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1.
Oxidation-reduction titrations have been carried out on the wild-type, ferredoxin-dependent sulfite reductase from maize and two site-specific variants of the enzyme. E(m) values have been determined for the siroheme and [4Fe-4S] cluster prosthetic groups of the enzyme, which titrate as independent, one-electron carriers. Visible-region difference spectra suggest that reduction of the [4Fe-4S] cluster significantly perturbs the spectrum of the reduced siroheme group of the enzyme. The effects of siroheme axial ligation, by either cyanide or phosphate ligands, on the redox properties of sulfite reductase have also been examined. For comparison, the effects of phosphate and cyanide on the redox properties of the ferredoxin-dependent nitrite reductase of spinach chloroplasts, an enzyme with the same prosthetic group arrangement as sulfite reductase, have been examined. 相似文献
2.
Interactions of ferredoxin-linked nitrite reductase (NiR) from spinach with its substrate were studied by spectrophotometry and electron spin resonance (ESR) spectroscopy. Siroheme was extractable from NiR with 2.5% (W/V) trichloroacetic acid (TCA) and with acetone containing 0.01 N HCl. The addition of nitrite or sulfite to these extracts resulted in shifts of the absorption spectra of siroheme. The HCl-acetone extract showed ESR signals of symmetrical high spin heme, which disappeared on addition of nitrite. Spectral titration indicated a high affinity of extracted siroheme to nitrite and sulfite. The addition of nitrite or sulfite to protoheme dissolved in 0.01 N HCl-acetone did not cause a shift of the absorption spectrum. The extractability of siroheme with 0.01 N HCl-acetone was suppressed by the addition of nitrite to the NiR preparation. Moreover, a substrate-induced difference spectrum with peaks at about 295 and 287 nm was observed on addition of nitrite to NiR. These observations indicated an intrinsic strong affinity of siroheme to nitrite and sulfite, formation of rhombicity of siroheme by binding to the protein moiety, and also a probable conformational change of NiR on binding to the substrate. In agreement with previous reports, ESR signals of the heme-NO complex were observed with NiR in the presence of nitrite, methyl viologen (MV), and dithionite. In the present study, the same signals of similar intensity were also observed on omission of MV, under which conditions no catalytic reduction of nitrite occurred. Furthermore, the signal of the heme-NO complex was not observed when MV was replaced by spinach ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
N P Hilliard M Hirasawa D B Knaff R W Shaw 《Archives of biochemistry and biophysics》1991,291(1):195-199
Recent preparations of nitrite reductase do not display the heterodimeric quaternary structure obtained previously (total molecular weight 85,000; subunit molecular weights 24,000 and 61,000), but rather yield only the 61,000 molecular weight subunit, even when buffers containing the protease inhibitor phenylmethylsulfonyl fluoride are used. Nevertheless, such preparations retain the high ratio of ferredoxin-linked to methyl viologen-linked enzyme activity which has been previously taken as a characteristic of only the heterodimeric form. These preparations display a siroheme prosthetic group to protein ratio of 1.1. When nitrite reductase samples are frozen during the purification scheme, even though the ferredoxin-linked specific activity does not significantly decrease, enzyme activity-stained native gel electrophoresis of the subsequently purified protein reveals that gels with several bands of activity can be obtained. Further evidence of protein heterogeneity in these preparations comes from N-terminal amino acid analysis which reveals that even nonfrozen preparations contain two major peptides with valine and cysteine as the N-termini. Formation of complexes of purified nitrite reductase with ferredoxin resulted in siroheme difference electronic spectra which resembled those observed previously for monomeric preparations. However, the siroheme midpoint potential of recent preparations of nitrite reductase (-287 mV) is close to that of the heterodimeric preparations. Ultrafiltration studies of crude extracts of the enzyme indicate that, at least at certain stages of the preparation, higher molecular weight forms of the enzyme may exist. We conclude that the 24,000 molecular weight polypeptide is a contaminant and that the heterodimeric quaternary structure model for spinach nitrite reductase is incorrect. Furthermore, the monomeric preparations we do obtain display both significant protein heterogeneity and facile loss of siroheme upon gel filtration. 相似文献
4.
