Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
J Biol Inorg Chem ; 17(4): 647-62, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22382353

ABSTRACT

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).


Subject(s)
Cytochromes a1/biosynthesis , Cytochromes a1/chemistry , Cytochromes c1/biosynthesis , Cytochromes c1/chemistry , Nitrate Reductases/biosynthesis , Nitrate Reductases/chemistry , Shewanella/enzymology , Adsorption , Crystallography, X-Ray , Cytochromes a1/genetics , Cytochromes a1/isolation & purification , Cytochromes c1/genetics , Cytochromes c1/isolation & purification , Electrodes , Kinetics , Models, Molecular , Nitrate Reductases/genetics , Nitrate Reductases/isolation & purification , Protein Conformation , Shewanella/cytology , Spectrophotometry, Ultraviolet , Surface Properties
2.
Methods Enzymol ; 496: 399-422, 2011.
Article in English | MEDLINE | ID: mdl-21514473

ABSTRACT

Cytochrome c nitrite reductase, NrfA, catalyzes the six-electron reduction of nitrite, NO(2)(-), to ammonium, NH(4)(+), as the final enzymatic step in the dissimilatory metabolic pathway of nitrite ammonification within the biogeochemical nitrogen cycle. NrfA is a 55-65kDa protein that binds five c-type heme groups via thioether bonds to the cysteines of conserved CXXCH heme attachment motifs. Four of these heme groups are considered to be electron transfer centers, with two histidine residues as axial ligands. The remaining heme group features an unusual CXXCK-binding motif, making lysine the proximal axial ligand and leaving the distal position for the substrate binding site located in a secluded binding pocket within the protein. The substrate nitrite is coordinated to the active site heme iron though the free electron pair at the nitrogen atom and is reduced in a consecutive series of electron and proton transfers to the final product, the ammonium ion. While no intermediates of the reaction are released, NrfA is able to reduce various other nitrogen oxides such as nitric oxide (NO), hydroxylamine (H(2)NOH), and nitrous oxide (N(2)O), but notably also sulfite, providing the only known direct link between the nitrogen and sulfur cycles. NrfA invariably forms stable homodimers, but there are at least two distinct electron transfer systems to the enzyme. In many enterobacterial species, NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a soluble electron carrier, NrfB, that in turn interacts with a membrane-integral quinol dehydrogenase, NrfCD. In δ- and ε-proteobacteria, the dimeric NrfA forms a complex with a small quinol dehydrogenase of the NapC/NirT family, NrfH, allowing a more efficient electron transfer.


Subject(s)
Bacteria/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cytochrome c Group/chemistry , Cytochrome c Group/metabolism , Cytochromes a1/chemistry , Cytochromes a1/metabolism , Cytochromes c1/chemistry , Cytochromes c1/metabolism , Nitrate Reductases/chemistry , Nitrate Reductases/metabolism , Bacterial Proteins/isolation & purification , Catalytic Domain , Crystallography, X-Ray , Cytochrome c Group/isolation & purification , Cytochromes a1/isolation & purification , Cytochromes c1/isolation & purification , Heme/chemistry , Nitrate Reductases/isolation & purification , Nitrogen Cycle , Protein Conformation , Structure-Activity Relationship , Substrate Specificity , Wolinella/enzymology
3.
Methods Enzymol ; 437: 63-77, 2008.
Article in English | MEDLINE | ID: mdl-18433623

ABSTRACT

The periplasmic cytochrome c nitrite reductase (Nrf) system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. Nitric oxide (NO) is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate. This chapter describes the method used to assay Nrf-catalyzed NO reduction in whole cells of E. coli and the procedures for preparing highly purified NrfA suitable for use in kinetic, spectroscopic, voltammetric, and crystallization studies.


