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1.
Chromate-Reducing Properties of Soluble Flavoproteins from Pseudomonas putida and Escherichia coli 总被引:1,自引:0,他引:1
D. F. Ackerley C. F. Gonzalez C. H. Park R. Blake II M. Keyhan A. Matin 《Applied microbiology》2004,70(2):873-882
Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins—ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)—were examined. Both are dimers and reduce chromate efficiently to Cr(III) (kcat/Km = ~2 × 104 M−1·s−1). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR. 相似文献
2.
Chromate [Cr(VI)] is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen-insensitive nitroreductase of Escherichia coli, is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single-electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one-electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni- and di-valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH-catalysed chromate reduction reaction decreased ROS generation by c. 65, 40, or 20%, respectively, suggesting that these enzymes can pre-empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound. 相似文献
3.
Puzon GJ Petersen JN Roberts AG Kramer DM Xun L 《Biochemical and biophysical research communications》2002,294(1):76-81
Biological reduction of carcinogenic chromate has been extensively studied in eukaryotic cells partly because the reduction produces stable chromium(III)-DNA adducts, which are mutagenic. Microbial reduction of chromate has been studied for bioremediation purposes, but little is known about the reduction mechanism. In eukaryotic cells chromate is mainly reduced non-enzymatically by ascorbate, which is usually absent in bacterial cells. We have characterized the reduction of chromate by a flavin reductase (Fre) from Escherichia coli with flavins. The Fre-flavin system rapidly reduced chromate, whereas chemical reduction by NADH and glutathione was very slow. Thus, enzymatic chromate reduction is likely the dominant mechanism in bacterial cells. Furthermore, the end-product was a soluble and stable Cr(III)-NAD(+) complex, instead of Cr(III) precipitate. Since intracellularly generated Cr(III) forms adducts with DNA, protein, glutathione, and ascorbate in eukaryotic cells, we suggest that the produced Cr(III) is primarily complexed to NAD(+), DNA, and other cellular components inside bacteria. 相似文献
4.
Eswaramoorthy S Poulain S Hienerwadel R Bremond N Sylvester MD Zhang YB Berthomieu C Van Der Lelie D Matin A 《PloS one》2012,7(4):e36017
The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 Å resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction. 相似文献
5.
The homodimeric flavoprotein FerB of Paracoccus
denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate.
The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was
found to be 1.53 ± 0.09 (O2 absent) or > 2 (O2 present), the apparent K
M value for chromate amounted to 70 ± 10 μM with the maximum rate of 2.9 ± 0.3 μmol NADH s−1 (mg protein)−1. Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being
due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain.
Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin,
suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted
culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly
reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a
strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate. 相似文献
6.
Gonzalez CF Ackerley DF Lynch SV Matin A 《The Journal of biological chemistry》2005,280(24):22590-22595
Most bacteria contain soluble quinone-reducing flavoenzymes. However, no biological benefit for this activity has previously been demonstrated. ChrR of Pseudomonas putida is one such enzyme that has also been characterized as a chromate reductase; yet we propose that it is the quinone-reducing activity of ChrR that has the greatest biological significance. ChrR reduces quinones by simultaneous two-electron transfer, avoiding formation of highly reactive semiquinone intermediates and producing quinols that promote tolerance of H(2)O(2). Expression of chrR was induced by H(2)O(2), and levels of chrR expression in overexpressing, wild type, and knock-out mutant strains correlated with the H(2)O(2) tolerance and scavenging ability of each strain. The chrR expression level also correlated with intracellular H(2)O(2) levels as measured by protein carbonylation assays and fluorescence-activated cell scanning analysis with the H(2)O(2)-responsive dye H(2)DCFDA. Thus, enhancing the activity of ChrR in a chromate-remediating bacterial strain may not only increase the rate of chromate transformation, it may also augment the capacity of these cells to withstand the unavoidable production of H(2)O(2) that accompanies chromate reduction. 相似文献
7.
