Identification of a copper protein as part of the nitrous oxide-reducing system in nitrite-respiring (denitrifying) pseudomonads

Copper is the essential transition element for nitrous oxide respiration in Pseudomonas perfectomarinus. Two novel kinds of copper proteins were detected in this organism. Their distribution was studied under different growth conditions and in other pseudomonads, as well as their association with N₂O reduction of intact cells. A low molecular mass copper protein (M _r 38,000) with a single absorption band at 340 nm (oxidized form), was found only in P. perfectomarinus and was not required for N₂O reduction. N₂O respiration was consistently associated with a high molecular mass copper protein (M _r 120,000) in P. perfectomarinus, Pseudomonas stutzeri, and in strains of Pseudomonas fluorescens that were capable of this type of respiration. The oxidized protein was violet to pink with absorption bands at 350, 480, 530, 620, and 780 nm. Pseudomonas chlororaphis and Pseudomonas aureofaciens which did not respire with N₂O as electron acceptor, did not contain the novel type of copper protein. Cytochrome patterns were compared in these denitrifying pseudomonads to search for the physiological electron carrier to N₂O reductase. The content and nature of the soluble c-type cytochromes depended strongly on the species and the particular growth condition.Abbreviations M _r relative molecular mass     

Identifying sources of nitrous oxide emission in anoxic/aerobic sequencing batch reactors (A/O SBRs) acclimated in different aeration rates

Both long term and batch experiments were carried out to identify the sources of the N₂O emission in anoxic/aerobic sequencing batch reactors (A/O SBRs) under different aeration rates. The obtained results showed that aeration rate has an important effect on the N₂O emission of A/O SBR and most of the N₂O was emitted during the aerobic phase. During the anoxic phase, nitrate ammonification was the major source of N₂O emission while denitrification performed as a sink of N₂O, in all three bioreactors. The N₂O emission mechanisms during the aerobic phase differed with the aeration rate. At low and high aeration rates (Run 1 and Run 3), both coupled-denitrification and nitrifier denitrification were ascribed to be the source of N₂O emission. At mild aeration rate (Run 2), nitrifier denitrification by Nitrosomonas-like ammonia oxidizing-bacterial (AOB) was responsible for N₂O emission while coupled-denitrification turned out to be a sink of N₂O because of the presence of inner anaerobic region in sludge flocs.     

Sequential reconstitution of copper sites in caeruloplasmin

Titration of apo-caeruloplasmin employing substoichiometric concentrations of [Cu(I)-(thiourea)₃]Cl was performed to elucidate possible sequential incorporation of copper into the different specific binding sites. The successful reconstitution was monitored by A₆₁₀ absorption, EPR spectroscopy and oxidase activity. Maximum activity and final absorption at 610 nm were reached after 20 min. When both A₆₁₀, indicative for type 1 copper, and oxidase activity were expressed per g-atom of copper, a sequential insertion was found. Owing to the specific data at the beginning, some type 3 copper appeared to be preferentially incorporated. After 3–4 g-atoms (including most of type 1 and type 2 copper), both absorption and oxidase activity surpassed transient maxima. Then type 3 and 4 copper were further bound to reach the known stoichiometry of six copper atoms per mole of protein.     

Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-asenosylmethionine metabilism in the rat

Inhalation of nitrous oxide oxidises cobalamin and, in turn, inactivates methionine synthetase which forms methionine from homocysteine and which requires cob[I]alamin as a co-factor. This study was planned to determine the effect of virtual cessation of methionine synthesis via a cobalamn-dependeent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N₂O but was restored to pre-N₂O levels after 6 days despite continuing N₂O exposure. Brain methionine fell within 12 h of N₂O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homoysteine methyltransferase in liver but this enzyme was not detected in brain. The activity of methionine synthetase remained very low in both liver and brain as long as N₂O inhalation was continued. There was an initial rise in liver S-adenosyl-methionine levels followed by a steady fall to 40% of its initial level after 11 days of N₂O exposure. However, there was no change in the level of S-adenosylmethionine in brain during this period. The data indicate that either brain meets its requirement by increased methionine uptake from plasma or that there are alternate pathways in brain for methionine synthesis other than those requiring a cobalamin coenzyme.     

