A study on the reaction mechanism of adenosine 5′-phosphosulfate reductase from Thiobacillus thioparus, an iron-sulfur flavoprotein

1977 ◽  
Vol 55 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Kazuo Adachi ◽  
Isamu Suzuki
Keyword(s):  
Reaction Mechanism ◽  
Cytochrome C ◽  
Direct Reduction ◽  
Potassium Phosphate ◽  
Difference Spectrum ◽  
Order Rate Constant ◽  
Horse Heart Cytochrome ◽  
Thiobacillus Thioparus ◽  
Aps Reductase ◽  
Concomitant Reduction

The reaction mechanism of adenosine 5′-phosphosulfate (APS) reductase (EC 1.8.99.2) from Thiobacillus thioparus was studied using difference spectrum and stopped-flow techniques. The enzyme-bound FAD was rapidly reduced by sulfite with a first order rate constant of 97.1 s−1. The addition of AMP induced further spectral changes in the reduced enzyme which were consistent with the oxidation of FADH2 to the red (anionic) semiquinone FADH∙) and the concomitant reduction of nonheme iron to the ferrous state. Superoxide dismutase (EC 1.15.1.1) or anaerobiosis inhibited the reduction of cytochrome c by the enzyme only to the extent of 25–35%, indicating the existence of a direct reduction of cytochrome c by the enzyme without involving O2−. The activity of enzyme with cytochrome c was inhibited by increasing the potassium phosphate concentration, the inhibition being more pronounced with horse heart cytochrome c than with Candida krusei cytochrome c.

Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. II. Properties of adenosine-5′-phosphosulfate reductase

1970 ◽  
Vol 48 (3) ◽  
pp. 344-354 ◽  
Author(s):  
Ronald M. Lyric ◽  
Isamu Suzuki
Keyword(s):  
Molecular Weight ◽  
Cytochrome C ◽  
Electron Acceptor ◽  
Desulfovibrio Desulfuricans ◽  
Thiobacillus Denitrificans ◽  
Ph Optimum ◽  
Thiobacillus Thioparus ◽  
Aps Reductase

Adenosine-5′-phosphosulfate (APS) reductase was purified from Thiobacillus thioparus extracts 25- to 46-fold and the properties were studied. The molecular weight was 170 000 and the enzyme had 1 mole of FAD, 8–10 moles of iron, and 4–5 moles of labile sulfide. Cytochrome c as well as ferricyanide served as the electron acceptor. The pH optimum shifted from 7.4 to 9.5 when cytochrome c was used instead of ferricyanide. The Km values for sulfite and AMP were reduced from 2.5 mM and 100 μM to 17 μM and 2.5 μM, respectively, with cytochrome c as electron acceptor. Properties of the T. thioparus enzyme were compared to those of APS reductase isolated from Thiobacillus denitrificans and Desulfovibrio desulfuricans.

The Sulfite Oxidase of Thiobacillus ferrooxidans (Ferrobacillus ferrooxidans)

1971 ◽  
Vol 49 (10) ◽  
pp. 1125-1130 ◽  
Author(s):  
J. Robie Vestal ◽  
D. G. Lundgren
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Cytochrome C ◽  
Energy Production ◽  
Thiobacillus Ferrooxidans ◽  
Sulfite Oxidase ◽  
Electron Acceptors ◽  
Horse Heart Cytochrome ◽  
Thiobacillus Thioparus ◽  
Horse Heart Cytochrome C

The sulfite oxidase (sulfite: cytochrome c oxidoreductase) from sulfur-grown Thiobacillus ferrooxidans was isolated and partially purified, and its properties were studied. The enzyme was purified 7.3-fold and was 75–85% of the protein present. Sulfite oxidase required SO32− for activity, and could use horse heart cytochrome c and ferricyanide as electron acceptors. The molecular weight was 41 500. The enzyme had a Km for sulfite of 0.58 mM with either ferricyanide or cytochrome c as the electron acceptor. The Km for ferricyanide was 0.25 mM. 5′-AMP did not stimulate enzyme activity. Other properties of the enzyme were similar to the enzyme from Thiobacillus thioparus and Thiobacillus novellus. A metabolic scheme of sulfur utilization for energy production in Thiobacillus ferrooxidans is presented.

