Species-specific differences in covalently crosslinked complexes of yeast cytochrome C peroxidase with horse and yeast ISO-1 ferricytochromes C

1. 1. The results of chemically crosslinking yeast cytochrome c peroxidase with both horse and yeast iso-1 ferricytochromes c have been studied by a combination of gel electrophoresis and proton NMR spectroscopy. 2. 2. The complexes were formed at a variety of potassium phosphate concentrations ranging from 10 to 300 mM using the water soluble crosslinking agent, EDC (l-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide). 3. 3. The primary crosslinking product in both cases is the 1:1 covalent complex, but, for each pair of partner proteins the yield of the 1:1 crosslinked complex varies with the salt concentration. 4. 4. Furthermore, at low salt concentrations the yield of the 1:1 covalent complex involving horse cytochrome c is much larger than the yield of the 1:1 covalent complex formed with yeast iso-1 cytochrome c, whereas at high salt concentrations the situation is reversed. 5. 5. Proton NMR spectroscopy, in combination with gel electrophoresis, provides evidence for the formation of different types of 1:1 complexes for the peroxidase/yeast cytochrome c pair and has been used to study the effect of changes in the solution ionic strength upon both the peroxidases/horse cytochrome c and the peroxidase/yeast cytochrome c complexes. 6. 6. This work indicates that electrostatic interactions between proteins play a dominant role in formation of complexes between cytochrome c peroxidase and horse ferricytochrome c, whereas the hydrophobic effect plays a comparatively larger role in stabilizing complexes between cytochrome c peroxidase and yeast iso-1 ferricytochrome c.