Enzymic activities of covalent 1:1 complexes of cytochrome c and cytochrome c peroxidase.
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 c peroxidase. (4) An allosteric or electrostatic effect exists between the two substrate binding sites, the binding of cytochrome c to the high-affinity site decreasing the binding affinity of the low-affinity site to cytochrome c. The higher the equilibrium binding affinity of the mutant cytochrome c to the peroxidase, the larger the apparent allosteric/electrostatic effect when that mutant protein is covalently bound to the high-affinity site of the enzyme. Furthermore, different locations of the covalently bound cytochrome c at the high-affinity site on the enzyme surface result in different degrees of allosteric/electrostatic effect. The presence of two active sites on the enzyme allows a simple interpretation of some of the differences in the steady-state kinetic behavior of cytochrome c peroxidase with horse and yeast iso-1 cytochrome c.
Department of Biological Sciences, University of Illinois at Chicago 60607-7020.
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Wang
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Margoliash
D000494:Allosteric Regulation; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics
D001386:Azides; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D003578:Cytochrome-c Peroxidase; D003545:Cysteine; C011171:4-azidophenacyl bromide