Nitrous oxide productivity of soil fungi along a gradient of cattle impact

The objective of the study was to identify N₂O-producing fungi isolated from six qualitatively different sections of an overwintering pasture with substantial cattle impact. 80 out of 164 fungal isolates were considered as N₂O-producers in nitrite-containing medium, representing 33 fungal species of 23 different genera. Ability to produce N₂O was newly reported in eight genera: Arthrinium, Gibellulopsis, Ilyonectria, Lichtheimia, Paraphaeosphaeria, Purpureocillium, Tolypocladium and Westerdykella. Three levels of fungal N₂O-productivity were assigned according to the fraction of nitrite-N transformed into N₂O–N: < 1%, 1–10%, over 10%. Fungi capable of high and moderate transformation rates were predominantly isolated from sections under current or past cattle impact, where they contributed with a maximum of 65% of the total N₂O emissions. There was no significant effect of cultivation conditions on the fraction of N₂O-producing fungi. The results demonstrate that N₂O-producing fungi are a common constituent of fungal communities in soils impacted by overwintering cattle.     

Mechanism of nitrite reduction to nitrous oxide in a photodenitrifier,Rhodopseudomonas sphaeroide forma sp. denitrificans

The mechanism of anaerobic reduction of NO₂^? to N₂O in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, was investigated. With ascorbate-reduced phenazine methosulfate (PMS) as the electron donor, the nitrite reductase of this photodenitrifier reduced NO₂^? to NO and a trace amount of N₂O. With dithionite-reduced benzyl viologen as the electron donor, the major product of NO₂^? reduction was NH₂OH, and a trace amount of N₂O was also produced. The nitrate reductase itself had no NO reductase activity with ascorbate-reduced PMS. It was concluded that the essential product of NO₂^? reduction by the purified nitrite reductase is NO. Chromatophore membranes stoichiometrically produced N₂O from NO₂^? with any electron donor, such as dithionite-redduced benzyl viologen, ascorbate-reduced PMS or NADH/FMN. The membranes also contrained activity of NO reduction of N₂O with either ascorbate-reduced PMS or duroquinol. The NO reductase activity with duroquinol was inhibited by antimycin A. Stoichiometric production of N₂O from N₂^? also was observed in the reconstituted NO₂^? reduction system which contained the cytochrome bc₁ complex, cytochrome c₂, the nitrite reductase and duroquinol as the electron donor. The preparation of the cytochrome bc₁ complex itself contianed NO reductase activity. From these results the mechanism of NO₂^? reduction to N₂O in this photodenitrifier was determined as the nitrite reductase reducing NO₂^? to NO with electrons from the cytochrome bc₁ complex, and NO subsequently being reduced, without release, to N₂O with electrons from the cytochrome bc₁ complex by the NO reductase, which is closely associated with the complex.     

The copper site in nitrous oxide reductase

Summary The properties of the novel copper enzyme nitrous oxide reductase from denitrifyingPseudomonas stutzeri are described. Multifrequency electron paramagnetic resonance spectroscopy is used to characterize the various forms of the enzyme. The features observed at 2.4, 3.4, 4.5, 9.31 and 35 GHz are explained by a mixed-valence \s[Cu(1.5)\3. Cu(1.5)\s]S=\12 species with the unpaired electron delocalized between the two Cu nuclei. This site is also present in the catalytically inactive derivative of nitrous oxide reductase which was obtained from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The resemblance of the low-frequency electron paramagnetic resonance spectra to the spectra for the so-called Cu_A of cytochromec oxidase can be taken as a first indication that the Cu_A may have a structural and electronic arrangement similar to the electron-paramagnetic-resonance-detectable copper in nitrous oxide reductase. Results from oxidation/reduction experiments, and from a quantitative determination of sulfhydryl and disulfide residues in the various forms of nitrous oxide reductase, suggest the involvement of the redox-couple cysteine/cystine in the structural organization of the active site of nitrous oxide reductase.     

