Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12

Morpeth, F.F.; Boxer, David H.

doi:10.1021/bi00322a007

Cited by 102 publications

(112 citation statements)

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“…The NarI mutant membranes were also assayed for the NO 3 Ϫ -dependent reoxidation of reduced benzyl viologen (BV . ϩ ) for which NarI is not required (2,24). Wild-type NarGHI has a specific activity of 100 mol of nitrate reduced min Ϫ1 (mg of NarGH) Ϫ1 (Table II).…”

Section: Resultsmentioning

confidence: 99%

“…We have previously shown that the BV . ϩ :NO 3 Ϫ oxidoreductase activity appears to be independent of the presence of NarI (2,24). This prompted us to investigate whether the NarI-H56R mutation has any effect on the assembly or conformation of the NarH [Fe-S] clusters.…”

Section: Fig 3 Redox Titration Of Narghi-enriched Membranesmentioning

confidence: 99%

“…NarGHI is a complex of three nonidentical subunits, NarG (150 kDa), NarH (60 kDa), and NarI (25 kDa), in the ratio of 1:1:1 as predicted by the operon organization. This complex is arranged in two catalytic domains: the NarGH subunits constitute the cytoplasmic domain and NarI the membrane anchor domain (2,3). The NarGH domain contains four [Fe-S] centers (4,5) and a molybdenum cofactor which is the site of nitrate reduction by the enzyme (6).…”

mentioning

confidence: 99%

“…The NarGH domain contains four [Fe-S] centers (4,5) and a molybdenum cofactor which is the site of nitrate reduction by the enzyme (6). The NarI subunit (cytochrome b nr ) is a diheme b-type cytochrome required for attachment of the cytoplasmic domain to the inner side of the cytoplasmic membrane (3) and it mediates electron transfer from the membrane quinol pool (menaquinol or ubiquinol) to the molybdenum cofactor (2,7). Optical redox titrations demonstrate the presence of two hemes in NarGHI with midpoint potentials (E m,7 values) of ϩ17 and ϩ122 mV (8).…”

mentioning

confidence: 99%

“…The hydropathy plot 2 and sequence analysis of NarI using the "positive inside" rule of von Heijne (12) leads to a topological model in which five transmembrane ␣-helical segments (helices I-V) are arranged with a periplasmic N terminus and a cytoplasmic C terminus (13). Five His residues are present in predicted transmembrane segments, at positions 56, 66, 74, 187, and 205.…”

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confidence: 99%

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Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

Magalon

Lemesle-Meunier²,

Rothery³

et al. 1997

Journal of Biological Chemistry

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Nitrate reductase A (NarGHI) 1 is the predominant respiratory complex in the membrane of Escherichia coli when the cells are grown under anaerobic conditions in the presence of nitrate. This complex catalyzes electron transfer from quinol to nitrate coupled to proton release into the periplasm and the generation of a concomitant transmembrane proton electrochemical potential (1). NarGHI is a complex of three nonidentical subunits, NarG (150 kDa), NarH (60 kDa), and NarI (25 kDa), in the ratio of 1:1:1 as predicted by the operon organization. This complex is arranged in two catalytic domains: the NarGH subunits constitute the cytoplasmic domain and NarI the membrane anchor domain (2, 3). The NarGH domain contains four [Fe-S] centers (4, 5) and a molybdenum cofactor which is the site of nitrate reduction by the enzyme (6). The NarI subunit (cytochrome b nr ) is a diheme b-type cytochrome required for attachment of the cytoplasmic domain to the inner side of the cytoplasmic membrane (3) and it mediates electron transfer from the membrane quinol pool (menaquinol or ubiquinol) to the molybdenum cofactor (2, 7). Optical redox titrations demonstrate the presence of two hemes in NarGHI with midpoint potentials (E m,7 values) of ϩ17 and ϩ122 mV (8).Of the well characterized membrane-intrinsic cytochromes b, the hemes are found to have bis-histidine ligation (9 -11), and it is very likely that this is also the case with the hemes of NarGHI. The hydropathy plot 2 and sequence analysis of NarI using the "positive inside" rule of von Heijne (12) leads to a topological model in which five transmembrane ␣-helical segments (helices I-V) are arranged with a periplasmic N terminus and a cytoplasmic C terminus (13). Five His residues are present in predicted transmembrane segments, at positions 56, 66, 74, 187, and 205. Sequence alignment of all known sequences of NarI from various bacterial membrane-bound nitrate reductases (4, 13, 14) reveals only four totally conserved histidines (His-56,His-66 in helix II and His-187,His-205 in helix V) as potential heme ligands, thereby excluding His-74 within helix II in heme ligation (13). Overall, this strongly suggests ligation of each heme by two histidine residues (13). Because of the different spacing of the conserved histidines on helix II (9 residues) and V (17 residues), the hemes probably lie in two planes with different angles relative to the plane of the membrane. Recently, an alternative model for heme ligation in NarI has been proposed by van der Oost et al. (15) on the basis of subunit stoichiometry and sequence analysis of various b-type cytochromes. This model predicts that the hemes are sandwiched in a NarI dimer and ligated only by His-187 and His-205 of helix V of each NarI subunit. Genetic procedures are, therefore, required for identifying the heme ligands and to distinguish the different models.In the present work, we have used site-directed mutagenesis, as well as optical and EPR spectroscopies, to test whether the two conserved histidine residues within helix II (Hi...

