1cyf
From Proteopedia
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'''IDENTIFYING THE PHYSIOLOGICAL ELECTRON TRANSFER SITE OF CYTOCHROME C PEROXIDASE BY STRUCTURE-BASED ENGINEERING''' | '''IDENTIFYING THE PHYSIOLOGICAL ELECTRON TRANSFER SITE OF CYTOCHROME C PEROXIDASE BY STRUCTURE-BASED ENGINEERING''' | ||
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[[Category: Kraut, J.]] | [[Category: Kraut, J.]] | ||
[[Category: Miller, M A.]] | [[Category: Miller, M A.]] | ||
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Revision as of 10:14, 2 May 2008
IDENTIFYING THE PHYSIOLOGICAL ELECTRON TRANSFER SITE OF CYTOCHROME C PEROXIDASE BY STRUCTURE-BASED ENGINEERING
Overview
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.
About this Structure
1CYF is a Single protein structure of sequence from Saccharomyces cerevisiae. Full crystallographic information is available from OCA.
Reference
Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering., Miller MA, Geren L, Han GW, Saunders A, Beasley J, Pielak GJ, Durham B, Millett F, Kraut J, Biochemistry. 1996 Jan 23;35(3):667-73. PMID:8547245 Page seeded by OCA on Fri May 2 13:14:41 2008
