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1.
In eukaryotes, small amounts of nitrite confer cytoprotection against ischemia/reperfusion‐related tissue damage in vivo, possibly via reduction to nitric oxide (NO) and inhibition of mitochondrial function. Several hemeproteins are involved in this protective mechanism, starting with deoxyhemoglobin, which is capable of reducing nitrite. In facultative aerobic bacteria, such as Pseudomonas aeruginosa, nitrite is reduced to NO by specialized heme‐containing enzymes called cd1 nitrite reductases. The details of their catalytic mechanism are summarized below, together with a hypothesis on the biological role of the unusual d1‐heme, which, in the reduced state, shows unique properties (very high affinity for nitrite and exceptionally fast dissociation of NO). Our results support the idea that the nitrite‐based reactions of contemporary eukaryotes are a vestige of earlier bacterial biochemical pathways. The evidence that nitrite reductase activities of enzymes with different cellular roles and biochemical features still exist today highlights the importance of nitrite in cellular homeostasis.  相似文献   

2.
The cd1 NiRs (nitrite reductases) are enzymes catalysing the reduction of nitrite to NO (nitric oxide) in the bacterial energy conversion denitrification process. These enzymes contain two distinct redox centres: one covalently bound c-haem, which is reduced by external electron donors, and another peculiar porphyrin, the d1-haem (3,8-dioxo-17-acrylate-porphyrindione), where nitrite is reduced to NO. In the present paper, we summarize the most recent results on the mechanism of nitrite reduction by the cd1 NiR from Pseudomonas aeruginosa. We discuss the essential catalytic features of this enzyme, with special attention to the allosteric regulation of the enzyme's activity and to the mechanism employed to avoid product inhibition, i.e. trapping of the active-site reduced haem by the product NO. These results shed light on the reactivity of cd1 NiRs and assign a central role to the unique d1-haem, present only in this class of enzymes.  相似文献   

3.
真菌异化硝酸盐还原机理的研究进展   总被引:1,自引:0,他引:1  
真菌异化硝酸盐还原途径的发现打破了反硝化仅存在于原核细胞这一传统观念。真菌异化硝酸盐还原途径是在环境中氧供给受限的情况下发生的, 包括反硝化和氨的发酵。硝酸盐能诱导产生反硝化作用的酶, 其中, 硝酸盐还原酶与亚硝酸还原酶位于线粒体中, 它们所催化的酶促反应能偶联呼吸链ATP合成酶合成ATP, 同时产生NO。与参与反硝化作用前两个酶不同, 真菌NO还原酶能以NADH为直接电子供体将NO还原为N2O, 在NAD+的再生和自由基NO的脱毒中起着重要作用。氨发酵则将硝酸盐还原成NH4+, 同时偶联乙酸的生成和底物水平磷酸化。此文从参与该过程的关键酶、关键酶的表达调节、真菌与细菌异化硝酸盐还原的比较等角度综述了真菌异化硝酸盐还原的最新研究进展。  相似文献   

4.
Formation and possible roles of nitric oxide in plant roots   总被引:12,自引:0,他引:12  
Nitric oxide has been reported to act as a signalling molecule in different plant tissues and to participate in a variety of physiological processes. It is produced by different enzymes and sources. The root-specific plasma membrane-bound enzymes forming NO from the substrates nitrate and nitrite are of particular interest because roots serve as interfaces between plants and the soil. The co-ordinated activity of the root-specific plasma membrane-bound nitrate reductase (PM-NR) and nitrite:NO reductase (NI-NOR) suggests that NO might also be involved in root signalling and development. The rate of enzymatic production of this NO depends largely on the environmental conditions, mainly the availability of nitrate and oxygen and it is proposed that this NO plays a role during anoxia as an indicator of the external nitrate availability and in regulating symbiotic interactions at the root surface.  相似文献   

5.
Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K-12 and Wolinella succinogenes were studied by low-temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high-spin and low-spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme-NO complexes with distinct EPR spectral characteristics. The heme-NO complexes formed with the two enzymes differed, however, in g values and line-shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme-NO complexes. These results suggested the involvement of an enzyme-bound NO intermediate during the six-electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme-NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.  相似文献   