M Hirasawa R W Shaw G Palmer D B Knaff 《The Journal of biological chemistry》1987,262(26):12428-12433
Chemical analysis of the ferredoxin-dependent native form (Mr = 85,000) of spinach nitrite reductase has demonstrated a siroheme content that approaches 2 mol of siroheme/mol of enzyme. A widely studied modified (Mr = 61,000) form of nitrite reductase, that has lost much of the native enzyme's ability to use ferredoxin as an electron donor, contains approximately 1 mol of siroheme/mol of enzyme. Quantitation of the high spin ferri-siroheme EPR signals and of nitrite-binding sites of the two preparations confirmed that the native enzyme's siroheme content is approximately twice that of the modified enzyme. Plots of nitrite and cyanide binding to the native enzyme versus ligand concentration are sigmoidal, with Hill coefficients of 1.6-1.8 and 2.3-2.8, respectively. Plots of enzyme activity versus nitrite concentration for the native enzyme are sigmoidal with a Hill coefficient of 2.4. Cyanide inhibition of enzymatic activity was shown to be not competitive. Addition of cyanide to the native enzyme resulted in a diminution of the high spin ferri-siroheme EPR signal and produced EPR signals with g values of 2.71, 2.33, and 1.49 due to low spin ferri-siroheme. 相似文献
5.
Nitrite reductases found in plants, algae, and cyanobacteria catalyze the six-electron reduction of nitrite to ammonia with reduced ferredoxin serving as the electron donor. They contain one siroheme and one [4Fe-4S] cluster, acting as separate one-electron carriers. Nitrite is thought to bind to the siroheme and to remain bound until its complete reduction to ammonia. In the present work the enzyme catalytic cycle, with ferredoxin reduced by photosystem 1 as an electron donor, has been studied by EPR and laser flash absorption spectroscopy. Substrate depletion during enzyme turnover, driven by a series of laser flashes, has been demonstrated. A complex of ferrous siroheme with NO, formed by two-electron reduction of the enzyme complex with nitrite, has been shown to be an intermediate in the enzyme catalytic cycle. The same complex can be formed by incubation of free oxidized nitrite reductase with an excess of nitrite and ascorbate. Hydroxylamine, another putative intermediate in the reduction of nitrite catalyzed by nitrite reductase, was found to react with oxidized nitrite reductase to produce the same ferrous siroheme-NO complex, with a characteristic formation time of about 13 min. The rate-limiting step for this reaction is probably hydroxylamine binding to the enzyme, with the conversion of hydroxylamine to NO at the enzyme active site likely being much faster. 相似文献
6.
The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis. 总被引:2,自引:2,他引:0
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K Ogawa E Akagawa K Yamane Z W Sun M LaCelle P Zuber M M Nakano 《Journal of bacteriology》1995,177(5):1409-1413
Bacillus subtilis can use either nitrate or nitrite as a sole source of nitrogen. The isolation of the nasABCDEF genes of B. subtilis, which are required for nitrate/nitrite assimilation, is reported. The probable gene products include subunits of nitrate/nitrite reductases and an enzyme involved in the synthesis of siroheme, a cofactor for nitrite reductase. 相似文献
7.
Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure 总被引:1,自引:0,他引:1
Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia. 相似文献
8.
Ling Kang Jean LeGall Andrzej T. Kowal Michael K. Johnson 《Journal of inorganic biochemistry》1987,30(4):273-290
Siroheme has been extracted from sulfite reductases and its properties in aqueous solution have been investigated by optical absorption, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MDC) spectroscopy. The absorption spectrum of siroheme exhibits a marked pH dependence, and two pK values, 4.2 and 9.0, were determined by pH titration in the range 2–12. The first pK (4.2) is thought to correspond to the ionization of the carboxylic acid side-chains on the tetrapyrrole rings, and the second pK (9.0) is attributed to displacement of the axial ligand chloride by hydroxide. The binding of the strong field ligands, CO, NO, and cyanide, were investigated by UV-visible absorption and, in the case of the cyanide complex, by low-temperature EPR and MCD spectroscopies. CO and NO were able to reduce and bind to siroheme without additional reducing agent. The EPR spectrum of the isolated siroheme (chloride-ferrisiroheme) exhibits an axial signal with gXXX = 6.0 and g= 2.0, typical of high-spin ferric hemes (S = 5/2), whereas the cyanide-complexed siroheme exhibits an approximately axial signal with gXXX = 2.38 and g = 1.76 that is indicative of a low-spin ferric heme (S = 1/2). The low-temperature MCD spectra and magnetization data for the as-isolated and cyanide-complexed ferrisiroheme are entirely consistent with the interpretation of the EPR spectra. The results for ferrosiroheme indicate that the siroheme remains high spin (S = 2) and low spin (S = 0) on reduction of the as-isolated and cyanide-complexed siroheme, respectively. The isolated siroheme expressed sulfite reductase activity but the assessable catalytic cycle was much less than that of the native enzyme, showing the importance of the protein environment. 相似文献
9.
