Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase.
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.
D000596:Amino Acids / Q000032:analysis; D003574:Cytochrome c Group / Q000302:isolation & purification / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000302:isolation & purification / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D010047:Ovalbumin / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D013053:Spectrophotometry
D000596:Amino Acids; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D010047:Ovalbumin; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D004229:Dithiothreitol