The oxidation of ferrocytochrome c in nonbinding buffer

1977 ◽  
Vol 55 (8) ◽  
pp. 796-803 ◽  
Author(s):  
B. F. Peterman ◽  
R. A. Morton
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Equilibrium Constant ◽  
Oxidation Reaction ◽  
Potassium Phosphate ◽  
Ionic Strength Dependence ◽  
Horse Heart Cytochrome ◽  
Apparent Equilibrium Constant ◽  
Diffusion Limited ◽  
Apparent Equilibrium

The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1–200 mM using Tris–cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox(~ 9 × 109 M−1 s−1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at μ = 0 was the order of 6 × 103), and was not consistent with diffusion-limited kinetics.

scholarly journals The reduction of carboxymethyl-cytochrome c by chromous ions

1974 ◽  
Vol 141 (2) ◽  
pp. 455-461 ◽  
Author(s):  
Thomas Brittain ◽  
Michael T. Wilson ◽  
Colin Greenwood
Keyword(s):  
Cytochrome C ◽  
Difference Spectrum ◽  
Order Rate Constant ◽  
Stopped Flow ◽  
Methionine Residue ◽  
Ferricytochrome C ◽  
A Charge ◽  
Kinetics Of ◽  
Dependent Processes ◽  
Kinetics Of Reduction

The reduction of ferricytochrome c and ferricytochrome c carboxymethylated at the haem-linked methionine (residue 80) by Cr2+ ions was studied by stopped-flow techniques. At pH6.2 the kinetics of reduction of ferricytochrome c are simple and correspond to a second-order rate constant of 1.21×103m-1·s-1. Under identical conditions the kinetics of reduction of the carboxymethyl derivative, carboxymethyl-cytochrome c, are complex; two Cr2+-concentration-dependent processes (1.5×104m-1·s-1 and 1.3×103m-1·s-1) lead to the formation of an intermediate which decays in monomolecular fashion (0.15s-1) to form the normal fully reduced material. The kinetic difference spectrum for the overall process corresponds to that found statically, whereas the kinetic difference spectrum of the intermediate minus the oxidized form resembles that of the low-spin ferrous form of carboxymethyl-cytochrome c minus oxidized carboxymethyl-cytochrome c. A model is proposed in which the reduction of low-spin ferric carboxymethyl-cytochrome c to high-spin ferrous carboxymethyl-cytochrome c involves a low-spin ferrous intermediate. The monomolecular step involving the decay of this low-spin ferrous intermediate is associated with an activation energy of approx. 126kJ·mol-1 and is thought to involve both a change of spin state and a protein-conformational event. Although carboxymethyl-cytochrome c represents a mixture of species separable on a charge basis, the above observations were independent of which species was chosen for study.

Kinetics and mechanism of the reaction between oxidized cytochrome c and ascorbic acid

1991 ◽  
Vol 56 (2) ◽  
pp. 478-490 ◽  
Author(s):  
Joaquin F. Perez-Benito ◽  
Conchita Arias
Keyword(s):  
Ascorbic Acid ◽  
Cytochrome C ◽  
Redox Reactions ◽  
Arrhenius Equation ◽  
Horse Heart Cytochrome ◽  
First Order ◽  
Acidic Form ◽  
Ph Range ◽  
Horse Heart Cytochrome C ◽  
Mechanism Of The Reaction

The reaction between horse-heart cytochrome c and ascorbic acid has been investigated in the pH range 5.5 – 7.1 and at 10.0 – 25.0 °C. The rate shows a first-order dependence on the concentration of cytochrome c, it increases in a non-linear way as the concentration of ascorbic acid increases, it increases markedly with increasing pH and, provided that the ionic strength of the medium is high enough, it fulfills the Arrhenius equation. The apparent activation energy increases as the pH of the solution increases. The results have been explained by means of a mechanism that includes the existence of an equilibrium between two forms (acidic and basic) of oxidized cytochrome c: cyt-H+ -Fe3+ + OH- cyt -Fe3+ + H2O, whose equilibrium constant is (6.7 ± 1.4). 108 at 25.0 °C, the acidic form being more reducible than the basic one. It is suggested that there is a linkage of hydrogenascorbate ion to both forms of cytochrome c previous to the redox reactions. Two possibilities for the oxidant-reductant linkage (binding and adsorption) are discussed in detail.

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