The ionic strength dependence of the reduction of cytochrome c peroxidase compound I by horse heart ferrocytochrome c
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The ionic strength dependence of the reduction of cytochrome c peroxidase compound I by horse cytochrome c has been studied, using stopped-flow technigue, in pH 7.5, potassium phosphate/nitrate buffer. The temperature was set at 25 ± 1° C. The wavelength was monitored at 4 32 nm, an isobestic point for ferri-/ferrocytochrome c, so only the absorbance change of the reduction of cytochrome c peroxidase compound I to compound II can be observed. The observed rate constant, kobs, as a function of the concentration of ferrocytochrome c shows a non-linear increase with increasing ionic strength. A two-parameter eguation is needed to fit the data at low ionic strength, 20 mM to 40 mM, while a three-parameter equation is needed at high ionic strength, 65 mM and above. The maximum rates of these reductions also show two different types of ionic strength dependence. At 20 mM to 40 mM ionic strength, the maximum rate of reduction decreases slightly, within experimental error, with increasing ionic strength. At high ionic strength, 65 mM to 300 mM, the reduction rate does not saturate at the highest cytochrome c concentration used. After testing different mechanisms, the simplest mechanism which can explain the data obtained at the whole ionic strength range, is a two-binding-site mechanism.