Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.     

Stoichiometry, ATP/2e values, and energy requirements for reactions catalyzed by nitrogenase from Azotobacter vinelandii.

The stoichiometry of the nitrogenase ATP-dependent H2 evolution and ecetylene reduction reactions using S2O4(2-) as an electron source was studied by various techniques. For each mole of S2O4(2-) oxidized to 2SO3(2-) by the enzyme-catalyzed reactions at 25 degrees and pH 8, 1 mol of H2 (1 mol of ethylene for acetylene reduction) and two protons are produced. Under these conditions, 4.5 mol of ATP was hydrolyzed to ADP and inorganic phosphate for each S2O4(2-) oxidized. ATP/S2O4(2-) (ATP/2e) values determined at 5 degree intervals from 10 to 35 degrees were found to go through a minimum at 20 degrees. This effect is explained in terms of possible enzyme structure modifications. Calorimetric measurements for the enzyme-catalyzed H2 evolution and acetylene reduction reactions gave deltaH values of -32.4 and -75.1 kcal/mol of S2O4(2-), respectively.     

The role of photoinduced electron transfer processes in photodegradation of the [Fe4(μ3-S)3(NO)7] cluster

Spectroscopic and electrochemical study of the [Fe(4)(mu(3)-S)(3)(NO)(7)](-) photochemical reaction and thermodynamic calculations of relevant systems demonstrate the redox character of this process. The photoinduced electron transfer between substrate clusters in excited and ground state (probably via exciplex formation) results in dismutation yielding unstable [Fe(4)(mu(3)-S)(3)(NO)(7)](2-) and [Fe(4)(mu(3)-S)(3)(NO)(7)](0). Back electron transfer between the primary products is responsible for fast reversibility of the photochemical reaction in deoxygenated solutions. In the presence of an electron acceptor (such as O(2), MV(2+) or NO) an oxidative quenching of the (*)[Fe(4)(mu(3)-S)(3)(NO)(7)](-) is anticipated, although NO seems to participate as well in the reductive quenching. The electron acceptors can also regenerate the substrate from its reduced form ([Fe(4)(mu(3)-S)(3)(NO)(7)](2-)), whereas the other primary product ([Fe(4)(mu(3)-S)(3)(NO)(7)](0)) decomposes to the final products. The suggested mechanism fits well to all experimental observations and shows the thermodynamically favored pathways and explains formation of all major (Fe(2+), S(2-), NO) and minor products (N(2)O, Fe(3+)). The photodissociation of nitrosyl ligands suggested earlier as the primary photochemical step cannot be, however, definitely excluded and may constitute a parallel pathway of [Fe(4)(mu(3)-S)(3)(NO)(7)](-) photolysis.     

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome p-450 rat liver microsomes.

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15--37 degrees C. The reactions were characterized by the values of the aniline oxidation rate constants, k2 = V/E0, where E0 is the initial concentration of cytochrome P-450: K 1/2 = 1.60 - 10(8) EXP (-13 400/RT) sec-1, k 2/2 = 1.66 - 10(9) exp (-14 500/RT) sec-1, k 3/2 = 6.83 - 10(9) exp (-15 300/RT) sec-1. The values of delta H0 and delta S0, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.     

Steady-state and transient kinetics of Escherichia coli nitric-oxide dioxygenase (flavohemoglobin). The B10 tyrosine hydroxyl is essential for dioxygen binding and catalysis

Escherichia coli expresses an inducible flavohemoglobin possessing robust NO dioxygenase activity. At 37 degrees C, the enzyme shows a maximal turnover number (V(max)) of 670 s(-1) and K(m) values for NADH, NO, and O(2) equal to 4.8, 0.28, and approximately 100 microM, respectively. Individual reduction, ligand binding, and NO dioxygenation reactions were examined at 20 degrees C, where V(max) is approximately 94 s(-1). Reduction by NADH occurs in two steps. NADH reduces bound FAD with a rate constant of approximately 15 microM(-1) s(-1), and heme iron is reduced by FADH(2) with a rate constant of 150 s(-1). Dioxygen binds tightly to reduced flavohemoglobin, with association and dissociation rate constants equal to 38 microM(-1) s(-1) and 0.44 s(-1), respectively, and the oxygenated flavohemoglobin dioxygenates NO to form nitrate. NO also binds reversibly to reduced flavohemoglobin in competition with O(2), dissociates slowly, and inhibits NO dioxygenase activity at [NO]/[O(2)] ratios of 1:100. Replacement of the heme pocket B10 tyrosine with phenylalanine increases the O(2) dissociation rate constant approximately 80-fold and reduces NO dioxygenase activity approximately 30-fold, demonstrating the importance of the tyrosine hydroxyl for O(2) affinity and NO scavenging activity. At 37 degrees C, V(max)/K(m)(NO) is 2,400 microM(-1) s(-1), demonstrating that the enzyme is extremely efficient at converting toxic NO into nitrate under physiological conditions.     

Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar spectra, O(2), NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V(max)) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme(-1) s(-1), respectively, at 37 degrees C with 200 microm O(2). The K(M) values for NO are low and range from 0.1 to 0.25 microm. V(max)/K(M)(NO) ratios of 900-2900 microm(-1) s(-1) indicate an extremely efficient dioxygenation mechanism. Approximate K(M) values for O(2) range from 60 to 90 microm. NO inhibits the dioxygenases at NO:O(2) ratios of > or =1:100 and makes true K(M)(O(2)) values difficult to determine. High and roughly equal second order rate constants for O(2) and NO association with the reduced flavoHbs (17-50 microm(-1) s(-1)) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O(2) (K(I)(CO) = approximately 1 microm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O(2) from inhibitory NO and CO.     

Respiration in relation to ion uptake in the crayfish Austropotamobius pallipes (Lereboullet).

1. O2 uptake was determined for periods of 23-46 h in salt-depleted crayfish held in deionized water (DW) or Na-free media at 10 degrees C. These media were replaced by artificial lakewater media (ALW) containing 0-2-0-6 mM Na and O2 uptake was again determined for periods of 24-66 h. 2. During net ion uptake in ALW the metabolic rate was either elevated or depressed. Standard metabolism in ALW altered by amounts equivalent to 0-1 - 15-5% (mean 6-4 (15) +/- 4-4% S. D.) of the metabolic rate measured during salt-depletion. On three occasions the metabolic rate was elevated by 22-0 - 66-7%, but some of this increase may have been due to locomotor activity. 3. The calculated values for thermodynamic work involved in ion transport were 0-056 - 0-268 J/10 g. h at 10 degrees C, or 1-5 - 7-2% of the mean standard metabolic rate. Most of the observed changes in metabolic rate lie within the limits of experimental error (ca. +/- 7%). Hence the energetic cost of ion transport is too small for direct measurement in intact crayfish.     

元素硫和双氰胺对蔬菜地土壤硝态氮淋失的影响

采用温室盆栽淋洗试验,以NH₄HCO₃为氮肥源,研究了元素硫(S₀)和双氰胺（DCD）对种葱和不种作物土壤NO₃^--N淋失量和NO₃^--N、NH₄⁺-N浓度的影响.结果表明,在12周试验期间,与对照相比,S₀+DCD和S₀处理NO₃^--N淋失量分别低83%～86%和83%;NH₄⁺-N淋失量分别高16.8～21.0 mg·盆^-1和20.4～25.0 mg·盆^-1;而同期无机氮（NO₃^--N、NH₄⁺-N）淋失量则低60%.试验结束后,,S₀+DCD和S₀处理土壤无机氮含量分别比对照高79.9%～85.4%和74.9%～82.6%,以NH₄⁺-N为主.S₀+DCD处理无机氮淋失量比S₀和DCD处理分别低4.6%～14.4%和15.4%～30.1%;试验结束后土壤无机氮分别高6.1%和16.8～36.0%.在Na₂S₂O₃+DCD、Na₂S₂O₃和DCD处理中也发现类似结果.可见S₀施入土壤具有与DCD同样的氨稳定和硝化抑制作用.S₀与DCD配合施用可使DCD的硝化抑制性增强,其作用机理是S₀氧化中间体S₂O₃^2-、S₄O₆^2-,具有抑制硝化和DCD降解作用,延缓DCD硝化抑制效果.S0与DCD配合施用可用于延缓太湖流域蔬菜地土壤NH₄⁺-N向NO₃^--N转化,减少氮向水体迁移风险.     

