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Journal article
Probing the Cytochrome c Peroxidase−Cytochrome c Electron Transfer Reaction Using Site Specific Cross-Linking
Biochemistry (Easton), v 35(15), pp 4837-4845
16 Apr 1996
PMID: 8664274
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Abstract
Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase−cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H. S., & Poulos, T. L. (1995) Biochemistry 34, 6573−6580] to mimic the known crystal structure of the peroxidase−cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748−1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be ≈0.5−1 s-1 in complex 3 and ≈800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 103 in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.
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Details
- Title
- Probing the Cytochrome c Peroxidase−Cytochrome c Electron Transfer Reaction Using Site Specific Cross-Linking
- Creators
- Helen S. Pappa - University of California, IrvineSherareh Tajbaksh - University of California, IrvineAleister J. Saunders - University of California, IrvineGary J. Pielak - University of California, IrvineThomas L. Poulos - University of California, Irvine
- Publication Details
- Biochemistry (Easton), v 35(15), pp 4837-4845
- Publisher
- American Chemical Society; Washington, DC
- Number of pages
- 9
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biology
- Web of Science ID
- WOS:A1996UF52100022
- Scopus ID
- 2-s2.0-0029865348
- Other Identifier
- 991021448036304721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology