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1.
The multiheme enzyme hydroxylamine oxidoreductase from the autotrophic bacteria Nitrosomonas europaea catalyzes the conversion of hydroxylamine to nitrite, with a complicate arrangement of heme groups in three subunits. As a distinctive feature, the protein has a covalent linkage between a tyrosyl residue of one subunit and a meso carbon atom of the heme active site of another. We studied the influence of this bond in the catalysis from a theoretical perspective through electronic structure calculations at the density functional theory level, starting from the crystal structure of the protein. Geometry optimizations of proposed reaction intermediates were used to calculate the dissociation energy of different nitrogen containing ligands, considering the presence and absence of the meso tyrosyl residue. The results indicate that the tyrosine residue enhances the binding of hydroxylamine, and increases the stability of a FeIIINO intermediate, while behaving indifferently in the FeIINO form. The calculations performed on model systems including neighboring aminoacids revealed the probable formation of a bidentate hydrogen bond between the FeIIIH2O complex and Asp 257, in a high-spin aquo complex as the resting state. Characterization of non-planar heme distortions showed that the meso-substituent induces significant ruffling in the evaluated intermediates. 相似文献
2.
Oxidation of hydroxylamine to nitrite catalyzed by hydroxylamine oxidoreductase purified fromNitrosomonas europaea 总被引:1,自引:0,他引:1
The oxidation of hydroxylamine to nitrite, which had been catalyzed by hydroxylamine oxidoreductase purified fromNitrosomonas europaea, was studied. The enzyme oxidized hydroxylamine almost completely to nitrite under aerobic conditions if sufficient amount
of cytochromec or ferricyanide was added and the reaction was performed in phosphate buffer. Even under anaerobic conditions, hydroxylamine
was oxidized to nitrite by the enzyme, but nitrite, once formed, disappeared when the reaction was continued for more than
several minutes. 相似文献
3.
Michael Hoppert Timothy J. Mahony Frank Mayer David J. Miller 《Archives of microbiology》1995,163(4):300-306
The hydroxylamine oxidoreductase from Nitrosomonas europaea was prepared to apparent electrophoretic homogeneity. Electron microscopy of negatively stained preparations of the sample revealed an overall diameter of about 8.8 nm of the enzyme particle. The native structure was determined as a tetrahedron-like assembly of identical subunits exhibiting four protein masses.Abbreviations
ESI
Electron spectroscopic imaging
-
HAO
Hydroxylamine oxidoreductase 相似文献
4.
During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Spectroscopic characterization of the number and type of iron-sulfur clusters in NADH:ubiquinone oxidoreductase 总被引:2,自引:0,他引:2
A T Kowal J E Morningstar M K Johnson R R Ramsay T P Singer 《The Journal of biological chemistry》1986,261(20):9239-9245
The number and type of iron-sulfur clusters present in the NADH dehydrogenase of the mammalian respiratory chain were studied by a combination of low temperature magnetic circular dichroism (MCD) and quantitative electron paramagnetic resonance spectroscopies. MCD was used with the high molecular weight, soluble enzyme, and EPR was used with both the purified enzyme and Complex I (NADH:ubiquinone oxidoreductase). The results of the EPR experiments of the two types of preparations agreed with each other, as well as with the data in the literature for various types of membrane-bound preparations. The two methods gave concordant results showing the presence of one binuclear and of three tetranuclear NADH-reducible iron-sulfur clusters. Earlier studies using the cluster extrusion technique indicated a higher ratio of binuclear to tetranuclear clusters which may be explained by cluster interconversion during the extrusion process. 相似文献
8.
Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation 总被引:14,自引:0,他引:14
In this study a novel hydroxylamine oxidoreductase (HAO) was purified and characterized from an anaerobic ammonium-oxidizing (Anammox) enrichment culture. The enzyme, which constituted about 9% of the protein mass in the soluble fraction of the cell extract, was able to oxidize hydroxylamine and hydrazine. When phenazine methosulfate and methylthiazolyltetrazolium bromide were used as electron acceptors, a V(max) [21 and 1.1 micromol min(-)(1) (mg of protein)(-)(1)] and K(m) (26 and 18 microM) for hydroxylamine and hydrazine were determined, respectively. The hydroxylamine oxidoreductase is a trimer and contains about 26 hemes per 183 kDa. As deduced from UV/vis spectra, hydroxylamine reduced more and different cytochromes than hydrazine. The dithionite-reduced spectrum showed an unusual 468 nm peak. Inhibition experiments with H(2)O(2) showed that hydroxylamine bound to this P-468 cytochrome, which is assumed to be the putative substrate binding site. Cyanide and hydrazine inhibited the oxidation of hydroxylamine. The amino acid sequences of several peptide fragments of HAO from Anammox showed a clear difference with the deduced amino acid sequence of HAO from the aerobic ammonia-oxidizing bacterium Nitrosomonas europaea. In EPR spectra of the Anammox HAO, two g-values (g(z)() = 2.37 and 2.42) were observed, which were not present in HAO of N. europaea. 相似文献
9.