Subject(s)
Cytochrome c Group/physiology , Escherichia coli/enzymology , Crystallography, X-Ray , Cytochrome c Group/chemistry , Cytochrome c Group/isolation & purification , Cytochrome c Group/metabolism , Cytochromes a1/chemistry , Cytochromes a1/isolation & purification , Cytochromes a1/metabolism , Cytochromes a1/physiology , Cytochromes c1/chemistry , Cytochromes c1/isolation & purification , Cytochromes c1/metabolism , Cytochromes c1/physiology , Escherichia coli/growth & development , Models, Molecular , Nitrate Reductases/chemistry , Nitrate Reductases/isolation & purification , Nitrate Reductases/metabolism , Nitrate Reductases/physiology , Nitric Oxide/metabolism
4.
Biochemistry (Mosc) ; 73(2): 164-70, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18298372

ABSTRACT

A new procedure for isolation of cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens increasing significantly the yield of the purified enzyme is presented. The enzyme is isolated from the soluble fraction of the cell extract as a hexamer, as shown by gel filtration chromatography and small angle X-ray scattering analysis. Thermostability of the hexameric form of the nitrite reductase is characterized in terms of thermoinactivation and thermodenaturation.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Cytochromes a1/chemistry , Cytochromes a1/isolation & purification , Cytochromes c1/chemistry , Cytochromes c1/isolation & purification , Ectothiorhodospiraceae/enzymology , Nitrate Reductases/chemistry , Nitrate Reductases/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , Enzyme Stability , Scattering, Small Angle , Temperature , X-Ray Diffraction
5.
FEBS Lett ; 581(2): 284-8, 2007 Jan 23.
Article in English | MEDLINE | ID: mdl-17207484

ABSTRACT

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.


Subject(s)
Bacterial Proteins/chemistry , Cytochromes a1/chemistry , Cytochromes c1/chemistry , Desulfovibrio desulfuricans/enzymology , Heme/chemistry , Nitrate Reductases/chemistry , Bacterial Proteins/isolation & purification , Binding Sites , Catalysis , Cytochromes a1/isolation & purification , Cytochromes c1/isolation & purification , Nitrate Reductases/isolation & purification , Potentiometry , Protons
6.
Biochemistry ; 43(47): 15086-94, 2004 Nov 30.
Article in English | MEDLINE | ID: mdl-15554716

ABSTRACT

Cytochrome c nitrite reductase is a dimeric decaheme-containing enzyme that catalyzes the reduction of nitrite to ammonium. The contrasting effects of two inhibitors on the activity of this enzyme have been revealed, and defined, by protein film voltammetry (PFV). Azide inhibition is rapid and reversible. Variation of the catalytic current magnitude describes mixed inhibition in which azide binds to the Michaelis complex (approximately 40 mM) with a lower affinity than to the enzyme alone (approximately 15 mM) and leads to complete inhibition of enzyme activity. The position of the catalytic wave reports tighter binding of azide when the active site is oxidized (approximately 39 microM) than when it is reduced. By contrast, binding and release of cyanide are sluggish. The higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase is immediately revealed, as is the presence of two sites for cyanide binding and inhibition of the enzyme. Formation of the monocyano complex by reduction of the enzyme followed by a "rapid" scan to high potentials captures the activity-potential profile of this enzyme form and shows it to be distinct from that of the uninhibited enzyme. The biscyano complex is inactive. These studies demonstrate the complexity that can be associated with inhibitor binding to redox enzymes and illustrate how PFV readily captures and deconvolves this complexity through its impact on the catalytic properties of the enzyme.


Subject(s)
Cytochrome c Group/metabolism , Cytochromes a1/antagonists & inhibitors , Cytochromes a1/metabolism , Cytochromes c1/antagonists & inhibitors , Cytochromes c1/metabolism , Nitrate Reductases/antagonists & inhibitors , Nitrate Reductases/metabolism , Potentiometry , Amino Acid Motifs , Amino Acid Sequence , Azides/chemistry , Binding Sites , Catalysis , Cyanides/chemistry , Cytochromes a1/chemistry , Cytochromes a1/isolation & purification , Cytochromes c1/chemistry , Cytochromes c1/isolation & purification , Dimerization , Electrochemistry , Enzyme Activation , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Heme/chemistry , Kinetics , Models, Molecular , Nitrate Reductases/chemistry , Nitrate Reductases/isolation & purification , Nitrites/metabolism , Oxidation-Reduction , Spectrophotometry
SELECTION OF CITATIONS
SEARCH DETAIL
...