scholarly journals On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes

1987 ◽  
Vol 245 (3) ◽  
pp. 773-779 ◽  
Author(s):  
C J A Wallace ◽  
A E I Proudfoot
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Biological Activities ◽  
Amino Acid Residues ◽  
Redox Potentials ◽  
Peptide Bonds ◽  
Oxidation Reduction ◽  
Oxidation Reduction Potential ◽  
Stable Association ◽  
The Relationship

We have confirmed the propensity of fragments of cytochrome c to form complexes that reproduce the structure and, in part, the functionality, of the native protein by preparing four novel complexes. We have used trypsin under three different sets of conditions in sequence to prepare a contiguous two-fragment complex (1-55).(56-104). One of the intermediates is a stable overlapping complex (1-65).(56-104). Conditions for limited acid hydrolysis of peptide bonds in cytochrome c have been developed that optimize the yield of fragments (1-50) and (51-104). These two fragments also form a stable association, as do (1-50) and (56-104). These complexes are potentially useful for the semisynthesis of analogues modified in the region of the cleavage sites, which include a number of highly conserved amino acid residues, and are being used for studies of protein folding, interactions with oxidase, cytochrome c immunogenicity and of artificially induced spontaneous resyntheses between complexing fragments. Like other known two-fragment complexes of cytochrome c, they exhibit normal visible spectra, including the presence of the 695 nm band, indicative of a functional haem crevice. Studies of their biological activities and redox potentials lead to a number of conclusions on structure-function relationships in cytochrome c. Most significantly there is a linear relationship between the logarithm of electron-transfer rates from cytochrome c reductase and redox potential in this series of analogues, indicating that such transfer is thermodynamically controlled. This discovery contributes to our understanding of the interaction of cytochrome and reductase. Since the relationship is obeyed by other types of analogues, except for those that involve modification of the active site of cytochrome c, we have a useful diagnostic for those residues that participate directly in electron transfer.

scholarly journals The oxidation-state-dependent ATP-binding site of cytochrome c. Implication of an essential arginine residue and the effect of occupancy on the oxidation-reduction potential

1988 ◽  
Vol 252 (2) ◽  
pp. 349-355 ◽  
Author(s):  
B E Corthésy ◽  
C J Wallace
Keyword(s):  
Cytochrome C ◽  
Oxidation State ◽  
Dependent Manner ◽  
Redox Potentials ◽  
Redox Systems ◽  
Atp Binding Site ◽  
Oxidation Reduction ◽  
State Dependent ◽  
Oxidation Reduction Potential

Arg-91 is not part of the active site of cytochrome c that mediates binding and electron transfer, yet it is absolutely conserved in eukaryotic cytochromes c, indicating a special function. The physicochemical properties of analogues are unaffected by the modification of this residue, so they can be used with confidence to study the role of Arg-91. We have established limiting conditions under which this residue alone is specifically modified by cyclohexane-1,2-dione, and have subsequently shown that ATP, and to a lesser extent ADP or Pi, protects it from the action of the reagent in an oxidation-state-dependent manner. These observations strongly support the idea that this site exerts a controlling influence on cytochrome c activity in the electron transport or other cellular redox systems, and we have commenced a study of how that influence might operate. We find that the redox potentials of both cytochrome c and analogue are little affected by changing ATP or Pi concentrations.

scholarly journals Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase

1982 ◽  
Vol 201 (1) ◽  
pp. 9-18 ◽  
Author(s):  
G W Pettigrew ◽  
S Seilman
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Peroxidase ◽  
Sodium Phosphate Buffer ◽  
Oxidation Reduction ◽  
Purification And Properties ◽  
Lysine Residues ◽  
The Cross ◽  
Nadh Cytochrome ◽  
Stable Association ◽  
Covalently Linked

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.

Microbial Biomass and Enzyme Activities in Chromium and Lead-Contaminated Sediments

2013 ◽  
Vol 798-799 ◽  
pp. 1139-1143
Author(s):  
Chao Wang ◽  
Shuai Cheng ◽  
Pei Fang Wang ◽  
Yan Yan Ma
Keyword(s):  
Heavy Metals ◽  
Microbial Biomass ◽  
Enzyme Activities ◽  
Chemical Properties ◽  
Nitrogen And Phosphorus ◽  
Oxidation Reduction ◽  
Oxidation Reduction Potential ◽  
Organic Carbon Oxidation ◽  
The Relationship ◽  
Cr Contamination

The relationship between microbial biomass and enzyme activities under heavy metal pollution had attracted much attention in ecology. The experimental sediment samples were supplemented with Pb and Cr and incubated at room temperature for a month. Microbial properties such as microbial biomass, urease, catalase and cellulase activities, together with several chemical properties such as pH, total organic carbon , oxidation-reduction potential, total nitrogen and phosphorus were measured to evaluate changes in sediment qualities. Our results demonstrate that heavy metals would inhibit sediment microbe biomass and enzyme activities. Such decreases in sediments microbial biomass and enzyme activities by Pb and Cr contamination may help to evaluate heavy metals contaminated soil ecologies.

Purification and Characterization of Cytochrome 553 from the Chrysophycean Alga Monochrysis lutheri

1971 ◽  
Vol 49 (6) ◽  
pp. 641-646 ◽  
Author(s):  
M. V. Laycock ◽  
J. S. Craigie
Keyword(s):  
Amino Acid ◽  
Higher Plants ◽  
Sedimentation Coefficient ◽  
Cytochrome F ◽  
Reduced Form ◽  
Amino Acid Residues ◽  
Oxidation Reduction ◽  
Oxidation Reduction Potential ◽  
Equilibrium Centrifugation

Cytochrome 553, an electron carrier in photosynthesis and a functional analogue of cytochrome f of higher plants, was isolated from Monochrysis lutheri and purified by salt fractionation, chromatography, and isoelectric focussing. Absorption maxima occurred at 275, 320, 416, 523, and 553 nm in the reduced form. The α-absorption peak was symmetrical and had an extinction coefficient of 25.9 mM−1 cm−1. The molecular weight was 11 100 by equilibrium centrifugation, 12 400 from iron determinations, and 10 300 from the amino acid composition. The molecule consisted of one heme group and 91 amino acid residues including two cysteine residues and one each of histidine, arginine, tryptophan, methionine, and proline. Other physical properties measured were: the sedimentation coefficient, 1.3 S; the normal oxidation reduction potential, + 0.388 V; and the isoelectric point, pH 3.75.

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