Siroheme, the cofactor for sulfite and nitrite reductases, is formed by methylation, oxidation, and iron insertion into the tetrapyrrole uroporphyrinogen III (Uro-III). The CysG protein performs all three steps of siroheme biosynthesis in the enteric bacteria Escherichia coli and Salmonella enterica. In either taxon, cysG mutants cannot reduce sulfite to sulfide and require a source of sulfide or cysteine for growth. In addition, CysG-mediated methylation of Uro-III is required for de novo synthesis of cobalamin (coenzyme B(12)) in S. enterica. We have determined that cysG mutants of the related enteric bacterium Klebsiella aerogenes have no defect in the reduction of sulfite to sulfide. These data suggest that an alternative enzyme allows for siroheme biosynthesis in CysG-deficient strains of Klebsiella. However, Klebsiella cysG mutants fail to synthesize coenzyme B(12), suggesting that the alternative siroheme biosynthetic pathway proceeds by a different route. Gene cysF, encoding an alternative siroheme synthase homologous to CysG, has been identified by genetic analysis and lies within the cysFDNC operon; the cysF gene is absent from the E. coli and S. enterica genomes. While CysG is coregulated with the siroheme-dependent nitrite reductase, the cysF gene is regulated by sulfur starvation. Models for alternative regulation of the CysF and CysG siroheme synthases in Klebsiella and for the loss of the cysF gene from the ancestor of E. coli and S. enterica are presented. 相似文献
10.
S H Han J F Madden R G Thompson S H Strauss L M Siegel T G Spiro 《Biochemistry》1989,28(13):5461-5471
Resonance Raman (RR) spectra are reported for the hemoprotein subunit (SiR-HP) of Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2) in various ligation and redox states. Comparison of the RR spectra of extracted siroheme and the mu-oxo FeIII dimer of octaethylisobacteriochlorin with those of mu-oxo FeIII octaethylchlorin dimer and mu-oxo FeIII octaethylporphyrin dimer demonstrates that many siroheme bands can be correlated with established porphyrin skeletal modes. Depolarization measurements are a powerful tool in this correlation, since the 45 degrees rotation of the C2 symmetry axis of the isobacteriochlorin ring relative to the chlorin system results in reversal of the polarization properties (polarized vs anomalously polarized) of bands correlating with B1g and B2g modes of porphyrin. Various SiR-HP adducts (CO, NO, CN-, SO3(2-] show upshifted high-frequency bands, characteristic of the low-spin state and consistent with the expected core size sensitivity of the skeletal modes. Fully reduced unliganded SiR-HP (both siroheme and Fe4S4 cluster reduced) in liquid solution displays RR features comparable to those of high-spin ferrous porphyrins; on freezing, the RR spectrum changes, reflecting an apparent mixture of siroheme spin states. At intermediate reduction levels in solution a RR species is observed whose high-frequency bands are upshifted relative to oxidized and fully reduced SiR-HP. This spectrum, thought to arise from the "one-electron" state of SiR-HP (siroheme reduced, cluster oxidized), may be due to S = 1 FeII siroheme. 相似文献
11.
Shilpa Bali Sarah Rollauer Pietro Roversi Evelyne Raux‐Deery Susan M. Lea Martin J. Warren Stuart J. Ferguson 《Molecular microbiology》2014,92(1):153-163
It has recently been shown that the biosynthetic route for both the d1‐haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative‐haem‐synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1‐haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin–ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild‐type phenotype of an Escherichia coli mutant‐strain lacking sirohydrochlorin–ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α‐proteobacteria as the terminal enzyme of siroheme biosynthesis. 相似文献
12.
Purified spinach nitrite reductase, a protein that contains siroheme, is characterized by absorption maxima in the visible region at 385 and 573 nm. On addition of the substrate nitrite, the bands shift to 360 and 570 nm. Dithionite also causes shifts in the maxima of the visible absorption region. Electron paramagnetic resonance studies show that the untreated enzyme contains a high-spin Fe3+ heme and that the addition of cyanide, an inhibitor that is competitive with nitrite, results in a spin-state change of the heme. Electron paramagnetic resonance analysis of the enzyme in the presence of dithionite or dithionite plus cyanide indicates the presence of a reduced iron-sulfur center with rhombic symmetry (g-values of 2.03, 1.94, and 1.91). In contrast, when the enzyme is treated with dithionite plus nitrite, the EPR spectrum of an NO-heme complex (g-values of 2.07 and 2.00) is observed. The presence of an iron-sulfur center has also been confirmed by chemical analyses of the nonheme iron and acid-labile sulfide in nitrite reductase. These results are discussed in terms of a mechanism for nitrite reduction that involves electron transfer between the iron-sulfur center and siroheme. 相似文献
13.