6.
Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.  相似文献   

7.
Tischner R  Planchet E  Kaiser WM 《FEBS letters》2004,576(1-2):151-155
Wild type (WT), and nitrate reductase (NR)- and nitrite-reductase (NiR)-deficient cells of Chlorella sorokiniana were used to characterize nitric oxide (NO) emission. The NO emission from nitrate-grown WT cells was very low in air, increased slightly after addition of nitrite (200 microM), but strongly under anoxia. Importantly, even completely NR-free mutants, as well as cells grown on tungstate, emitted NO when fed with nitrite under anoxia. Therefore, this NO production from nitrite was independent of NR and other molybdenum cofactor enzymes. Cyanide and inhibitors of mitochondrial complex III, myxothiazol or antimycin A, but not salicylhydroxamic acid (inhibitor of alternative oxidase) inhibited NO production by NR-free cells. In contrast, NiR-deficient cells growing on nitrate accumulated nitrite and emitted NO at very high equal rates in air and anoxia. This NO emission was 50% inhibited by salicylhydroxamic acid, indicating that in these cells the alternative oxidase pathway had been induced and reduced nitrite to NO.  相似文献   

8.
Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.  相似文献   

9.
Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.  相似文献   

10.
Nitric oxide (NO) is a gaseous signalling molecule which becomes very toxic due to its ability to react with multiple cellular targets in biological systems. Bacterial cells protect against NO through the expression of enzymes that detoxify this molecule by oxidizing it to nitrate or reducing it to nitrous oxide or ammonia. These enzymes are haemoglobins, c-type nitric oxide reductase, flavorubredoxins and the cytochrome c respiratory nitrite reductase. Expression of the genes encoding these enzymes is controlled by NO-sensitive regulatory proteins. The production of NO in rhizobia-legume symbiosis has been demonstrated recently. In functioning nodules, NO acts as a potent inhibitor of nitrogenase enzymes. These observations have led to the question of how rhizobia overcome the toxicity of NO. Several studies on the NO response have been undertaken in two non-dentrifying rhizobial species, Sinorhizobium meliloti and Rhizobium etli, and in a denitrifying species, Bradyrhizobium japonicum. In the present mini-review, current knowledge of the NO response in those legume-associated endosymbiotic bacteria is summarized.  相似文献   

11.
Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)  相似文献   

12.
The physiological functions of nitric oxide (NO) are well established. The finding that the endothelium-derived relaxing factor (EDRF) is NO was totally unexpected. It was shown that NO is a reaction product of an enzymatically catalyzed, overall, 5-electron oxidation of guanidinium nitrogen from L-arginine followed by the release of the free radical species NO. NO is synthesized by a single protein complex supported by cofactors, coenzymes (such as tetrahydrobiopterin) and cytochrome P450. The latter can uncouple from substrate oxidation producing O2*- radicals. The research groups of Richter [Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett 1997; 418: 291-296.] and Boveris [Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem 1998; 273: 11038-11043.] identified a mitochondrial NO synthase (NOS). There are, however, increasing reports demonstrating that mitochondrial NO is derived from cytosolic NOS belonging to the Ca2+-dependent enzymes. NO was thought to control cytochrome oxidase. This assumption is controversial due to the life-time of NO in biological systems (millisecond range). We found a nitrite reductase in mitochondria which is of major interest. Any increase of nitrite in the tissue which is the first oxidation product of NO, for instance following NO donors, will stimulate NO-recycling via mitochondrial nitrite reductase. In this paper, we describe the identity and the function of mitochondrial nitrite reductase and the consequences of NO-recycling in the metabolic compartment of mitochondria.  相似文献   

13.
Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.  相似文献   

14.
Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR—the nitrite reduction—and propose a new class of molybdenum-containing nitrite reductases.  相似文献   

15.
Nitrate reductases (NRs) are enzymes that catalyze reduction of nitrate to nitrite using a molybdenum cofactor. In an alternative reaction, plant NRs have also been shown to catalyze reduction of nitrite to nitric oxide, and this appears to be a major source of nitric oxide synthesis in plants, although other pathways have also been shown. Here, density functional theory (DFT) results are shown, indicating that although nitrate is thermodynamically the preferred substrate for the NR active site, both nitrite and nitrate are easily reduced to nitrite and NO, respectively. These mechanisms require a Mo(IV) state. Additionally, in the case of the nitrite, linkage isomerism is at work and controlled by the metal oxidation state, and reduction is, unlike in the nitrate case, dependent on protonation. The data may be relevant to other molybdenum enzymes with similar active sites, such as xanthine oxidase.  相似文献   