Excessive use of nitrogen in Chinese agriculture results in high N2O/(N2O+N2) product ratio of denitrification,primarily due to acidification of the soils

China is the world's largest producer and consumer of fertilizer N, and decades of overuse has caused nitrate leaching and possibly soil acidification. We hypothesized that this would enhance the soils' propensity to emit N₂O from denitrification by reducing the expression of the enzyme N₂O reductase. We investigated this by standardized oxic/anoxic incubations of soils from five long‐term fertilization experiments in different regions of China. After adjusting the nitrate concentration to 2 mM, we measured oxic respiration (R), potential denitrification (D), substrate‐induced denitrification, and the denitrification product stoichiometry (NO, N₂O, N₂). Soils with a history of high fertilizer N levels had high N₂O/(N₂O+N₂) ratios, but only in those field experiments where soil pH had been lowered by N fertilization. By comparing all soils, we found a strong negative correlation between pH and the N₂O/(N₂O+N₂) product ratio (r² = 0.759, P < 0.001). In contrast, the potential denitrification (D) was found to be a linear function of oxic respiration (R), and the ratio D/R was largely unaffected by soil pH. The immediate effect of liming acidified soils was lowered N₂O/(N₂O+N₂) ratios. The results provide evidence that soil pH has a marginal direct effect on potential denitrification, but that it is the master variable controlling the percentage of denitrified N emitted as N₂O. It has been known for long that low pH may result in high N₂O/(N₂O+N₂) product ratios of denitrification, but our documentation of a pervasive pH‐control of this ratio across soil types and management practices is new. The results are in good agreement with new understanding of how pH may interfere with the expression of N₂O reductase. We argue that the management of soil pH should be high on the agenda for mitigating N₂O emissions in the future, particularly for countries where ongoing intensification of plant production is likely to acidify the soils.     

Impact of COD/N ratio on nitrous oxide emission from microcosm wetlands and their performance in removing nitrogen from wastewater

Constructed wetlands (CWs) are considered to be important sources of nitrous oxide (N₂O). In order to investigate the effect of influent COD/N ratio on N₂O emission and control excess emission from nitrogen removal, free water surface microcosm wetlands were used and fed with different influent. In addition, the transformation of nitrogen was examined for better understanding of the mechanism of N₂O production under different operating COD/N ratios. It was found that N₂O emission and the performance of microcosm wetlands were significantly affected by COD/N ratio of wastewater influent. Strong relationships exist between N₂O production rate and nitrite (r = 0.421, p < 0.01). During denitrification process, DO concentration crucially influences N₂O production rate. An optimal influent COD/N ratio was obtained by adjusting external carbon sources for most effective N₂O emission control and best performance of the CWs in nitrogen removal from wastewater. It is concluded that under the operating condition of COD/N ratio = 5, total N₂O emission is minimum and the microcosm wetland is most effective in wastewater nitrogen removal.     

Characterization of a new type of dissimilatory sulfite reductase present in Thermodesulfobacterium commune

A new type of dissimilatory bisulfite reductase, desulfofuscidin, was isolated from the nonsporeforming thermophilic sulfate-reducing microorganism Thermodesulfobacterium commune. The molecular weight of the enzyme was estimated at 167,000 by sedimentation equilibrium, and the protein was pure by both disc electrophoresis and ultracentrifugation. The bisulfite reductase was a tetramer and had two types of subunits with an α₂β₂ structure and an individual molecular weight of 47,000. The enzyme exhibited absorption maxima at 576, 389, and 279 nm, with a weak band at 693 nm. Upon the addition of dithionite, the absorption maxima at 576 and 693 nm were weakened, and a new band appeared at 605 nm. The protein reacted with CO in the presence of dithionite to give a complex with absorption peaks at 593, 548, and 395 nm. The extinction coefficients of the purified enzyme at 576, 389, and 279 nm were 89,000, 310,000, and 663,000 M⁻¹ cm⁻¹, respectively. Siroheme was detected as the prosthetic group. The protein contains 20 to 21 nonheme iron atoms and 16 to 17 acid-labile sulfur groups per molecule. The data suggest the presence of four sirohemes and probably four (4Fe-4S) centers per molecule by comparison with desulfoviridin, the dissimilatory sulfite reductase from Desulfovibrio species. The protein contains 36 cysteine residues and is high in acidic and aromatic amino acids. The N-terminal amino acids of the α and β subunits were threonine and serine, respectively. With reduced methyl viologen as electron donor, the major product of sulfite reduction was trithionate, and the pH optimum for activity was 6.0. The enzyme was stable to 70°C and denatured rapidly above this temperature. The dependence of T. commune bisulfite reductase activity on temperature was linear between 35 and 65°C, and the Q₁₀ values observed were above 3. The presence of this new type of dissimilatory bisulfite reductase in T. commune is discussed in terms of taxonomic significance.     