Dipalmitoylphosphatidylcholine-palmitic acid phase diagram studied by 13C nuclear magnetic resonance

The phase diagram of dipalmitoylphosphatidylcholine (DPPC) and palmitic acid mixtures in excess D2O was studied by 13C-NMR. Phase boundaries were determined from plots of apparent spin-spin relaxation time T2 (for both choline methyl and fatty acid chain carbons) versus temperature. A peritectic transition in the 1-10 mol% region, whose existence has been theoretically inferred from the Gibbs phase rule but which was undetectable by differential thermal analysis (DTA) (S.E. Schullery et al. Biochemistry, 20 (1981) 6818-6824), was located by NMR at 41.6 degrees C. A second, nearby peritectic line at 44 degrees C, which had been shown by DTA to extend from about 3-25 mol% palmitic acid, was seen by NMR only above 10 mol%. The palmitic acid/DPPC complex (2:1), with a sharp melting point at 64 degrees C, reported in earlier studies, was also seen by NMR. A phase diagram including both NMR and DTA results is presented. Important general conclusions from this study are: (i) NMR and scanning thermal analysis are complementary techniques for phase studies; each can see transitions that are invisible to the other. (ii) The case for the applicability of the Gibbs phase rule to lipid bilayer systems has been strengthened by the observance of two predicted, close-spaced boundaries. (iii) Low concentrations of fatty acids and related molecules can not be assumed to disperse as simple ideal solutes in the bilayer matrix.     

Effect of secondary substrate binding in penicillopepsin: contributions of subsites S3 and S2' to kcat

The kinetic parameters kcat, KM, and kcat/KM were determined at 25 degrees C and pH 4.5, 5.5, and 6.0 for the series of penicillopepsin substrates Ac-Alam-Lys-(NO2)Phe-Alan-amide, where (NO2)Phe is p-nitrophenylalanine and m and n equal 0-3. KM values at pH 6.0 were the same for all 12 peptides and averaged 0.088 +/- 0.02 mM but increased to different degrees at lower pH. In contrast, kcat values increased with increasing chain length. At pH 6 and at the pH optimum of kcat, the largest increases (about 37-fold on average) were obtained when alanine residues were added in positions P2' and P3. Only 1-2-fold increases were observed for positions P2, P3', P4, and P4'. These results show that occupation of subsites S2' and S3 is largely responsible for the rate enhancements caused by secondary substrate interactions with this series of peptides. Additional support for an important role of subsite S3 comes from the observation that the two peptides where m = 1 and n = 1 or 2, respectively, are cleaved not only between lysine and p-nitrophenylalanine but also between the latter and alanine, suggesting that occupation of subsite S3 by the N-terminal alanine overcomes the unfavorable interaction of alanine in subsite P1'. Subsite S3 is also important in the binding of pepstatin analogues and in transpeptidation reactions. It is proposed that the roles of subsites S3 and S2' are to facilitate the conversion of the first enzyme-substrate complex into a productive complex and to assist in the distortion of the scissile bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of the effects of molsidomine and 3-morpholinosydnonimine on the redox status of rat erythrocytes and reticulocytes

After enzymic biotransformation, molsidomine (MO) acts via the metabolite 3-morpholinosydnonimine (SIN-1) through spontaneous liberation of nitric oxide (NO) and superoxide (O(2)(.-)). The aim of this study was to compare the effects of MO and its active metabolite SIN-1 on the redox status of rat erythrocytes and reticulocytes. Rat erythrocyte as well as reticulocyte-rich red blood cell (RBC) suspensions were aerobically incubated (2 h, 37 degrees C) without (control) or in the presence of different concentrations of MO or SIN-1. In rat erythrocytes, biotransformation of MO resulted in the production of NO and nitroxyl (NO(-)). Endogenous superoxide anion (O(2)(.-)) participated in peroxynitrite generation. SIN-1 simultaneously liberated NO and O(2)(.-), which formed peroxynitrite (at least in part), but the liberated NO predominantly reacted with haemoglobin, forming methaemoglobin in erythrocytes. In reticulocytes, MO and SIN-1 caused an increase in the levels of both nitrite and 3-nitrotyrosine (an indicator of peroxynitrite), whereas they decreased the level of O(2)(.-). In reticulocytes, MO was metabolized into SIN-1 which led to the generation of NO, which reacted with O(2)(.-) (endogenous or exogenous) forming reactive nitrogen species. In conclusion, there are two metabolic pathways for MO biotransformation: one causing NO and NO(-) generation predominantly in erythrocytes and the other, via SIN-1 metabolism, in reticulocytes. The main difference between the action of MO and SIN-1 was that the latter caused oxidative damage in RBCs.     