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Section: Resultsmentioning

confidence: 99%

Section: Fig 3 Redox Titration Of Narghi-enriched Membranesmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

Magalon

Lemesle-Meunier²,

Rothery³

et al. 1997

Journal of Biological Chemistry

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Kinetics of microbial bromate reduction in a hydrogen‐oxidizing, denitrifying biofilm reactor

Downing

Nerenberg

2007

Biotech & Bioengineering

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Bromate (BrO(3)(-)) is an oxidized contaminant produced from bromide (Br(-)) during ozonation and advanced oxidation of drinking water. Previous research shows that denitrifying bioreactors can reduce bromate to innocuous bromide. We studied a hydrogen-based, denitrifying membrane-biofilm reactor (MBfR) for bromate reduction, and report the first kinetics for a hydrogen-based bromate reduction process. A mixed-culture MBfR reduced up to 1,500 microg/L bromate to below 10 microg/L with a 50-min hydraulic residence time. Kinetics were determined using short-term tests on a completely mixed MBfR at steady state with an influent of 5 mg N/L nitrate plus 100 microg/L bromate. Short-term tests examined the impact of pH, nitrite, nitrate, and bromate on bromate reduction rates in the MBfR. Kinetic parameters for the process were estimated based on the short-term bromate tests. The q(max) for bromate reduction was 0.12 mg BrO(3)(-) x mg(x)(-1) x day(-1), and the K was 1.2 mg BrO(3)(-)/L. This q(max) is 2-3 times higher than reported for heterotrophic enrichments, and the K is the first reported in the literature. Nitrite and nitrate partially inhibited bromate reduction, with nitrite exerting a stronger inhibitory effect. Bromate was self-inhibitory at concentrations above 15 mg/L, but up to 50 mg/L of bromate had no inhibitory effect on denitrification. The optimum pH was approximately 7. We also examined the performance of an MBfR containing pure culture of the denitrifying bacterium Ralstonia eutropha. Under conditions similar to the mixed-culture tests, no bromate reduction was detected, showing that not all denitrifying bacteria are active in bromate reduction. Our results suggest the presence of specialized, dissimilatory bromate-reducing bacteria in the mixed-culture MBfR.

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Effect of operating conditions on Bromate Removal Efficiency in BAC filters

Kirisits¹,

Snoeyink²,

Chee‐Sanford³

et al. 2002

Journal AWWA

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This study examined the use of biologically active carbon (BAC) filters for microbial reduction of bromate to bromide. In these filters, abiotic reduction by the activated carbon did not play a significant role in bromate removal. Bromate removal in a BAC filter decreased by 30% after a system perturbation, suggesting that filter history affects bromate removal. Although 25 and 51 mm (1 and 2 in.) inner‐diameter BAC filters provided comparable bromate removals for the tested contact times, the larger filter exhibited nitrite production; therefore, system performance must be monitored to prevent such undesirable compounds from entering the water. The mass of bromate removed increased as the influent bromate concentration increased, whereas bromate removal did not change after backwashing. Experimental evidence indicated that increased regrowth would not be observed in the distribution system following BAC filtration with a low influent dissolved oxygen concentration (2 mg/L) in the tested water.

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Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12

Cited by 102 publications

References 32 publications

Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

Kinetics of microbial bromate reduction in a hydrogen‐oxidizing, denitrifying biofilm reactor

Effect of operating conditions on Bromate Removal Efficiency in BAC filters

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