Variable-temperature spectroelectrochemical study of horseradish peroxidase.
The reduction potentials of the compound II/ferric and compound I/compound II couples have been studied, using potassium hexachloroiridate as 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 degrees' (compound I/compound II) = 897.9 +/- 3 mV (NHE) and E degrees'-(compound II/ferric) = 869.1 +/- 2 mV. Redox thermodynamic parameters, obtained from the temperature dependences of the reduction potentials of both couples, are reported. The reaction entropies (delta S degrees rc) for the compound II/ferric and compound I/compound II couples are 19.8 +/- 3.9 and 12.1 +/- 3.7 eu, respectively. This result indicates that the reorganization energy for the macrocycle-centered couple is lower than that for the metal-centered one. Together with our observation that E degrees' for the former is ca. 30 mV greater than that for the latter, these results suggest that compound I is more reactive toward outer-sphere reductants than compound II. In particular, the electron self-exchange rates for the compound I/compound II and compound II/ferric couples are estimated to be 4.4 x 10(-1) and 4.9 x 10(-4) M-1 s-1, respectively. Surprisingly, the formation of compound I from ferric HRP is accompanied by an almost zero standard entropy (delta S degrees') change.
Farhangrazi
Z S
ZS
National Center for the Design of Molecular Function, Utah State University, Logan 84322.
Fossett
M E
ME
Powers
L S
LS
Ellis
W R
WR
D004563:Electrochemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics
D007527:Isoenzymes; D006735:Horseradish Peroxidase