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1.
Membrane introduction mass spectrometry was used to investigate the inhibitory effect of acetylene on the nitrous oxide reductase activity of intact cells of Pseudomonas nautica. We studied the effects of the concentrations of nitrate and sulfide, and the redox potential, which have all been implicated in causing a decrease in the inhibitory effects of acetylene during measurements of denitrification in natural environments. There was no evidence that the concentration of nitrate influenced the effect of acetylene. Lowering the redox potential with the reductant Ti(III)-nitrilotriacetate caused a slight alleviation of acetylene inhibition. Much greater effects at the same redox potential were obtained with concentrations of sulfide in the range 1-10 microM.  相似文献   

2.
The copper site in nitrous oxide reductase   总被引:2,自引:0,他引:2  
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 CuA of cytochromec oxidase can be taken as a first indication that the CuA 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.  相似文献   

3.
The mechanism for the reduction of nitric oxide to nitrous oxide and water in an A-type flavoprotein (FprA) in Moorella thermoacetica, which has been proposed to be a scavenging type of nitric oxide reductase, has been investigated using density functional theory (B3LYP). A dinitrosyl complex, [{FeNO}7]2, has previously been proposed to be a key intermediate in the NO reduction catalyzed by FprA. The electrons and protons involved in the reduction were suggested to “super-reduce” the dinitrosyl intermediate to [{FeNO}8]2 or the corresponding diprotonated form, [{FeNO(H)}8]2. In this type of mechanism the electron and/or proton transfers will be a part of the rate-determining step. In the present study, on the other hand, a reaction mechanism is suggested in which N2O can be formed before the protons and electrons enter the catalytic cycle. One of the irons in the diiron center is used to stabilize the formation of a hyponitrite dianion, instead of binding a second NO. Cleaving the N–O bond in the hyponitrite dianion intermediate is the rate-determining step in the proposed reaction mechanism. The barrier of 16.5 kcal mol−1 is in good agreement with the barrier height of the experimental rate-determining step of 14.8 kcal mol−1. The energetics of some intermediates in the “super-reduction” mechanism and the mechanism proceeding via a hyponitrite dianion are compared, favoring the latter. It is also discussed how to experimentally discriminate between the two mechanisms. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.  相似文献   

4.
Abstract: A component of Hamilton Harbour sediment prevented nitrous oxide (N2O) reduction in denitrification assays with a mixed population of endogenous bacteria and a pure culture (HH1) isolated from the sediment. A 5% (v/v) concentration of sediment in nutrient broth caused near maximum inhibition of N2O reduction. Sediment taken from a site closer to pollution sources (Site 906) was twice as inhibitory (as measured by N2O accumulation) as sediment from Site 910, further from pollution sources. N2O persistence was associated with the particulate sediment fraction only. Several heavy metals were tested at in situ concentrations, and ionic cadmium (Cd) and chromium (Cr) caused N2O accumulation. Ashed sediment did not cause N2O accumulation, but did decrease initial nitrate reduction rates with HH1.  相似文献   

5.
Characteristics of dissimilatory nitrate reduction by Propionibacterium acidi-propionici, P. freudenreichii, P. jensenii, P. shermanii and P. thoenii were studied. All strains reduced nitrate to nitrite and further to N2O. Recovery of added nitrite-N as N2O-N approached 100%, so that no other end product existed in a significant quantity. Specific rates of N2O production were 3 to 6 orders of magnitude lower than specific rates of N2 production by common denitrifiers. Oxygen but not acetylene inhibited N2O production in P. acidi-propionici and P. thoenii. Nitrite reduction rates were generally higher than nitrate reduction rates. The enzymes involved in nitrate and nitrite reduction were either constitutive or derepressed by anacrobiosis. Nitrate stimulated synthesis of nitrate reductase in P. acidi-propionici. Specific growth rates and growth yields were increased by nitrate. At 10 mM, nitrite was toxic to all strains, and at 1 mM its effect ranged from none to total inhibition. No distinction was obvious between incomplete forms of denitrification and dissimilatory nitrate reduction to ammonia. N2O production from nitrite by propionibacteria may represent a detoxication mechanism rather than a part of an energy transformation system.  相似文献   

