxidation reaction is not stimulated by a superoxide generating system nor inhibited by SDM.     

Structure elucidation of a 6-methylbenzo[a]pyrene-DNA adduct formed by horseradish peroxidase in vitro and mouse skin in vivo

Activation of polycyclic aromatic hydrocarbons (PAH) by horseradish peroxidase (HRP) with H₂O₂ has been studied as a model system for one-electron oxidation. This peroxidase has been used to catalyze binding of $\text{6-}\text{[}{}^{14}\text{C]methylbenzo}\text{[a]}\text{pyrene}$ (BP-6-CH₃) to DNA, which was purified, hydrolyzed to deoxyribonucleosides and analyzed by high pressure liquid chromatography (HPLC). The predominant hydrocarbon-DNA adduct observed was identified as BP-6-CH₃ bound at the 6-methyl group to the 2-amino group of dG, confirming that activation by HRP occurs by one-electron oxidation. When DNA from mouse skin treated in vivo with [¹⁴C]BP-6-CH₃ was purified, hydrolyzed and analyzed by HPLC, a profile was observed which was qualitatively similar to that from the peroxidase system. In particular, the identified adduct with the hydrocarbon bound at the 6-methyl group to the 2-amino group of dG was obtained. These results demonstrate that one-electron oxidation is the mechanism of activation by HRP for aromatic hydrocarbons and indicate that the same mechanism may occur in mouse skin, a target tissue for hydrocarbon carcinogenesis.     

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.     

In vivo regulation of adipose tissue protein kinase by adenosine 3',5'-monophosphate mediated glucagon stimulation.

The cytochrome P-450 content of primary hepatocyte cultures was maintained at levels close to those found $\text{in vivo}$ by using a defined medium containing testosterone, thyroxine, hydrocortisone, estradiol, glucagon, insulin, linoleic acid and oleic acid. Using these cultures, [¹⁴C]aflatoxin B₁, a potent liver carcinogen, was metabolized primarily to water-soluble metabolites. In agreement with $\text{in vivo}$ results, aflatoxin M₁ was the only nonpolar metabolite detected. In addition, a significant portion of radioactivity was covalently bound to cell constituents. These results suggest that primary hepatocyte cultures may be a good model of the liver for studying the metabolism and mechanism of action of toxic chemicals.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Incorporation of 18O2 and absence of stereospecificity in primary product formation during fungal metabolism of a lignin model compound

The metabolism of a lignin substructure model compound, 1,2-bis(3-methoxy-4-ethoxyphenyl)propane-1,3-diol (Ia) in ligninolytic cultures of Phanerochaete chrysosporium was studied to help elucidate the biochemical mechanism of lignin degradation. The primary reaction was cleavage of the model compound between C₁ and C₂ of the propane moiety to produce 1-(3-methoxy-4-ethoxyphenyl)ethane-1,2-diol and a C₆-C₁ product (probably 3-methoxy-4-ethoxybenzaldehyde). Other identified products arose secondarily; all were further metabolized. Even though the model compound was a mixture of four stereoisomers, no stereoselectivity was observed in its metabolism. In cultures under ¹⁸O₂, the initial cleavage produced the diol product with ≈70% enrichment by ¹⁸O in the benzyl alcohol group. The diol was a mixture of the two possible enantiomers, and the O₂-derived hydroxyl was incorporated at the asymmetric (benzyl) carbon. (Limited optical activity in the diol was traced to selective further metabolism of the D form.) These results show that the primary cleavage reaction lacked stereospecificity and was primarily oxygenative, implicating a nonspecific oxygenase or a nonenzymatic reaction involving activated oxygen. Preliminary experiments demonstrated no cell homogenate activity against Ia.     

Influence of culture parameters on lignin metabolism byPhanerochaete chrysosporium

Culture parameters influencing metabolism of synthetic¹⁴C-lignins to¹⁴CO₂ in defined media have been studied in shallow batch cultures of the ligninolytic wood-destroying HymenomycetePhanerochaete chrysosporium Burds. Study of the effect of O₂ concentration in the gas phase above non-agitated cultures indicated essentially complete absence of attack on the lignin polymer at 5% O₂ in N₂, and a 2- to 3-fold enhancement by 100% O₂ as compared to air (21% O₂). Agitation of the cultures resulting in the formation of mycelial pellets greatly suppressed lignin decomposition. The optimum culture pH for lignin decomposition was 4 to 4.5, with marked suppression above 5.5 and below 3.5. The source of nutrient nitrogen (NO ₃ ^– , NH ₄ ⁺ , amino acids) had little influence on lignin decomposition, but the concentration of nitrogen was critical; decomposition at 24 mM was only 25–35% of that at 2.4 mM N. Thiamine was the only vitamin required for growth and lignin decomposition. Under the optimum conditions developed, decomposition of 5 mg of synthetic lignin was accompanied by utilization of approximately 100 mg of glucose. The influence of the various culture parameters was analogous for metabolism of synthetic lignin labeled in the ring-,side chain-, and methoxyl carbon atoms.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

Invivo conversion of 3-ketoceramide to ceramide in rat liver

A doubly labeled 3-ketoceramide, [1-¹⁴C] lignoceroyl [1-³H₂] 3-ketosphingosine ($\text{3H}{}^{14}\text{C}$ ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal $\text{3H}\text{>}{}^{14}\text{C}$ ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver.     

Aryl alpha-haloalkyl ketoximes as enzyme modifying agents. The reaction of alpha-haloacetophenone oximes with the active site of papain

$\text{Syn}$-α-chloroacetophenone oxime has been found to inactivate papain rapidly at pH 7 and 25.0^O in a 1:1 stoichiometric fashion as measured by rate assays with $\text{p}$-nitrophenyl $\text{N}$-benzyloxycarbonylglycinate and sulfhydryl group titrations with 5,5′-dithiobis-(2-nitrobenzoic acid). By the use of a ¹⁴C label in the halomethyl function a similar stoichiometric reaction with papain could be demonstrated for $\text{syn}$-α-bromoacetophenone oxime despite the rapidity of the competitive nonenzymatic solvolysis of the latter compound under the conditions employed. These results indicate that a new class of reactive modifying agents, α-haloalkyl oximes, can be used for the selective alkylation of catalytically essential functional groups in enzyme active sites.     

The relative effectiveness of .OH,H2O2,O2−, and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using selective free radical scavengers

The relative effectiveness of oxidizing (^.OH, H₂O₂), ambivalent (O₂^?) and reducing free radicals (e^? and CO₂^?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ($\text{R}$(enz)) were measured and the rates of damage to membranes ($\text{R}$(mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. ^.OH, O₂^? and H₂ O₂ were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e^? and CO₂^? were the least effective. $\text{R}$(enz) values of O₂^? and H₂O₂ were 10-times and of ^.OH 15-times that of e^?. $\text{R}$(mb) values were quite similar for e^? and H₂O₂ (about twice that of O₂^?), while that of ^.OH was 3-times that of O₂^?. Hence, with respect to $\text{R}$(mb): ${}^{\mathrm{.}}\text{OH}\text{>}\text{e}{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{O}{}_2{}^{\mathrm{?}}$ , and with respect to $\text{R}$(enz): ${}^{\mathrm{.}}\text{OH}\text{>}\text{O}{}_2{}^{\mathrm{?}}\text{=}\text{H}{}_2\text{O}{}_2\text{>}\text{e}{}^{\mathrm{?}}$. The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H₂O₂ added as a chemical reagent and H₂O₂ formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H₂O₂ but markedly susceptible to the latter.