16.
An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.  相似文献   

17.
The purL gene of Bacillus subtilis is associated with nematicidal activity   总被引:1,自引:0,他引:1  
Xia Y  Xie S  Ma X  Wu H  Wang X  Gao X 《FEMS microbiology letters》2011,325(2):99-107
Major questions concerning the sources and mechanisms of the reduction of nitric oxide by enteric bacteria remain unresolved. The membrane-associated nitrate reductase is the major source of NO generated from nitrite, but at least one other source remains to be identified. Nitrite reductases are primarily detoxification systems that decrease rather than increase the accumulation of NO in the cytoplasm. Whether they also catalyze NO formation is unresolved. The FNR protein that regulates transitions between aerobic and anaerobic growth is inactivated as a consequence of nitrosative damage, but we challenge the idea that FNR is a physiologically relevant sensor of NO, except under the most severe nitrosative stress. As none of the three enzymes that reduce NO account for the majority of the rate of NO reduction, additional mechanisms remain to be discovered. Little is known about the biochemistry of damage repair. Whatever the growth conditions and however severe the nitrosative stress, groups of proteins are synthesized to protect the bacterial cytoplasm against the side effects of nitrate and nitrite reduction. The enigmatic hybrid cluster protein is more likely to be part of a repair pathway than a hydroxylamine reductase, as annotated in many genome databases.  相似文献   

18.
Nitrite is an important species in the global nitrogen cycle, and the nitrite reductase enzymes convert nitrite to nitric oxide (NO). Recently, it has been shown that hemoglobin and myoglobin catalyze the reduction of nitrite to NO under hypoxic conditions. We have determined the 1.20 A resolution crystal structure of the nitrite adduct of ferric horse heart myoglobin (hh Mb). The ligand is bound to iron in the nitrito form, and the complex is formulated as MbIII(ONO-). The Fe-ONO bond length is 1.94 A, and the O-N-O angle is 113 degrees . In addition, the nitrite ligand is stabilized by hydrogen bonding with the distal His64 residue. We have also determined the 1.30 A resolution crystal structures of hh MbIINO. When hh MbIINO is prepared from the reaction of metMbIII with nitrite/dithionite, the FeNO angle is 144 degrees with a Fe-NO bond length of 1.87 A. However, when prepared from the reaction of NO with reduced MbII, the FeNO angle is 120 degrees with a Fe-NO bond length of 2.13 A. This difference in FeNO conformations as a function of preparative method is reproducible, and suggests a role of the distal pocket in hh MbIINO in stabilizing local FeNO conformational minima.  相似文献   

19.
Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.  相似文献   

20.
Nitric oxide (NO) is a gas displaying multiple physiologicalfunctions in plants, animals and bacteria. The enzymes nitratereductase and NO synthase have been suggested to be involvedin the production of NO in plants and algae, but the implicationof those enzymes in NO production under physiological conditionsremains obscure. Symbiodinium microadriaticum, commonly referredto as zooxanthellae, is a marine microalga commonly found insymbiotic association with a cnidarian host including reef-buildingcorals. Here we demonstrate NO production in zooxanthellae uponsupplementation of either sodium nitrite or L-arginine as asubstrate. The nitrite-dependent NO production was detectedelectrochemically and confirmed by the application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO), a specific NO scavenger. Cells stained with the diaminofluorescein,DAF-2 DA, an NO fluorescent probe, showed an increase in fluorescenceintensity upon supplementation of both sodium nitrite and L-arginine.Microscopic observations of DAF-stained cells verified thatNO was produced inside the cells. NO production in S. microadriaticumwas found to increase upon exposure of cells to an acute heatstress which also caused a decline in the photosynthetic efficiencyof PSII (Fv/Fm). This study provides substantial evidence toconfirm that zooxanthellae can synthesize NO even when theyare not in a symbiotic association with a coral host. The increasein NO production at high temperatures suggests that heat stressstimulates the microalgal NO production in a temperature-dependentmanner. The implications of these findings are discussed inthe light of the coral bleaching phenomenon which is associatedwith elevated sea surface temperature due to global warming.  相似文献   

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