VARIABLE-TEMPERATURE SPECTROELECTROCHEMICAL STUDY OF HORSERADISH-PEROXIDASE

The reduction potentials of the compound II/ferric and compound I/comp ound II couples have been studied, using potassium hexachloroiridate a s a mediator titrant, by thin-layer spectroelectrochemistry. Compound I, which is 2 equiv more oxidized than the ferric (i.e., resting) form of the enzyme, was reversibly formed via a compound II intermediate; no evidence for a ferric porphyrin pi-cation radical intermediate was obtained. At 25 degrees C, E(o)'(compound I/compound II) = 897.9 +/- 3 mV (NHE) and E(o)'(compound II/ferric) = 869.1 +/- 2 mV. Redox thermo dynamic parameters, obtained from the temperature dependences of the r eduction potentials of both couples, are reported, The reaction entrop ies (Delta S-rc(o)) for the compound II/ferric and compound I/compound II couples are 19.8 +/- 3.9 and 12.1 +/- 3.7 eu, respectively. This r esult indicates that the reorganization energy for the macrocycle-cent ered couple is lower than that for the metal-centered one. Together wi th our observation that E(o)' for the former is ca. 30 mV greater than that for the latter, these results suggest that compound I is more re active toward outer-sphere reductants than compound II. In particular, the electron self-exchange rates for the compound I/compound II and c ompound II/ferric couples are estimated to be 4.4 x 10(-1) and 4.9 x 1 0(-4) M(-1) s(-1), respectively. Surprisingly, the formation of compou nd I from ferric HRP is accompanied by an almost zero standard entropy (Delta S-o') change.