Fu J Yamamoto K Guan ZW Kimura S Iyanagi T 《Archives of biochemistry and biophysics》2004,427(2):180-187
We have analyzed the mechanism of one-electron reduction of adriamycin (Adr) using recombinant full-length human neuronal nitric-oxide synthase and its flavin domains. Both enzymes catalyzed aerobic NADPH oxidation in the presence of Adr. Calcium/calmodulin (Ca(2+)/CaM) stimulated the NADPH oxidation of Adr. In the presence or absence of Ca(2+)/CaM, the flavin semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by Adr. The FAD-NADPH binding domain did not significantly catalyze the reduction of Adr. Neither the FAD semiquinone (FADH*) nor the air-stable semiquinone (FAD-FMNH*) reacted rapidly with Adr. These data indicate that the fully reduced species of FMN (FMNH(2)) donates one electron to Adr, and that the rate of Adr reduction is stimulated by a rapid electron exchange between the two flavins in the presence of Ca(2+)/CaM. Based on these findings, we propose a role for the FAD-FMN pair in the one-electron reduction of Adr. 相似文献
8.
Environmental protection through biological mechanisms that aid in the reductive immobilization of toxic metals (e.g., chromate and uranyl) has been identified to involve specific NADH-dependent flavoproteins that promote cell viability. To understand the enzyme mechanisms responsible for metal reduction, the enzyme kinetics of a putative chromate reductase from Gluconacetobacter hansenii (Gh-ChrR) was measured and the crystal structure of the protein determined at 2.25 Å resolution. Gh-ChrR catalyzes the NADH-dependent reduction of chromate, ferricyanide, and uranyl anions under aerobic conditions. Kinetic measurements indicate that NADH acts as a substrate inhibitor; catalysis requires chromate binding prior to NADH association. The crystal structure of Gh-ChrR shows the protein is a homotetramer with one bound flavin mononucleotide (FMN) per subunit. A bound anion is visualized proximal to the FMN at the interface between adjacent subunits within a cationic pocket, which is positioned at an optimal distance for hydride transfer. Site-directed substitutions of residues proposed to involve in both NADH and metal anion binding (N85A or R101A) result in 90–95% reductions in enzyme efficiencies for NADH-dependent chromate reduction. In comparison site-directed substitution of a residue (S118A) participating in the coordination of FMN in the active site results in only modest (50%) reductions in catalytic efficiencies, consistent with the presence of a multitude of side chains that position the FMN in the active site. The proposed proximity relationships between metal anion binding site and enzyme cofactors is discussed in terms of rational design principles for the use of enzymes in chromate and uranyl bioremediation. 相似文献
9.
Chad A Haynes Ronald L Koder Anne-Frances Miller David W Rodgers 《The Journal of biological chemistry》2002,277(13):11513-11520
The crystal structure of the nitroreductase enzyme from Enterobacter cloacae has been determined for the oxidized form in separate complexes with benzoate and acetate inhibitors and for the two-electron reduced form. Nitroreductase is a member of a group of enzymes that reduce a broad range of nitroaromatic compounds and has potential uses in chemotherapy and bioremediation. The monomers of the nitroreductase dimer adopt an alpha+beta fold and together bind two flavin mononucleotide prosthetic groups at the dimer interface. In the oxidized enzyme, the flavin ring system adopts a strongly bent (16 degrees ) conformation, and the bend increases (25 degrees ) in the reduced form of the enzyme, roughly the conformation predicted for reduced flavin free in solution. Because free oxidized flavin is planar, the induced bend in the oxidized enzyme may favor reduction, and it may also account for the characteristic inability of the enzyme to stabilize the one electron-reduced semiquinone flavin, which is also planar. Both inhibitors bind over the pyrimidine and central rings of the flavin in partially overlapping sites. Comparison of the two inhibitor complexes shows that a portion of helix H6 can flex to accommodate the differently sized inhibitors suggesting a mechanism for accommodating varied substrates. 相似文献
10.
Analysis of novel soluble chromate and uranyl reductases and generation of an improved enzyme by directed evolution 总被引:1,自引:0,他引:1
Barak Y Ackerley DF Dodge CJ Banwari L Alex C Francis AJ Matin A 《Applied and environmental microbiology》2006,72(11):7074-7082
Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, (Tyr)128(Asn), was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased. 相似文献
11.
Jirí Mazoch Radek Tesarík Vojtech Sedlácek Igor Kucera Jaroslav Turánek 《European journal of biochemistry》2004,271(3):553-562
Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzoquinone. 相似文献
12.
Mechanisms of bacterial resistance to chromium compounds 总被引:1,自引:0,他引:1
Martha I. Ramírez-Díaz César Díaz-Pérez Eréndira Vargas Héctor Riveros-Rosas Jesús Campos-García Carlos Cervantes 《Biometals》2008,21(3):321-332
Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this
heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species,
the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species.
At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually
present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated
with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported.
The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III).
Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus
metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative
ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by
chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized
enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems
protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate
involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of
iron and sulfur. 相似文献
13.
Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response. 相似文献
14.
Analysis of Novel Soluble Chromate and Uranyl Reductases and Generation of an Improved Enzyme by Directed Evolution
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Y. Barak D. F. Ackerley C. J. Dodge L. Banwari C. Alex A. J. Francis A. Matin 《Applied microbiology》2006,72(11):7074-7082
Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, Tyr128Asn, was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased. 相似文献
15.
Mechanisms of chromium toxicity in mitochondria 总被引:2,自引:0,他引:2
The oxygen consumption of isolated rat heart mitochondria was potently depressed in presence of 10-50 microM Na2CrO4 when NAD-linked substrates were oxidized. The succinate stimulated respiration and the oxidation of exogeneous NADH in sonicated mitochondria were not affected by chromate at this concentration range. A rapid and persistent drop (40% in 2 min) in the mitochondrial NADH level was observed after chromate addition (30 microM) under conditions which generally should promote regeneration of NADH. Experiments with bis-(2-ethyl-2-hydroxybutyrato)oxochromate(V) and vanadyl induced reduction of Cr(VI) in presence of excess NADH were performed. These experiments indicated that NADH may be directly oxidized by Cr(V) at physiological pH. The activity of 10 different enzymes were measured after lysis of intact mitochondria pretreated with chromate (1-100 microM). Na2CrO4 at a very low level (3-5 microM) was sufficient for 50% inhibition of alpha-ketoglutarate dehydrogenase. Higher concentrations (20-70 microM) was necessary for similar effect on beta-hydroxybutyrate and pyruvate dehydrogenase. The other enzymes tested were unaffected. Thus, the chromate toxicity in mitochondria may be due to NADH depletion as a result of direct oxidation by Cr(V) as well as reduced formation of NADH due to specific enzyme inhibition. 相似文献
16.
Minyan He Xiangyang Li Liang Guo Susan J Miller Christopher Rensing Gejiao Wang 《BMC microbiology》2010,10(1):221
Background
Chromium is a toxic heavy metal, which primarily exists in two inorganic forms, Cr(VI) and Cr(III). Chromate [Cr(VI)] is carcinogenic, mutational, and teratogenic due to its strong oxidizing nature. Biotransformation of Cr(VI) to less-toxic Cr(III) by chromate-resistant and reducing bacteria has offered an ecological and economical option for chromate detoxification and bioremediation. However, knowledge of the genetic determinants for chromate resistance and reduction has been limited so far. Our main aim was to investigate chromate resistance and reduction by Bacillus cereus SJ1, and to further study the underlying mechanisms at the molecular level using the obtained genome sequence. 相似文献17.
Park CH Keyhan M Wielinga B Fendorf S Matin A 《Applied and environmental microbiology》2000,66(5):1788-1795
Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, we purified to homogeneity (>600-fold purification) and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation (55 to 70%), anion-exchange chromatography (DEAE Sepharose CL-6B), chromatofocusing (Polybuffer exchanger 94), and gel filtration (Superose 12 HR 10/30). The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 degrees C and 5, respectively; and the K(m) was 374 microM, with a V(max) of 1.72 micromol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 degrees C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction. 相似文献
18.
Bacterial strains, previously isolated from a chromium-polluted soil, were identified on the basis of Gram reaction and biochemical
characteristics (Biolog system). Moreover, chromate MICs, chromate reduction capability, multiple heavy metal tolerance, and
antibiotic susceptibility were tested for each isolate. All strains but one were Gram-positive and resistant to high concentrations
of chromate. The most Cr(VI)-resistant isolate (22mM) was identified as Corynebacterium hoagii. All Cr(VI)-resistant strains except the isolate ChrC20 were capable of catalyzing the reduction of Cr(VI) to Cr(III), a
less toxic and less water-soluble form of chromium. The only isolate Cr(VI)-sensitive, attributed to the Pseudomonas genus, also exhibited Cr(VI)-reduction. Isolates were also screened for the presence of plasmid DNA. The strains ChrC20 and
ChrB20 harbored one and two plasmids of high molecular mass, respectively. This approach permitted selection of some bacterial
strains, which could be used for bioremediation of Cr(VI)-polluted environments.
Received: 21 February 2002 / Accepted: 27 March 2002 相似文献
19.
Timothy S. Magnuson Michael W. Swenson Andrzej J. Paszczynski Lee A. Deobald David Kerk David E. Cummings 《Biometals》2010,23(6):1129-1138
Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism. 相似文献
20.
The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ. 相似文献