Enzymic activities of covalent 1:1 complexes of cytochrome c and cytochrome c peroxidase

Biochemistry
Y Wang, Emanuel Margoliash

Abstract

We have obtained several cysteine mutants in or around the cytochrome c peroxidase binding domain of rat and yeast iso-1 cytochrome c by site-directed mutagenesis. These cysteine residues were specifically labeled with the bifunctional photoactive cross-linker 4-azidophenacyl bromide (APB). 1:1 covalent complexes of cytochrome c peroxidase and cytochrome c were generated by cross-linking these specifically labeled cytochromes c to cytochrome c peroxidase, and the 1:1 complexes were purified. Steady-state kinetic studies of the purified 1:1 complexes with free yeast and horse cytochromes c showed the following: (1) Cytochrome c peroxidase has two distinct catalytic sites--a high-affinity and a low-affinity site. (2) Other than the difference in affinity, the binding of substrate at the low-affinity site is similar to that at the high-affinity site, with yeast cytochrome c interacting more strongly than the horse protein, the binding of both substrates being sensitive to ionic strength, and both sites able to transfer electrons. (3) HPLC chromatography of purified 1:1 complex showed multiple forms of 1:1 complexes, supporting the idea of multiple possible interactions between cytochrome c and the high-affinity site on cytochrome ...Continue Reading

References

Sep 27, 1995·Biochimica Et Biophysica Acta·W QinEmanuel Margoliash
Jun 5, 2002·Biochimica Et Biophysica Acta·J E Erman, L B Vitello
Jan 12, 2002·Bioelectrochemistry·Katsumi NikiEmanuel Margoliash
May 7, 2002·Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire·Christian BlouinCarmichael J A Wallace
Oct 22, 2013·Biotechnology and Bioengineering·Feng JiaSurya Mallapragada
Aug 9, 2011·Biochimica Et Biophysica Acta·Alexander N VolkovJonathan A R Worrall

Related Concepts

Covalent Interaction
Complex (molecular entity)
Mutagenesis, Site-Directed
Chromatography
Peroxidases
Cytochrome c Group
4-azidophenacyl bromide
High Pressure Liquid Chromatography Procedure
Cytochrome c-551 Peroxidase
Cyct

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