pH-dependent spectral and kinetic properties of cytochrome c peroxidase: comparison of freshly isolated and stored enzyme.

The effect of long-term storage on the electronic absorption spectrum and the kinetic properties of cytochrome c peroxidase has been investigated. No detectable differences were observed between freshly isolated enzyme and enzyme stored below -20 degrees C, in the crystalline state, for up to 41 months. The electronic absorption spectrum and the rate of the enzyme-hydrogen peroxide reaction are essentially independent of pH in 0.1 M potassium phosphate buffers for both fresh and stored enzyme. In buffers containing KNO3, the absorption spectrum and the kinetic properties of both fresh and stored enzyme vary with pH, consistent with the titration of an ionizable group with an apparent pKa of 5.5 +/- 0.1. The differences between phosphate- and nitrate-containing buffers are attributed to specific ion effects. In KNO3-containing buffers, the high-pH form of the enzyme reacts rapidly with hydrogen peroxide while the low-pH form is unreactive. Evidence is presented which indicates that both the low-pH and high-pH forms of the enzyme in KNO3-containing buffers are 5-coordinate, high-spin Fe(III) species.