Nitrous oxide in brackish Lakes Shinji and Nakaumi, Japan

Nitrous oxide (N₂O) was measured monthly from September 1997 to August 1998 in the brackish Lakes Shinji and Nakaumi, Japan. N₂O (5–37 μg N l⁻¹) was supersaturated in the overlying water on lake sediments from October 1997 to January 1998. The N₂O concentration in the hypolimnion was higher than that in the epilimnion on 17 October 1997, when N₂O was first observed in a water column of Lake Nakaumi. Afterward, N₂O was almost uniform throughout the water column and then disappeared on 16 February 1998. On the one hand, large amounts of N₂O were found throughout the year in the interstitial water in Lake Shinji, where a high concentration of nitrate was discharged from the Hii River. On the other hand, in Lake Nakaumi, stratified by halocline, a high concentration of N₂O was observed in the interstitial water only from winter to spring. N₂O concentrations in the interstitial water were about 10 to 1000 times as large as those in the overlying water. These results imply that N₂O was mainly produced at the sediment-water interface and was diffused to the overlying water. It was also suggested that the accumulation of N₂O in the sediment-water system was accelerated by a high concentration of hydrogen sulfide. Received: July 6, 2000 / Accepted: November 30, 2000     

Homogeneous alkylcysteine lyase of Acacia farnesiana: Fresh seedlings vs. acetone powders

Alkyleysteine lyase (EC 4.4.1.6) was purified essentially to homogeneity from both fresh hypocotyls of 5- to 8-day-old etiolated seedlings of Acacia farnesiana and acetone powders of such hypocotyls. The enzyme from the fresh material had twice the specific activity of that from the acetone powder. Sodium dodecylsulphate gel electrophoresis showed that both enzymes were composed of a subunit of M_rca 42 000. The final enzyme solutions were quite different in their absorbance spectra. The fresh hypocotyl enzyme had an absorbance maximum at 425 nm in addition to the 280 nm protein absorbance. This maximum in the visible region is due to bound pyridoxal phosphate. The acetone powder enzyme had the same maxima and in addition peaks at 498 and 340 nm. The fresh enzyme contained 1.8 mol cofactor/mol enzyme and the acetone powder enzyme 1.0 mol/mol. The KK_m for the probable natural substrate L-djenkolate was the same for both enzymes, 0.8 mM, but the V_max for the fresh was twice that of the acetone powder enzyme. The common practice of using acetone powder preparations for starting material in enzyme purifications would appear to require some caution.     

Nitrous oxide in brackish Lake Nakaumi, Japan II: the role of nitrification and denitrification in N2O accumulation

In order to understand the role of nitrification and denitrification in the accumulation of nitrous oxide (N₂O) in the hypolimnetic water of brackish Lake Nakaumi, the effects of dissolved oxygen (DO) concentration on these activities were investigated by incubation experiments. N₂O was produced during the oxidation of NH₄ ⁺ to NO₂ ⁻ in nitrification and during the reduction of NO₃ ⁻ to N₂ in denitrification. N₂O-producing activity by nitrification (N₂O_N) increased markedly with decreasing concentrations of DO. Low DO (10%–30% saturation) induced high N₂O_N. In contrast to nitrification, N₂O-producing activity by denitrification (N₂O_D) decreased with decreasing concentrations of DO. Little N₂O was accumulated during denitrification under low-level conditions of DO (10%–30%), because of further reduction of N₂O to N₂. It can therefore be assumed that N₂O produced as the by-product of nitrification is concurrently reduced to N₂ by denitrification under low-DO conditions. This would result in no substantial accumulation of N₂O during active nitrification in the hypolimnetic water of Lake Nakaumi. Received: July 6, 2001 / Accepted: December 10, 2001     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a²⁺₃-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a²⁺₃-NO and recombination of NO with cytochrome a²⁺₃ (in the 30–70 K region) revealed no differences in structure between cytochrome a²⁺₃ in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a²⁺₃-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a²⁺₃-NO. Photodissociation of cytochrome a²⁺₃-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a²⁺₃ to cytochrome a³⁺. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20–40 K region) which differ completely from those observed in cytochrome a²⁺₃-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a²⁺₃-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu²⁺_B-NO compound. The triplet species with Δm_s = 2 EPR signals at g 4 and Δm_s = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

