Kinetics and Energetics of Intramolecular Electron Transfer in Yeast Cytochrome c Peroxidase

Pui S. Ho*, Natalie Solomon, Chae Hee Kang, Emanuel Margoliash, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yo-netani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D.C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618], The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) /. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration. This is slow relative to fluoride binding to the ferric enzyme, indicating that the rate-limiting step is the intramolecular transfer of oxidizing equivalents. Arrhenius plots of the observed rate constants for the Fe(IV) peroxidase and the Fe(III) enzyme yield some of the thermodynamic parameters that describe intermediates in the reaction pathway.

Original languageEnglish (US)
Pages (from-to)4122-4128
Number of pages7
JournalBiochemistry
Volume23
Issue number18
DOIs
StatePublished - Aug 1984

ASJC Scopus subject areas

  • Biochemistry

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