scholarly journals Kinetic studies on (N-formyltryptophyl)cytochrome c

1975 ◽  
Vol 149 (3) ◽  
pp. 713-717 ◽  
Author(s):  
T Brittain ◽  
C Greenwood
Keyword(s):  
Cytochrome C ◽  
Flash Photolysis ◽  
Reduction Process ◽  
Kinetic Studies ◽  
Tryptophan Residue ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Normal Environment ◽  
Two Phases ◽  
Flow Techniques

The formylation of the ring nitrogen atom of the tryptophan residue in cytochrome c was carried out and consequent changes in the kinetic properties of the protein were investigated. The reduction of formylated cytochrome c by Cr2+ was studied by stopped-flow techniques. At pH 6.5 the reduction process shows the presence of two phases. One phase (k = 4 × 104 M-1-s-1) is dependent on Cr2+ concentration and one phase (k = 5.0 s-1) is not. A study of the temperature dependence of the two phases yields values for their activation energies of 38.6kJ·mol-1 and 42.4kJ·mol-1 respectively. The reaction of the reduced formylated cytochrome c with CO was followed by means of both stopped-flow techniques and flash photolysis. The combination with CO at pH 6.8 measured in stopped-flow experiments shows two phases, both dependent on the concentration of CO (k1 = 1.8 × 102 M-1-s-1). If CO was dissociated from the protein by photolysis and then allowed to recombine with it, it was found to do so in a simple manner, at a rate which depended on the concentration of CO (k = 1.9 × 10M2 M-1-s-1). A tentative model which can accommodate these findings is proposed. The reaction of the oxidized form of formylated cytochrome c with NO was followed by means of stopped-flow techniques. The reaction was found to be biphasic with one phase dependent on the concentration of NO (k = 2.8 × 103 M-1-s-1) and one phase (k = 0.2s-1) independent of the concentration of NO. This behaviour is compared with that of the native molecule. A comparison of these kinetic observations with those on other tryptophan-specific modifications leads to the conclusion that the main alteration in kinetic properties is due, not to the nature of the modifying group, but rather to the disruption of the normal environment of the haem.

scholarly journals Kinetic studies on mammalian cytochrome c modified with 2-hydroxy-5-hydroxy-5-nitrobenzyl bromide

1975 ◽  
Vol 149 (1) ◽  
pp. 179-185 ◽  
Author(s):  
T Brittain ◽  
C Greenwood
Keyword(s):  
Cytochrome C ◽  
Flash Photolysis ◽  
Ph Dependence ◽  
Phase 1 ◽  
Kinetic Studies ◽  
Chromium Concentration ◽  
Stopped Flow ◽  
Phase 2 ◽  
Difference Spectra ◽  
Phase Phase

The reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c by the chromous ion was studied by stopped-flow techniques. At pH6.5 the reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c is complex, showing the presence of three distinct phases. Two chromium concentration-dependent phases are observed (1.1 × 105 M-1-S-1, phase 1; 1.25 × 104M-1-S-1, phase 2) and one slow first-order process (0.25S-1, phase 3). A comparison of the static and kinetic difference spectra, along with the data from the reduction of the reoxidized reduced protein, suggests that the slow chromium concentration-independent phase is due to a slow conformational event after fast reduction of the NO2 group. The rates of the chromium concentration-dependent phases show a marked variation with pH above 7.5. The activation energies for the three processes were also measured at 33.2, 38.6 and 69.7 kJ-mol-1 for phases 1, 2 and 3 respectively. The reaction of reduced 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c with CO was foollowed by means of both stopped-flow and flash photolysis. The combination with CO at pH 6.8 as measured in stopped-flow experiments showed two phases, one CO-dependent phase (phase 2, 2.4 × 102M-1-S-1) and one CO-independent phase (phase 1, 0.015S-1). Investigation of the pH-dependence of the phases showed both the rates and amounts of each phase to be pH-invariant. CO recombination, after photolytic removal, was found to be biphasic; a CO-dependent phase (phase 2, 2.4 × 102M-1-S-1) and a CO-independent phase (phase 1, 1.0s-1) were observed. A tentative model which can accommodate these observations is proposed.

scholarly journals Kinetic studies on [methionine sulphoxide] cytochrome c

1976 ◽  
Vol 159 (3) ◽  
pp. 621-626 ◽  
Author(s):  
T Brittain ◽  
C Greenwood
Keyword(s):  
Cytochrome C ◽  
Reduction Process ◽  
Order Reduction ◽  
Kinetic Studies ◽  
Stopped Flow ◽  
Chemically Modified ◽  
Neutral Ph ◽  
Recombination Processes ◽  
Reduced Forms ◽  
Flow Experiments

A cytochrome c haem ligand, methionine-80, was photo-oxidized to methionine sulphoxide and the subsequent changes in redox properties and ligand binding were monitored kinetically. Isoelectric focusing of the product showed the presence of a single oxidized species, capable of binding CO when reduced. The binding of CO to the reduced protein was followed in stopped-flow experiments, which revealed the presence of two binding processes, at neutral pH, with rate constants of K+1 = 3.4 × 10(3)M-1-S-1 and k+2 = 5.80 × 10(2)M-1-S-1. When CO was photolytically dissociated from the reduced protein two recombination processes were observed with rates almost identical with those observed in the stopped-flow experiments (k+1 = 3.3 × 10(3)M-1-S-1 and k+2 = 6.0 × 10(2)M-1-S-1). These findings provide evidence of two reduced forms of the protein. The reduction of [methionine sulphoxide]cytochrome c by Cr2+ at neutral pH in stopped-flow experiments showed the presence of a single second-order reduction process (k = 7.2 × 10(3)M-1-S-1, activation energy = 44kJ/mol) and one first-order process. This protein was compared with some other chemically modified cytochromes.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide

1975 ◽  
Vol 151 (1) ◽  
pp. 51-59 ◽  
Author(s):  
S R Parr ◽  
M T Wilson ◽  
C Greenwood
Keyword(s):  
Cytochrome Oxidase ◽  
Flash Photolysis ◽  
Order Rate Constant ◽  
Second Order ◽  
Stopped Flow ◽  
First Order ◽  
Order Rate ◽  
Co Concentration ◽  
Pseudo First Order ◽  
Two Phases

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 × 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 × 10(4)M-1-s-1 and 1.6 × 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.

scholarly journals Transient kinetic studies of Neurospora crassa cytochrome c oxidase

1983 ◽  
Vol 215 (2) ◽  
pp. 425-427 ◽  
Author(s):  
M Brunori ◽  
M C Silvestrini ◽  
M T Wilson ◽  
H Weiss
Keyword(s):  
Cytochrome C ◽  
Neurospora Crassa ◽  
Cytochrome C Oxidase ◽  
Flash Photolysis ◽  
Kinetic Studies ◽  
Dissociation Rate ◽  
Steady State Kinetic ◽  
Kinetic Investigations ◽  
Dissociation Rate Constants

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be ‘pulsed’, i.e. activated, like the mammalian enzyme. The ‘pulsed’ species is spectroscopically different from the ‘resting’ one, and the decay into the ‘resting’ state is fast (t1/2 approx. 3 min).

scholarly journals Transient kinetics of subunit-III-depleted cytochrome c oxidase

1986 ◽  
Vol 234 (3) ◽  
pp. 569-572 ◽  
Author(s):  
F Malatesta ◽  
G Antonini ◽  
P Sarti ◽  
M Brunori
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Flash Photolysis ◽  
Order Rate Constant ◽  
Kinetic Properties ◽  
Transient Kinetics ◽  
Rate Limiting Step ◽  
Subunit Iii ◽  
The Stability ◽  
Rate Limiting

Cytochrome c oxidase from ox heart was depleted of subunit III and its transient kinetic properties studied by stopped-flow and flash photolysis. It was found that the overall mechanism of electron transfer is very similar for subunit-III-depleted and native oxidase, although significant differences in some kinetic parameters have been detected. These include the second-order rate constant for cytochrome c oxidation and the rate-limiting step of the overall process. Moreover, at low cytochrome c/oxidase ratios (where the number of reducing equivalents is insufficient), the rate of reoxidation of cytochrome a was found to be very slow, even in air, and in fact for the subunit-III-depleted enzyme is even slower than for the native oxidase. The stability of reduced cytochrome a excludes the likelihood that removal of subunit III leads to a new O2-binding site, and the result may be relevant to the lowered vectorial H+/e- stoichiometry. The subunit-III-depleted oxidase can be pulsed under appropriate conditions and its combination with CO is unchanged, as shown by kinetic experiments and difference spectroscopy.

Kinetic studies on the reaction of NO with iron(ii) complexes using low temperature stopped-flow techniques

2020 ◽  
Vol 49 (27) ◽  
pp. 9480-9486
Author(s):  
Pascal Specht ◽  
Martin Oßberger ◽  
Peter Klüfers ◽  
Siegfried Schindler
Keyword(s):  
Low Temperature ◽  
Kinetic Study ◽  
Reaction Mechanisms ◽  
Kinetic Studies ◽  
Stopped Flow ◽  
Flow Techniques

Quite different reaction mechanisms were observed in a kinetic study of NO binding to iron(ii) complexes.

scholarly journals Kinetics of reaction of [nitrotyrosyl]cytochrome c with ligands

1976 ◽  
Vol 157 (1) ◽  
pp. 217-220
Author(s):  
A José do Nascimento ◽  
K Hishida do Nascimento
Keyword(s):  
Cytochrome C ◽  
Slow Phase ◽  
Stopped Flow ◽  
Order Process ◽  
First Order ◽  
Fast Phase ◽  
Kinetics Of Reaction ◽  
Kinetics Of ◽  
Flow Techniques

The reaction of [nitrotyrosyl]cytochrome c with ligands was studied by stopped-flow techniques. At pH 7.0 the reaction with imidazole shows two distinct phases, one fast phase being concentration-dependent and a slow phase being concentration-independent. The results are consistent with the existence of two forms of [nitrotyrosyl]cytochrome c in solutions [Schejter et al. (1970) Biochemistry 9, 5118-5122]; form I, the smaller fraction, seems to be responsible for the slow first-order process.

scholarly journals Pseudomonas cytochrome C-551 peroxidase. A purification procedure and study of CO-binding kinetics

1983 ◽  
Vol 209 (3) ◽  
pp. 701-707 ◽  
Author(s):  
N Foote ◽  
A C Thompson ◽  
D Barber ◽  
C Greenwood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cytochrome C ◽  
Isoelectric Focusing ◽  
Flash Photolysis ◽  
Cytochrome C Peroxidase ◽  
Acid Precipitation ◽  
Binding Kinetics ◽  
Stopped Flow ◽  
Purification Procedure ◽  
Kinetics Of

A procedure is described for the purification of cytochrome c peroxidase from Pseudomonas aeruginosa involving extraction by sonication, followed by acid precipitation and chromatography on only two types of gel. The final preparation had a purity ratio A407/A280 of 4.2, and was found to be essentially pure by isoelectric focusing. The enzyme was shown to be unstable during degassing under vacuum except in the presence of detergent. The kinetics of CO binding to dithionite-reduced peroxidase were studied with stopped-flow and flash-photolysis techniques, and the results obtained between pH 5 and 7 suggest the existence of two forms of dithionite-reduced enzyme in slow equilibrium.

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