Life on earth is dependent on sulphur (S) and nitrogen (N). In plants, the second step in the reduction of sulphate and nitrate are mediated by the enzymes sulphite and nitrite reductases, which contain the iron (Fe)-containing siroheme as a cofactor. It is synthesized from the tetrapyrrole primogenitor uroporphyrinogen III in the plastids via three enzymatic reactions, methylation, oxidation and ferrochelatation. Without siroheme biosynthesis, there would be no life on earth. Limitations in siroheme should have an enormous effect on the S- and Nmetabolism, plant growth, development, fitness and reproduction, biotic and abiotic stresses including growth under S, N and Fe limitations, and the response to pathogens and beneficial interaction partners. Furthermore, the vast majority of redoxreactions in plants depend on S-components, and S-containing compounds are also involved in the detoxification of heavy metals and other chemical toxins. Disturbance of siroheme biosynthesis may cause the accumulation of light-sensitive intermediates and reactive oxygen species, which are harmful, or they can function as signaling molecules and participate in interorganellar signaling processes. This review highlights the role of siroheme in these scenarios.Key words: iron, nitrogen, plant/microbe interaction, redox, siroheme, sulphur 相似文献
14.
J R Lancaster J M Vega H Kamin N R Orme-Johnson W H Orme-Johnson R J Krueger L M Siegel 《The Journal of biological chemistry》1979,254(4):1268-1272
EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis. 相似文献
15.
Masakazu Hirasawa-Soga Shigeo Horie Goro Tamura 《Bioscience, biotechnology, and biochemistry》2013,77(5):1319-1328
Ferredoxin-dependent nitrite reductase of spinach has been further characterized and the relationship between this enzyme and methyl viologen-dependent nitrite reductase studied.Purified ferredoxin nitrite reductase, having a molecular weight of 86,000, showed 2.5 times higher ferredoxin-dependent activity than methyl viologen-linked activity. Besides 4 mol of labile sulfide the enzyme contained about 2 mol of siroheme per mol. When dithionite, methyl viologen and nitrite were added, ESR signals of a heme nitrosyl complex at g = 2.14, 2.07 and 2.02 were observed. Moreover, hyperfine splitting of the signal due to 14N nuclear spin was also observed at 2.033, 2.023 and 2.013. The sole addition of hydroxylamine to the ferric enzyme also caused the same but much less intense signals with the hyperfine splitting.On treatment of the ferredoxin nitrite reductase (native enzyme) with DEAE-Sephadex A-50 chromatography, a modified nitrite reductase having a molecular weight of 61,000 and a protein fraction having an apparent molecular weight of 24,000 were separated. The modified enzyme contained about one mol of siroheme and 4 mol of labile sulfide per mol and showed essentially the same heme ESR signals as the native enzyme. Contrary to the native enzyme, this modified enzyme accepted electrons more efficiently from methyl viologen than ferredoxin and the reduction of nitrite to ammonia catalyzed by the modified enzyme was not stoichiometric. The observed nitrite to ammonia ratio was 1 to less than 0.6. Cyanide at concentrations between 0.02 to 0.2 mm inhibited the activity of the native enzyme almost completely but the modified enzyme was inhibited only partially.From the results obtained, it is suggested that the native ferredoxin-linked nitrite reductase consists of two components (or subunits) and removal of the light component results in formation of a modified enzyme with increased relative affinity to methyl viologen. 相似文献
16.
Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand. 相似文献
17.
18.
C L Liu D V DerVartanian H D Peck 《Biochemical and biophysical research communications》1979,91(3):962-970
The redox properties of purified bisulfite reductases from Desulfovibrio gigas, D. desulfuricans (Norway) and Desulfotomaculum ruminis, containing non-heme iron and siroheme have been studied by EPR spectroscopy. Each enzyme shows ferric siroheme EPR signals which are not completely reduced by dithionite after 20 min, but are readily reduced within 1 min by dithionite plus methyl viologen. With the latter reducing system, each reductase also reveals a variable Beinert “g=1.94” type iron-sulfur signal. Reaction of each reductase with reduced methyl viologen results in reduction of only the siroheme. These results suggest different redox potentials for the iron-sulfur and siroheme moieties, and indicate that their functional properties are similar for each reductase. 相似文献
19.
20.
The six-electron reductions of sulfite to sulfide and nitrite to ammonia, fundamental to early and contemporary life, are catalyzed by diverse sulfite and nitrite reductases that share an unusual prosthetic assembly in their active centers, namely siroheme covalently linked to an Fe4S4 cluster. The recently determined crystallographic structure of the sulfite reductase hemoprotein from Escherichia coli complements extensive biochemical and spectroscopic studies in revealing structural features that are key for the catalytic mechanism and in suggesting a common symmetric structural unit for this diverse family of enzymes. 相似文献