Journal article
Identifying the Physiological Electron Transfer Site of Cytochrome c Peroxidase by Structure-Based Engineering
Biochemistry (Easton), Vol.35(3), pp.667-673
23 Jan 1996
PMID: 8547245
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys of the parent enzyme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in the crystal structure. Cys 193 was then modified with a bulky sulfhydryl reagent, 3-(N-maleimidylpropionyl)biocytin (MPB), to prevent yCc from binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20−100-fold decreases in transient and steady-state ET reaction rates with yCc. The MPB modification causes only 2−3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies based on the crystal structures and indicates that solution phase ET complexes closely resemble the crystalline complexes. The low rate of catalysis of the MPB-enzyme was constant for yCc in buffers of 20−160 mM ionic strength. This indicates that the low affinity complex formed between CcP(MI) and yCc at low ionic strength is not reactive in ET.
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Details
- Title
- Identifying the Physiological Electron Transfer Site of Cytochrome c Peroxidase by Structure-Based Engineering
- Creators
- Mark A. Miller - University of Arkansas at FayettevilleLois Geren - University of Arkansas at FayettevilleGye Won Han - Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0317, Department of Chemistry, University of Arkansas, Fayetteville, Arkansas 72701-1201, and Departments of Chemistry and Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-3290Aleister Saunders - Drexel University, BiologyJames Beasley - University of North Carolina at Chapel HillGary J. Pielak - University of Arkansas at FayettevilleBill Durham - University of Arkansas at FayettevilleFrancis Millett - University of Arkansas at FayettevilleJoseph Kraut - Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0317, Department of Chemistry, University of Arkansas, Fayetteville, Arkansas 72701-1201, and Departments of Chemistry and Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-3290
- Publication Details
- Biochemistry (Easton), Vol.35(3), pp.667-673
- Publisher
- American Chemical Society (ACS)
- Number of pages
- 7
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biology
- Identifiers
- 991021906506604721
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- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology