PROBING THE CYTOCHROME-C PEROXIDASE CYTOCHROME-C ELECTRON-TRANSFER REACTION USING SITE-SPECIFIC CROSS-LINKING

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) an d yeast iso-1-cytochrome c have been used to generate site specificall y cross-linked peroxidase-cytochrome c complexes for the purpose of pr obing interaction domains and the intramolecular electron transfer rea ction. Complex 2 was designed earlier [Pappa, H. S., & Poulos, T. L. ( 1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp14 8 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase t ransfers an electron to peroxidase compound I is estimated to be appro ximate to 0.5-1 s(-1) in complex 3 and approximate to 800 s(-1) in com plex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyfer ryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic meas urements show that complex 3 behaves like the wild type enzyme when ei ther horse heart or yeast ferrocytochrome c is used as an exogenous su bstrate, indicating that the region blocked in complex 3 is not a func tionally important interaction site. In contrast, complex 2 is inactiv e toward horse heart ferrocytochrome c at all ionic strengths tested a nd yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does compl ex 2 give wild type enzyme activity. This observation indicates that i n complex 2 the primary site of interaction of CCP with horse heart an d yeast ferrocytochrome c at high ionic strengths is blocked. The rele vance of these results to the pathway versus distance models of electr on transfer and to the interaction domains between peroxidase and cyto chrome c is discussed.