Stability and sulfur-reduction activity in non-aqueous phase liquids of the hydrogenase from the hyperthermophile Pyrococcus furiosus.

Hydrogenase from the hyperthermophilic archaeon, Pyrococcus furiosus, catalyzes the reversible activation of H(2) gas and the reduction of elemental sulfur (S degrees ) at 90 degrees C and above. The pure enzyme, modified with polyethylene glycol (PEG), was soluble (> 5 mg/mL) in toluene and benzene with t(1/2) values of more than 6 h at 25 degrees C. At 100 degrees C the PEG-modified enzyme was less stable in aqueous solution (t(1/2) approximately 10 min) than the native (unmodified) enzyme (t(1/2) approximately 1 h), but they exhibited comparable H(2) evolution, H(2) oxidation, and S degrees reduction activities at 80 degrees C. The H(2) evolution activity of the modified enzyme was twice that of the unmodified enzyme at 25 degrees C. The PEG-modified enzyme did not catalyze S degrees reduction (at 80 degrees C) in pure toluene unless H(2)O was added. The mechanism by which hydrogenase produces H(2)S appears to involve H(2)O as the proton source and H(2) as the electron source. The inability of the modified hydrogenase to catalyze S degrees reduction in a homogeneous non-aqueous phase complicates potential applications of this enzyme.     

The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ > Mn2+ > Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophilus (T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization. The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase.     

Microscale biosensor for measurement of volatile fatty acids in anoxic environments

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO(-)(3) to N(2)O. A culture of denitrifying bacteria deficient in N(2)O reductase was immobilized in front of the tip of an electrochemical N(2)O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N(2)O sensor. The signal of the sensor, which corresponded to the rate of N(2)O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O(2), N(2)O, NO(2)(-), H(2)S, and NO(-)(3). Interference from NO(-)(3) was low and could be quantified and accounted for. The detection limit was equivalent to about 1 microM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32 per thousand. The functioning of the sensor over a temperature span of 7 to 30 degrees C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5 degrees C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.     

Presence of rhodanese in the cytosolic fraction of the fruit bat (Eidolon helvum) liver

Rhodanese was isolated and purified from the cytosolic fraction of liver tissue homogenate of the fruit bat, Eidolon helvum, by using ammonium sulphate precipitation and CM-Sephadex C-50 ion exchange chromatography. The specific activity was increased 130-fold with a 53% recovery. The K(m) values for KCN and Na(2)S(2)O(3) as substrates were 13.5 +/- 2.2mM and 19.5 +/- 0.7 mM, respectively. The apparent molecular weight was estimated by gel filtration on a Sephadex G-100 column to be 36,000 Da. The optimal activity was found at a high pH (pH 9.0) and the temperature optimum was 35 degrees C. An Arrhenius plot of the heat stability data consisted of two linear segments with a break occurring at 35 degrees C. The apparent activation energy values from these slopes were 11.5 kcal/mol and 76.6 kcal/mol. Inhibition studies on the enzyme with a number of cations showed that Mg(2+), Mn(2+), Ca(2+), and Co(2+) did not affect the activity of the enzyme, but Hg(2+) and Ba(2+) inhibited the enzyme.     

Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge

The objective of this study was to evaluate the performance characteristics of a hyperthermophilic digester system that consists of an acidogenic reactor operated at hyperthermophilic (70 degrees C) conditions in series with a methane reactor operated at mesophilic (35 degrees C), thermophilic (55 degrees C), and hyperthermophilic (65 degrees C) conditions. Lab-scale reactors were operated continuously, and were fed with co-substrates composed of artificial kitchen garbage (TS 9.8%) and excess sludge (TS 0.5%) at the volumetric ratio of 20:80. In the acidification step, COD solubilization was in the range of 22-46% at 70 degrees C, while it was 21-29% at 55 degrees C. The average protein solubilization was 44% at 70 degrees C. The double bond fatty acid removal ratio at 70 degrees C was much higher than at 55 degrees C. These results suggested that the optimal operation conditions for the acidogenic fermenter were about 3.1 days of HRT and 4 days of SRT at 70 degrees C. Methane conversion efficiency and the VS removal percentage in the methanogenic step following acidification was around 65% and 64% on average at 55 degrees C, respectively. The optimal operational conditions for this system are acidogenesis performed at 70 degrees C and methanogenesis at 55 degrees C. The key microbes determined in the hyperthermophilic acidification step were Anaerobic thermophile IC-BH at 6.4 days of HRT and Thermoanaerobacter thermohydrosulfuricus DSM 567 at 2.4 days of HRT. These results indicated that the hyperthermophilic system provides considerable advantages in treating co-substrates containing high concentrations of proteins, lipids, and nonbiodegradable solid matter.     

Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of approximately 155 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life of 11 min at this temperature indicates its thermostability. The enzyme is specific for NAD(H), and maximal specific activities were detected with L-threonine (10.3 U x mg(-1)) and acetoin (3.9 U x mg(-1)) in the oxidative and reductive reactions, respectively. Pf-TDH also utilizes L-serine and D-threonine as substrate, but could not oxidize other L-amino acids. The enzyme requires bivalent cations such as Zn2+ and Co2+ for activity and contains at least one zinc atom per subunit. Km values for L-threonine and NAD+ at 70 degrees C were 1.5 mm and 0.055 mm, respectively.     

The mode of decomposition of Angeli's salt (Na2N2O3) and the effects thereon of oxygen,nitrite, superoxide dismutase,and glutathione

The classical view of the aerobic decomposition of Angeli's salt is that it releases NO(2)(-) + NO(-)/HNO the latter then reacting with O(2) to yield ONOO(-). An alternative that has recently been proposed envisions electron transfer to O(2) followed by decomposition to NO(2)(-) + NO. The classical view is now strongly supported by the observation that the rates of decomposition of Angeli's salt under 20% O(2) or 100% O(2) were equal. Moreover, NO(2)(-), which inhibits this decomposition by favoring the back reaction, was more effective in the absence of agents that scavenge NO(-)/HNO. It is thus clear that Angeli's salt is a useful source of NO(-)/HNO for use in defined aqueous systems. The measurements made in the course of this work allowed approximation of the rate constants for the reactions of NO(-)/HNO with NO(2)(-), O(2), glutathione, or Cu, Zn superoxide dismutase. The likelihood of the formation of NO(-)/HNO in vivo is also discussed.     

Relation between redox potentials and rate constants in reactions coupled with the system oxygen-superoxide.

Univalent oxidation-reduction reactions coupled with the oxygen-superoxide system were investigated in the reactions shown in eq 3 and 8, where Q and Q.- stand for p-benzoquinone and p-benzosemiquinone, respectively. From kinetic experiments the following rate constants were obtained at pH 7.0:k3 = 4.5 x 10(4) M-1 sec-1 and k8 = 3 x 10(-2) M-1 sec-1. With known values of k-3 and k-8, and of E0' for the systems Q-Q.- (0.10 V) and Cyt c3+ - Cyt c2+ (0.255 V), the calculated values of E0(O2-O2.-) were found to lie in the range between -0.27 and -0.33 V.     

Anaerobic sulfide oxidation with nitrate by a freshwater Beggiatoa enrichment culture

A lithotrophic freshwater Beggiatoa strain was enriched in O2-H2S gradient tubes to investigate its ability to oxidize sulfide with NO3- as an alternative electron acceptor. The gradient tubes contained different NO3- concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O2, H2S, pH, and NO3- were determined with microsensors. The more NO3- that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO3- to oxidize sulfide at depths below the depth that O2 penetrated. In the presence of NO3- Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO3- was added. These thick mats spatially separated O2 and sulfide but not NO3- and sulfide, and therefore NO3- must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O2 flux and a twofold-higher sulfide flux into the NO3- -exposed mats compared to the fluxes for controls without NO3-. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S0 with NO3- as the electron acceptor.