6.
Nitric oxide is a denitrification intermediate which is produced from nitrite and then further converted via nitrous oxide to nitrogen. Here, the effect of low concentrations of the protonophore carbonylcyanide m-chlorophenylhydrazone on the time courses for dissolved gases was examined. While NO was found to oscillate, N(2)O only increased gradually as the reduction of nitrite progressed. The frequency and shape of protonophore-induced NO oscillations were influenced by temperature and the concentration of electron donor N,N,N',N'-tetramethyl-p-phenylene diamine (TMPD) in a manner compatible with the observed differential effects on the two involved enzyme activities. We demonstrated the existence of a pH interval, where [NO] oscillates even without uncoupler addition. Occurrence of nitric oxide oscillations in mixtures of a nitrite reductase mutant with a nitric oxide reductase mutant suggests that they cannot be due to a competition of the enzymes for redox equivalents from one common respiratory chain.  相似文献   

7.
Production of nitric oxide in Nitrosomonas europaea by reduction of nitrite   总被引:1,自引:0,他引:1  
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.  相似文献   

8.
Abstract NO production and consumption rates as well as N2O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N2O. Under aerobic conditions untreated soil showed very low release of NO and N2O but high consumption of NO. Fertilization with NH4+ or urea stimulated both NO and N2O production by nitrification. Addition of glucose at high soil moisture contents led to increased N2 and N2O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N2O production by denitrification. The production of NO and N2O was further stimulated at low moisture contents and after addition of glucose or NO3. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO3. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N2O release.  相似文献   

9.
Abstract NO production and consumption rates as well as N2O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N2O. Under aerobic conditions untreated soil showed very low release of NO and N2O but high consumption of NO. Fertilization with NH4+ or urea stimulated both NO and N2O production by nitrification. Addition of glucose at high soil moisture contents led to increased N2 and N2O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N2O production by denitrification. The production of NO and N2O was further stimulated at low moisture contents and after addition of glucose or NO3. Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO3. Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N2O release.  相似文献   

10.
Reduction of the four Fe centers is not required to initiate the reaction of the Halomonas halodenitrificans nitric oxide reductase (NOR) based on the facts that NOR in the form that ferric heme b(3) and non-heme iron (Fe(B)) are not bridged and/or the interaction between them is weakened and reversibly binds NO molecules, and that NOR in the form that only heme b(3) is oxidized reacts with NO molecules.  相似文献   

11.
The nitrous oxide (N2O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ (structural N2O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti. To aid homology studies, nosR from R. meliloti has also been sequenced. Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made. The heterologous expression of NosD, NosZ (N2O reductase), and NosL is also reported. A model of the CuA site in N2O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented. The model suggests that a His residue of the CuA domain may be a ligand to the catalytic CuZ site. In addition, the origin of the spectroscopically-observed Cys coordination to CuZ is discussed in terms of the sequence alignment of seven N2O reductases.  相似文献   

12.
李冀  朱莹  张晓君 《微生物学通报》2017,44(7):1714-1719
氧化亚氮(N_2O)是一种强力温室气体,能够破坏臭氧层。微生物含有的nosZ基因能够编码氧化亚氮还原酶,该酶可还原N_2O成为无害的N_2,因而对环境中nosZ基因的研究成为气候变化研究的一个热点。最近研究者对全基因组序列分析的结果揭示了一类新型nosZ基因(非典型nosZ Ⅱ基因)存在于更为广泛和多样的氮代谢微生物当中,这类nosZ编码的蛋白能够起到氧化亚氮还原酶的作用,并且广泛存在于多样的自然环境中。然而,针对含有非典型nosZ Ⅱ基因的微生物的相关研究还很不全面,这类微生物发挥作用的环境条件以及在N_2O还原过程中的特性仍然未知。本文主要综述了非典型nosZ Ⅱ基因与典型nosZ Ⅰ的主要差异、在环境中的分布情况以及未来研究方向的展望等。  相似文献   

13.
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.  相似文献   

14.
15.
16.
The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b3-FeB active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme FeB consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of CuB in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b3 and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an FeB (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of FeB the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b3 taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic FeB will form a μ-oxo bridge to heme b3 in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable μ-oxo bridge between heme b3 and FeB is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme FeB in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.  相似文献   

17.
《Process Biochemistry》2014,49(12):2228-2234
The competition for electrons has been recently demonstrated to affect the reduction rates of the nitrogen oxides in a methanol enriched denitrifying community. The aim of this study was to test if electron competition also occurred when other substrates were used for denitrification and if that could have an effect on the potential nitrous oxide (N2O) production and subsequent consumption. A denitrifying culture was developed in a sequencing batch reactor using nitrate as electron acceptor and a combination of acetate, ethanol and methanol as carbon sources. Four sets of batch tests were conducted using acetate, ethanol, methanol and a combination of the three carbon sources respectively. For each set the effect of nitrate, nitrite and nitrous oxide on each other reduction rates when present individually or in combination was assessed. Results show that reduction rates are affected by the type of substrate added, probably due to different microbial populations specialized with consuming a particular substrate. Also, N2O reduction rate is the most reduced under the different electron competition scenarios tested, which results in N2O accumulation in some cases. The effect of substrate limitation on N2O reduction was also assessed.  相似文献   

