scholarly journals Electron transfer in zinc-reconstituted nitrite reductase from Pseudomonas aeruginosa

1996 ◽  
Vol 319 (2) ◽  
pp. 407-410 ◽  
Author(s):  
Andrea BELLELLI ◽  
Peter BRZEZINSKI ◽  
Marzia ARESE ◽  
Francesca CUTRUZZOLÀ ◽  
Maria Chiara SILVESTRINI ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Rate Constant ◽  
Nitrite Reductase ◽  
Flow Analysis ◽  
Protoporphyrin Ix ◽  
Back Reaction ◽  
Stopped Flow ◽  
Triplet Excited State ◽  
Prosthetic Groups

1. The catalytic cycle of the haem-containing nitrite reductase (NIR) from Pseudomonas aeruginosa involves electron transfer between the two prosthetic groups of the enzyme, the c-haem and the d1-haem; this reaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d1-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d1-haem. 2. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem, with a rate constant of 7×105 s-1; since the intermediate with reduced c-haem is not significantly populated, we conclude that the back reaction is probably as fast. 3. Even taking into account that in the native NIR the driving force is close to zero, the rate constant for the c → d1 electron transfer, estimated from our experiments, is still much higher than that observed by stopped flow (k = 0.3 s-1) using reduced azurin as the electron donor. This finding may be a direct kinetic indication that reduction of the d1-haem is associated with a substantial reorganization of the co-ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR.

scholarly journals Kinetic studies on the nitrite reductase of Wolinella succinogenes

1986 ◽  
Vol 233 (2) ◽  
pp. 553-557 ◽  
Author(s):  
R Blackmore ◽  
T Brittain
Keyword(s):  
Rate Constant ◽  
Nitrite Reductase ◽  
Flash Photolysis ◽  
Spectral Characteristics ◽  
Order Rate Constant ◽  
Second Order ◽  
Stopped Flow ◽  
Dependent Manner ◽  
Wolinella Succinogenes ◽  
Order Rate

The six haem groups of the nitrite reductase enzyme isolated from Wolinella succinogenes are rapidly reduced by the addition of dithionite (S2O4(2-)). The reduction, however, is not homogeneous. Two of the haem groups, namely those that show spectral characteristics typical of five-co-ordinated haem groups, are reduced in a dithionite-concentration-dependent fashion with a rate limit of 1.5 S-1. The other four haem groups, which show spectral characteristics very similar to those of normal six-co-ordinate c-haem groups, reduce in a linear dithionite-concentration-dependent manner with a second-order rate constant of 150 M-1/2 X S-1. The ratio of the amplitudes of the two reduction phases observed in stopped-flow studies is found to be dependent on the concentration of dithionite used. A model is proposed to account for these observations, and computer simulations show that the model represents a good fit to the experimental data. The two haem groups with five-co-ordinate spectral characteristics bind CO. Flash photolysis of the CO complex exhibits one major recombination process with a linear dependence in rate on CO concentration with a second-order rate constant of 2 × 106 M-1 × S-1. By contrast, stopped-flow mixing of the reduced protein with CO shows a very complex pattern of combination, with most of the observed absorbance change associated with a concentration-independent step. These findings are rationalized in terms of structural changes in the protein consequent to ligand binding.

Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells

2012 ◽  
Vol 302 (2) ◽  
pp. C419-C428 ◽  
Author(s):  
Sandrine Genetet ◽  
Pierre Ripoche ◽  
Julien Picot ◽  
Sylvain Bigot ◽  
Jean Delaunay ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Hemolytic Anemia ◽  
Rate Constant ◽  
Rate Constants ◽  
Flow Analysis ◽  
Red Cells ◽  
Stopped Flow ◽  
Loss Of Function ◽  
Dramatic Decrease ◽  
Time Courses

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pHi) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH3transport through the channel and NH3diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rhnulllacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.

scholarly journals Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein

1992 ◽  
Vol 285 (2) ◽  
pp. 661-666 ◽  
Author(s):  
M C Silvestrini ◽  
F Cutruzzolà ◽  
R D'Alessandro ◽  
M Brunori ◽  
N Fochesato ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Recombinant Protein ◽  
Pseudomonas Putida ◽  
Molecular Mass ◽  
Nitrite Reductase ◽  
Water Soluble ◽  
Binding Kinetics ◽  
Cytochrome C551

Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).

scholarly journals Intramolecular Electron Transfer in Pseudomonas aeruginosa cd1 Nitrite Reductase: Thermodynamics and Kinetics

2009 ◽  
Vol 96 (7) ◽  
pp. 2849-2856 ◽  
Author(s):  
Ole Farver ◽  
Maurizio Brunori ◽  
Francesca Cutruzzolà ◽  
Serena Rinaldo ◽  
Scot Wherland ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Nitrite Reductase ◽  
Intramolecular Electron Transfer ◽  
Thermodynamics And Kinetics

Kinetics of electron transfer in Pseudomonas aeruginosa cytochrome cd1-nitrite reductase

1996 ◽  
Vol 243 (1-2) ◽  
pp. 25-31 ◽  
Author(s):  
Steven A. Schichman ◽  
Terry E. Meyer ◽  
Harry B. Gray
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Nitrite Reductase ◽  
Cytochrome Cd1 ◽  
Kinetics Of

Formation of CT complexes and electron transfer from excited molecules of anthracene derivatives to methylviologen in aqueous and micellar media

1987 ◽  
Vol 52 (7) ◽  
pp. 1658-1665
Author(s):  
Viktor Řehák ◽  
Jana Boledovičová
Keyword(s):  
Electron Transfer ◽  
Complex Formation ◽  
Fluorescence Quenching ◽  
Rate Constant ◽  
Formation Constants ◽  
Dynamic Quenching ◽  
Micellar Media ◽  
Complex Formation Constants ◽  
Anthracene Derivatives ◽  
Ct Complexes

Disodium 1,5- and 1,8-anthracenedisulphonate (ADS) and 9-acetylanthracene form coloured CT complexes with methylviologen (MV2+) in aqueous and micellar media. The complex formation constants and molar absorptivities were determined by the Benesi-Hildebrandt method. In the fluorescence quenching, its static component plays the major role. The dynamic quenching component is determined by the rate constant of electron transfer from the S1 state of ADS to MV2+.

The Effect of p-Toluidine on the Two-Step Electroreduction of Zn(II) in Water-Methanol and Water-Dimethylformamide

1999 ◽  
Vol 64 (4) ◽  
pp. 585-594 ◽  
Author(s):  
Barbara Marczewska
Keyword(s):  
Electron Transfer ◽  
Binary Mixtures ◽  
Rate Constant ◽  
Rate Constants ◽  
Electrode Surface ◽  
Mercury Electrode ◽  
Forward Rate ◽  
Solvent Molecules ◽  
Activated Complex

The acceleration effect of p-toluidine on the electroreduction of Zn(II) on the mercury electrode surface in binary mixtures water-methanol and water-dimethylformamide is discussed. The obtained apparent and true forward rate constants of Zn(II) reduction indicate that the rate constant of the first electron transfer increases in the presence of p-toluidine. The acceleration effect may probably be accounted for by the concept of the formation on the mercury electrode an activated complex, presumably composed of p-toluidine and solvent molecules.

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