Variable-Temperature Spectroelectrochemical Study of Horseradish Peroxidase

Z. Shadi Farhangrazi, Martin E. Fossett, Linda S. Powers, Walther R. Ellis

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89 Scopus citations


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 π-cation radical intermediate was obtained. At 25 °C, E°'(compound I/compound II) = 897.9 ± 3 mV (NHE) and E°'- (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 (AS°rc) for the compound /ferric and compound I/compound 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°’ 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 (∆S°') change.

Original languageEnglish (US)
Pages (from-to)2866-2871
Number of pages6
Issue number9
StatePublished - Mar 1995

ASJC Scopus subject areas

  • Biochemistry


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