Reaction of glycolaldehyde with the binary E-NADP complex of bovine kidney aldose reductase (ALR2) produces an enzyme-bound chromophore whose absorbance (lambd max 341 nm) and fluorescence (lambda ex max 341 nm; lambda emit max 421 nm) properties are distinct from those of NADPH or E.NADPH yet are consistent with the proposed covalent adduct structure [1,4-dihydro-4-(1-hydroxy-2-oxoethyl)nicotinamide adenine dinucleotide phosphate]. The kinetics of adduct formation, both in solution and at the enzyme active site, support a mechanism involving rate-determining enolization of glycolaldehyde at high [NADP+] or [E.NADP]. At low [NADP+] or [E.NADP] the reaction is second-order overall, but the ALR2-mediated reaction displays saturation by glycolaldehyde due to competition of the aldehyde (plus hydrate) and enol for E.NADP. Measurement of the pre-steady-state burst of E-adduct formation confirms that glycolaldehyde enol is the reactive species and gives a value of 1.3 x 10(-6) for Kenol = [enol]/[( aldehyde] + [hydrate]), similar to that determined by trapping the enol with I3-. At the ALR2 active site, the rate of adduct formation is enhanced 79,000-fold and the adduct is stabilized greater than or equal to 13,000-fold relative to the reaction with NADP+ in solution. A portion of this enhancement is ascribed to specific interaction of NADP+ with the enzyme since the 3-acetylpyridine analogue, (AP)ADP+, gives values that are 15-200-fold lower. Additional evidence for strong interaction of ALR2 with both NADP+ and NADPH is reported. Yet, because dissociation of adduct is slow, catalysis of the overall adduct formation reaction by ALR2 is less than or equal to 67-fold. 相似文献
10.
de Vries S Strampraad MJ Lu S Moënne-Loccoz P Schröder I 《The Journal of biological chemistry》2003,278(38):35861-35868
The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity. The enzyme displays MQH2:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio. The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (gz = 3.015; gy = 2.226; gx = 1.45), whereas the other heme adopts a high spin configuration. The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme. This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction. The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex. The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (Ki(NO) = 7 microm). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degrees C for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes Op1 and Op2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively. The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo3 and aa3 oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase. 相似文献
11.
The nitrifying bacterium Nitrosomonas europaea contains three copies of the gene (hao) encoding hydroxylamine oxidoreductase (HAO), the second enzyme in the nitrification pathway which oxidizes NH(2)OH to NO(2)(-). The nucleotide sequences of the hao genes differ by only one nucleotide. Two of the three gene copies have identical promoter sequences, while the third promoter has a different nucleotide sequence. Mutant strains with two of the three copies of hao inactivated were created by insertional inactivation, using DNA cassettes containing kanamycin- and gentamycin-resistance genes. All three double-mutant combinations were obtained. These double mutants were phenotypically identical under the conditions tested. Two of these double mutants were similar to wild-type cells or cells having a single hao copy inactivated regarding growth rates or hydroxylamine-dependent O(2) uptake activity, but had only about 50% of the wild-type level of in vitro HAO activity and hao mRNA. The third hao double mutant had an unstable genotype, resulting in recombination of the gentamycin marker into another copy of hao. The N. europaea genomic sequence was recently completed, revealing the locations of the copies of hao and other nitrification genes. Comparison with the arrangement of hao genes in the closely related strain, Nitrosomonas sp. strain ENI-11, showed a similar organization. 相似文献
12.
Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination. 总被引:2,自引:0,他引:2
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Mutagenesis of Nitrosomonas europaea was achieved by electroporation and recombination. To demonstrate this, an aminoglycoside 3'-phosphotransferase (kan) gene was specifically inserted into each of the three gene copies of hao individually. Southern hybridizations and PCR analysis showed the incorporation of the kan gene at the chosen genetic loci. The isolation of mutant strains was achieved in 7 to 14 days when the strains were grown on solid medium. The induced mutations were stable even in the absence of kanamycin-selective pressure for periods of up to 45 days in culture. The mutant strains did not show an observable phenotype different from that of the wild type when grown under the same conditions. 相似文献
13.
T Mikami N Tanaka T Sato H Moriyama M Numata T Fujiwara Y Fukumori T Yamanaka M Sato K Kakiuchi 《Journal of biochemistry》1991,110(5):681-682
Hydroxylamine oxidoreductase [EC 1.7.3.4] from Nitrosomonas europaea was crystallized by the microdialysis method using ammonium sulfate. Its space group is P63 with cell dimensions of a = b = 96.4 A and c = 266.2 A. Its molecular weight was determined to be 190,000-195,000 by the X-ray small angle scattering and ultracentrifugal methods. 相似文献
14.