18.
Nitric oxide (NO) and nitrous oxide (N2O) are climatically important trace gases that are produced by both nitrifying and denitrifying bacteria. In the denitrification pathway, N2O is produced from nitric oxide (NO) by the enzyme nitric oxide reductase (NOR). The ammonia-oxidizing bacterium Nitrosomonas europaea also possesses a functional nitric oxide reductase, which was shown recently to serve a unique function. In this study, sequences homologous to the large subunit of nitric oxide reductase (norB) were obtained from eight additional strains of ammonia-oxidizing bacteria, including Nitrosomonas and Nitrosococcus species (i.e., both beta- and gamma-Proteobacterial ammonia oxidizers), showing widespread occurrence of a norB homologue in ammonia-oxidizing bacteria. However, despite efforts to detect norB homologues from Nitrosospira strains, sequences have not yet been obtained. Phylogenetic analysis placed nitrifier norB homologues in a subcluster, distinct from denitrifier sequences. The similarities and differences of these sequences highlight the need to understand the variety of metabolisms represented within a "functional group" defined by the presence of a single homologous gene. These results expand the database of norB homologue sequences in nitrifying bacteria.  相似文献   

19.
Nitrous oxide in brackish Lakes Shinji and Nakaumi, Japan   总被引:1,自引:0,他引:1  
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  相似文献   

20.
The reactivity of nitrite towards the copper(II) and copper(I) centers of a series of complexes with tridentate nitrogen donor ligands has been investigated. The ligands are bis[(1-methylbenzimidazol-2-yl)methyl]amine (1-bb), bis[2-(1-methylbenzimidazol-2-yl)ethyl]amine (2-bb), and bis[2-(3,5-dimethyl-1-pyrazolyl)ethyl]amine (ddah) and carry two terminal benzimidazole (1-bb, 2-bb) or pyrazole (ddah) rings and a central amine donor residue. While 2-bb and ddah form two adjacent six-membered chelate rings on metal coordination, 1-bb forms two smaller rings of five members. The binding affinity of nitrite and azide to the Cu(II) complexes (ClO4 as counterion) has been determined in solution. The association constants for the two ligands are similar, but nitrite is a slightly stronger ligand than azide when it binds as a bidentate donor. The X-ray crystal structure of the nitrite complex [Cu(ddah)(NO2)]ClO4 (final R=0.056) has been determined: triclinic P1ˉspace group, a=8.200(2) ?, b=9.582(3) ?, c=15.541(4) ?. It may be described as a perchlorate salt of a “supramolecular” species resulting from the assembly of two complex cations and one sodium perchlorate unit. The copper stereochemistry in the complex is intermediate between SPY and TBP, and nitrite binds to Cu(II) asymmetrically, with Cu-O distances of 2.037(2) and 2.390(3) ? and a nearly planar CuO2N cycle. On standing, solutions of [Cu(ddah)(NO2)]ClO4 in methanol produce the dinuclear complex [Cu(ddah)(OMe)]2(ClO4)2, containing dibridging methoxy groups. In fact the crystal structure analysis (final R=0.083) showed that the crystals are built up by dinuclear cations, arranged on a crystallographic symmetry center, and perchlorate anions. Electrochemical analysis shows that binding of nitrite to the Cu(II) complexes of 2-bb and ddah shifts the reduction potential of the Cu(II)/Cu(I) couple towards negative values by about 0.3 V. The thermodynamic parameters of the Cu(II)/Cu(I) electron transfer have also been analyzed. The mechanism of reductive activation of nitrite to nitric oxide by the Cu(I) complexes of 1-bb, 2-bb, and ddah has been studied. The reaction requires two protons per molecule of nitrite and Cu(I). Kinetic experiments show that the reaction is first order in [Cu(I)] and [H+] and exhibits saturation behavior with respect to nitrite concentration. The kinetic data show that [Cu(2-bb)]+ is more efficient than [Cu(1-bb)]+ and [Cu(ddah)]+ in reducing nitrite. Received: 19 November 1999 / Accepted: 20 January 2000  相似文献   

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