ENZYMATIC-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.