Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.4 (nitrite reductase)
1,847 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dissimilatory nitrite reductase, cytochrome cd1, from Pseudomonas aeruginosa (ATCC 19429) was irreversibly inactivated by methyl- or phenylhydrazine but was only reduced by hydrazine itself. The reaction required oxygen and several turnovers, approximately four, of the cytochrome acting to transfer reducing equivalents from phenylhydrazine to oxygen. The reaction with methyl- or phenylhydrazine altered the visible spectrum of the cytochrome. Bands characteristic of reduced heme c appeared plus new features that were not characteristic of either oxidized or reduced heme d1. Extraction of the heme from phenylhydrazine-treated cytochrome yielded a covalently modified form of the original heme d1. Visible, 1H NMR, and mass spectra were obtained on the purified modified heme and on the metal-free esterified derivative. The spectroscopic data indicate that the modification was the regiospecific substitution of the 5 meso-proton by a phenyl group.
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PMID:Inactivation of cytochrome cd1 by hydrazines. 215 16

The biosynthetic origin of methyl groups in heme d1 isolated from the nitrite reductase cytochrome cd1 was investigated by a stable isotope labeling experiment. Pseudomonas aeruginosa (American Type Culture Collection strain 19429) was grown on a minimal medium supplemented with [13C]methionine. The enzyme was purified, the heme extracted, converted into the free base methyl ester derivative, and purified. 1H NMR and 13C NMR indicated that only the methyl groups attached to C2 and C7 are derived from methionine.
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PMID:C-methylation occurs during the biosynthesis of heme d1. 216 31

The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.
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PMID:Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase. 217 71

Mixtures of the dissimilatory nitrite reductase cytochrome cd1 from Pseudomonas aeruginosa and potential electron-donating proteins were prepared in both fully oxidized and fully reduced states and examined by 1H NMR spectroscopy. The relatively narrower lines of the donor proteins enabled them to be clearly observed in spectra in the presence of significant amounts of the high molecular weight cd1. Mixtures of the physiological donor (Pseudomonas ferrocytochrome c-551) and ferrocytochrome cd1 showed specific line-broadening effects on the resonances of c-551 that depended on the mole ratio of c-551 to cd1. The experimental broadening fit a model in which c-551 is in intermediate or fast exchange between free solution and a complex with cd1, with an association constant for the complex in excess of 10(4) M-1. The model yields a minimum estimate for the forward bimolecular rate constant of 5 X 10(7) M-1 s-1 and suggests that the actual value may be much larger. The complexation was independent of pH in the range of 6-8, was independent of ionic strength over a salt concentration range of 20-1000 mM, and possessed a low thermal activation barrier. Mixtures of ferricytochrome c-551 and ferricytochrome cd1 showed no observable NMR perturbations, indicating that any hypothetical complex involving the oxidized forms must follow different dynamical and/or equilibrium conditions. No observable NMR perturbations existed in spectra of mixtures of cd1 and mammalian cytochrome c or Pseudomonas azurin in either oxidation state.
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PMID:1H NMR investigation of cytochrome cd1: complexes with electron-donor proteins. 300 18

Heme d1 is the noncovalently associated heme prosthetic group of the bacterial nitrite reductase known as cytochrome cd1. Additional evidence has been obtained in support of a dioxoisobacteriochlorin, or 1,3-porphyrindione, skeleton for this heme. The new data include the natural abundance 13C NMR spectrum of the free base methyl ester derivative of d1, mass spectrometric determinations of the molecular mass of the free base methyl ester and the Cu and the Zn chelates, visible and 1H NMR spectral comparisons between d1 and synthetic porphyrindione model compounds, and the isolation and characterization of several byproducts formed during the purification of the free base methyl ester of d1. The accumulated evidence strongly supports the following structure for the skeleton of d1: 1-oxo-2-methyl-2'-acetyl-3-oxo-4-methyl-4'-acetyl-5-methyl-6-acrylyl+ ++-7- propionyl-8-methylporphyrin.
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PMID:Evidence that heme d1 is a 1,3-porphyrindione. 382 88