Hydroxylamine metabolism in Pseudomonas PB16: involvement of a novel hydroxylamine oxidoreductase 总被引:4,自引:0,他引:4
Pseudomonas strain PB16, a Gram-negative heterotrophic nitrifying bacterium closely related to Pseudomonas azalaica on the basis of 16 S rDNA analysis, was able to use hydroxylamine as an additional energy source during growth in acetate limited chemostat cultures giving an increased biomass yield. In aerobically growing cells of Pseudomonas PB16 only 50% of supplemented hydroxylamine could be recovered as nitrite. In addition to nitrite, N2O could be detected in the chemostat off-gas, indicating combined heterotrophic nitrification and aerobic denitrification. The maximum specific hydroxylamine oxidizing activity observed was 450 nmol per min per mg dry weight, with a Ks of approximately 40 µm. Upon addition of hydroxylamine to the medium, Pseudomonas PB16 induced a soluble 132 KDa dimeric hydroxylamine oxidoreductase. The enzyme had a pH optimum of 9, and did not contain spectroscopic features typical for cytochromes, which is in contrast to hydroxylamine oxidoreductases fou nd in autotrophic bacteria. 相似文献
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Zhao Bin Ran Xiao Chuan An Qiang Huang Yuan Sheng Lv Qing Hao Dan Qiao 《Bioprocess and biosystems engineering》2019,42(12):1983-1992
Bioprocess and Biosystems Engineering - N2O production from NH2OH oxidation involved in a heterotrophic nitrifier Alcaligenes faecalis strain NR was studied. 15N-labeling experiments showed that... 相似文献
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Ish K. Dhawan Roopali Roy Brian P. Koehler Swaranalatha Mukund Michael W. W. Adams Michael K. Johnson 《Journal of biological inorganic chemistry》2000,5(3):313-327
Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD)
spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (E
m=−368 mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of
W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed
upon oxidation, termed the “low-potential” W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/W(V) and
W(V)/W(VI) states at physiologically relevant potentials (E
m=−335 and −280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g=1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of −34 mV and persists
up to at least +300 mV. At potentials above 0 mV, a major “high-potential” W(V) signal, g=1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples
of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox
properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential
species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E
m=−265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; E
m=+65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total
W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential
species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for
each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts
of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible
for the catalytic activity in sulfide-activated samples of Tl FOR.
Received: 9 September 1999 / Accepted: 17 February 2000 相似文献
19.
We report results of continuum electrostatics calculations of the cofactor redox potentials, and of the titratable group pK(a) values, in hydroxylamine oxidoreductase (HAO). A picture of a sophisticated multicomponent control of electron flow in the protein emerged from the studies. First, we found that neighboring heme cofactors strongly interact electrostatically, with energies of 50-100 mV. Thus, cofactor redox potentials depend on the oxidation state of other cofactors, and cofactor redox potentials in the active (partially oxidized) enzyme differ substantially from the values obtained in electrochemical redox titration experiments. We found that, together, solvent-exposed heme 1 (having a large negative redox potential) and heme 2 (having a large positive redox potential) form a lock for electrons generated during the oxidation reaction The attachment of HAO's physiological electron transfer partner cytochrome c(554) results in a positive shift in the redox potential of heme 1, and "opens the electron gate". Electrons generated as a result of hydroxylamine oxidation travel to heme 3 and heme 8, which have redox potentials close to 0 mV versus NHE (this result is in partial disagreement with an existing experimental redox potential assignment). The closeness of hemes 3 and 8 from different enzyme subunits allows redistribution of the four electrons generated as a result of hydroxylamine oxidation, among the three enzyme subunits. For the multielectron oxidation process to be maximally efficient, the redox potentials of the electron-accepting cofactors should be roughly equal, and electrostatic interactions between extra electrons on these cofactors should be minimal. The redox potential assignments presented in the paper satisfy this general rule. 相似文献
20.
Kumke MU Dosche C Flehr R Trowitzsch-Kienast W Löhmannsröben HG 《Zeitschrift für Naturforschung. C, Journal of biosciences》2006,61(9-10):741-748
Siderophores play a very important role in the uptake process of iron by bacteria. Due to the so-called active transport the uptake of siderophores by bacteria is very specific, which makes the use of siderophores as effective shuttles for antibiotics in the treatment of infections and other diseases caused by bacteria highly attractive. In order to further investigate the transport and incorporation of siderophores into the bacteria cells, distinct molecular probes are needed. Especially artificial siderophores, that show a specific intrinsic fluorescence, are highly attractive for such monitoring purposes. A promising candidate of such a fluorescent artificial siderophore is bis-2,3-dihydroxybenzoyl-2,6-dimethylamino-pyridine (pyridinochelin, PY). The fluorescence properties of PY were investigated in different solvents and in the presence of different metal ions. It was found that PY in its free form shows a complex fluorescence behavior. In methanol a clear dual fluorescence is observed. In aqueous solution intermolecular interactions with water molecules are determining the intrinsic fluorescence. Upon complexation with metal ions (Me3+ = Eu3+, Tb3+, Al3+, Fe3+) the fluorescence characteristics changed. The fluorescence quantum yield of PY decreased upon addition of Me3+--except for Al3+, which showed no fluorescence quenching. The fluorescence decay of PY loaded with metal ions showed a nicely mono-exponential fluorescence decay, which was in contrast to PY in the absence of metal ions. This drastic change in the fluorescence properties of PY upon metal ion complexation makes PY highly attractive as a fluorescence probe for the investigation of siderophore action and siderophore-mediated transport processes. 相似文献