Complex-formation between cytochrome c and cytochrome c peroxidase. Equilibrium and titration studies.
1. Physical studies of complex-formation between cytochrome c and yeast peroxidase are consistent with kinetic predictions that these complexes participate in the catalytic activity of yeast peroxidase towards ferrocytochrome c. Enzyme-ferricytochrome c complexes have been detected both by the analytical ultracentrifuge and by column chromatography, whereas an enzyme-ferrocytochrome c complex was demonstrated by column chromatography. Estimated binding constants obtained from chromatographic experiments were similar to the measured kinetic values. 2. The physicochemical study of the enzyme-ferricytochrome c complex, and an analysis of its spectrum and reactivity, suggest that the conformation and reactivity of neither cytochrome c nor yeast peroxidase are grossly modified in the complex. 3. The peroxide compound of yeast cytochrome c peroxidase was found to have two oxidizing equivalents accessible to cytochrome c but only one readily accessible to ferrocyanide. Several types of peroxide compound, differing in available oxidizing equivalents and in reactivity with cytochrome c, seem to be formed by stoicheiometric amounts of hydrogen peroxide. 4. Fluoride combines not only with free yeast peroxidase but also with peroxidase-peroxide and accelerates the decomposition of the latter compound. The ligand-catalysed decomposition provides evidence for one-electron reduction pathways in yeast peroxidase, and the reversible binding of fluoride casts doubt upon the concept that the peroxidase-peroxide intermediate is any form of peroxide complex. 5. A mechanism for cytochrome c oxidation is proposed involving the successive reaction of two reversibly bound molecules of cytochrome c with oxidizing equivalents associated with the enzyme protein.
D002845:Chromatography; D003580:Cytochromes / Q000378:metabolism*; D004579:Electron Transport; D005295:Ferrocyanides; D005459:Fluorides; D006861:Hydrogen Peroxide; D007700:Kinetics; D008970:Molecular Weight; D010544:Peroxidases / Q000378:metabolism*; D010545:Peroxides; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D014461:Ultracentrifugation; D015003:Yeasts / Q000201:enzymology
D003580:Cytochromes; D005295:Ferrocyanides; D010545:Peroxides; D006861:Hydrogen Peroxide; D010544:Peroxidases; D005459:Fluorides