The substituents of the noncovalently associated heme prosthetic group of the bacterial nitrite reductase-cytochrome oxidase (EC 1.9.6.1 or EC 1.9.3.2.) from Pseudomonas aeruginosa (ATCC 19429) and Paracoccus denitrificans (ATCC 13456) have been identified. This was accomplished by 1H NMR, infrared, visible, and mass spectroscopic techniques applied to semi-purified heme and purified methyl ester derivatives of the iron-free porphyrin. The main structural features are as follows. 1) The macrocycle is a reduced porphyrin of the chlorin type, that is, one of the pyrrole rings has been saturated so that the beta-carbons have sp3-hybridization. 2) The chlorin is a tetracarboxylic acid. 3) The substituents of the saturated pyrrole ring are two methyl groups and two hydroxymethyl groups. 4) The substituents of the unsaturated pyrrole rings are: (i) two methyl groups (ii) a propionic acid (iii) an acrylic acid, and (iv) two directly bonded formic acid groups. No firm evidence has been obtained for the relative positions of these unsaturated beta-pyrrolic substituents around the macrocyclic ring, but a proposed structure will be discussed. Several previously inexplicable chemical properties of the heme in extracts or in the intact enzyme can be now interpreted in view of the proposed structure. The trivial name "acrylochlorin" is suggested for the macrocycle.
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PMID:Proposed structure for the noncovalently associated heme prosthetic group of dissimilatory nitrite reductases. Identification of substituents. 642 Apr 11

Nitrogen-15 nuclear magnetic resonance (15N NMR) spectroscopy at 30.4 MHz was employed to determine the interaction of the substrate nitrite (97.2% enriched) with bacterial nitrite reductase, denoted cytochrome cd1, from Pseudomonas aeruginosa. The addition of ferric enzyme to nitrite did not alter the chemical shift of the bulk nitrite resonance, nor was it possible to observe a new resonance from a hypothetical bound form. However, the spin-lattice relaxation time (T1) was lowered from 13.2 to 2.7 s, and the spin-spin relaxation time (T2) was halved. Values of T1 were measured by progressive saturation and values of T2 by line widths. Control experiments involving ferric cytochrome c and metmyoglobin demonstrated that the perturbations did not arise from the bulk paramagnetic properties of the protein solutions. Variable enzyme/substrate ratios were measured to assess the strength of interaction. The most reasonable model consistent with the data proposes a weak association between nitrite and ferric reductase with a value of 1.3 M-1 for the association constant.
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PMID:Nitrogen-15 nuclear magnetic resonance investigation of nitrite reductase-substrate interaction. 681 79

In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551 and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.
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PMID:Electron-transfer properties of Pseudomonas aeruginosa [Lys44, Glu64]azurin. 924 43

A molecular model of QH-ADH, the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni, has been built by homology modelling. Sequence similarity of N-terminal residues 1-570 with the alpha-subunit of quinoprotein methanol dehydrogenases (MDHs) from Methylophilus methylotrophus W3A1 and Methylobacterium extorquens provided a basis for the design of the PQQ-binding domain of QH-ADH. Minimal sequence similarity with cytochrome c551 from Ectothiorhodospira halophila and cytochrome c5 from Azotobacter vinelandii has been used to model the C-terminal haem c-binding domain, residues 571-677, absent in MDHs. Distance constraints inferred from 19F-NMR relaxation studies of trifluoromethylphenylhydrazine-derivatized PQQ bound to QH-ADH apoenzyme as well as theoretical relations for optimal electron transfer have been employed to position the haem- and PQQ-binding domains relative to each other. The homology model obtained shows overall topological similarity with the crystal structure of cd1-nitrite reductase from Thiosphera pantotropha. The proposed model accounts for the following: (i) the site that is sensitive to in vivo proteolytic attack; (ii) the substrate specificity in comparison with MDHs; (iii) changes of the spectral properties of the haem c upon reconstitution of apo-enzyme with PQQ; (iv) electronic interaction between haem and PQQ; and (v) enantioselectivity in the conversion of a chiral sec alcohol.
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PMID:Homology model of the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni. 961 42

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.
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PMID:Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase. 1103 20


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