Peroxidase activity as a tool for studying the folding of c-type cytochromes.
The peroxidase activity of c-type cytochromes increases substantially by unfolding. This phenomenon was used to study the equilibrium unfolding of ferricytochrome c. The peroxidase activity is already enhanced at low denaturant concentrations. The lowest free energy folding intermediate is easily detected by this method, while it is invisible using fluorescence or optical spectroscopy. The free energy difference between this folding intermediate and the native state depends on the strength of the sixth ligand of the heme-iron and the increase in peroxidase activity upon unfolding is shown to be a sensitive indicator of the strength of this ligand. Under fully denaturing conditions, the peroxidase activity is inhibited by protein-based ligands. It is shown that at least three different ligand groups can be responsible for this inhibition, and that at neutral or alkaline pH, the predominant ligand is not histidine. The use of peroxidase activity assays as a method to study the unfolding of cytochrome c is evaluated.
Diederix
Rutger E M
RE
Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Ubbink
Marcellus
M
Canters
Gerard W
GW
D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D019791:Guanidine / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D009206:Myocardium / Q000201:enzymology; D010230:Paracoccus / Q000201:enzymology; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000201:enzymology
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D007093:Imidazoles; D008024:Ligands; C029899:imidazole; C031732:cytochrome C-550; D009195:Peroxidase; D019791:Guanidine; D005998:Glycine