Production of nitric oxide in Nitrosomonas europaea by reduction of nitrite

Nitrosomonas europaea and Nitrosovibrio sp. produced NO and N₂O during nitrification of ammonium. Less then 15% of the produced NO was due to chemical decomposition of nitrite. Production of NO and especially of N₂O increased when the bacteria were incubated under anaerobic conditions at decreasing flow rates of air, or at increasing cell densities. Low concentrations of chlorite (10 M) inhibited the production of NO and N₂, but not of nitrite indicating that NO and N₂O were not produced during the oxidative conversion of ammonium to nitrite. NO and N₂O were produced during reduction of nitrite with hydrazine as electron donor in almost stoichiometric quantities indicating that reduction of nitrite was the main source of NO and N₂O.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Prevalence of nitrous oxide reducing capacity in denitrifiers from a variety of habitats

Summary A total of 81 strains isolated by T. N. Gamble from soils from eight countries, fresh water lake sediments and nitrified poultry manure were examined for their ability to grow on N₂O as their electron acceptor, as well as for their tendency to produce N₂O from NO ₃ ⁻ in the absence and presence of acetylene. Seventy-seven of the 81 strains were confirmed as denitrifiers. Fifty-nine of the 77 strains grew on N₂O, while 12 strains produced N₂O but could not utilize it. Six strains reduced NO ₃ ⁻ to N₂ but could not grow on N₂O, suggesting that even if N₂O is always an intermediate product of denitrification, it is not always a freely diffusible intermediate. The organisms, however, would consume N₂O that accumulated early in growth and accumulated N₂O in the presence of acetylene. Thus the total number of N₂O users was 65 strains or 83% of the total tested. This implies that the N₂O reducing capacity of denitrifiers occur widely in nature. A high proportion ofPseudomonas fluorescens biotype II reduced N₂O. The accumulation of N₂O from NO ₃ ⁻ in the presence of acetylene provides strong evidence that N₂O is generally an intermediate in denitrification as well as provides additional support for the usefulness of this chemical as a general inhibitor of N₂O reduction.     

Spectroscopic properties of spinach catalase

Spinach catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6) has been purified to homogeneity. The purified enzyme has a specific activity of 25 000 units per mg protein. The presence of 2-mercaptoethanol and phenylmethylsulfonyl fluoride (PMSF) were required for high yields of the enzyme. The molecular weight of the enzyme was estimated to be 125 000 by gel filtration. Subunit analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single peptide with M_r 55 000. The enzyme, which exhibits optical absorbance maxima at 279, 403, 542, 592 and 723 nm and shoulders at 290, 500 and 630 nm, contains 2 mol iron per mol protein. One of the two irons can be attributed to protoheme, while the other iron appears to be present in a novel heme. The oxidized catalase exhibited two sets of high-spin, ferriheme EPR signals.     

Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.     

Characterization of indoles by thin-layer chromatography and in situ fluorometry

The characterization of microgram quantities of a number of naturally occurring and synthetic indoles through a combination of thin-layer chromatography and in situ fluorescence spectroscopy is reported. Instrumental detection limits of 0.03–0.05 μg of the indoles are possible using the native fluorescence of the indoles in the ultraviolet range, with excitation maxima in the range 285–310 nm and emission maxima in the range 345–360 nm. Spraying with a dilute acid solution (0.1 N H₂SO₄ in methanol) produces an additional pair of maxima, with excitation at about 350 nm and emission at about 450 nm. The presence of a polar compound such as sulfuric acid or dimethyl sulfoxide in the spray produces an enhancement of the indole fluorescence. The procedure should find application in the determination of indoles in biological samples.