SITE-SPECIFIC CROSS-LINKING AS A METHOD FOR STUDYING INTRAMOLECULAR ELECTRON-TRANSFER

Site-directed mutagenesis has been used to introduce cysteine residues into yeast cytochrome c peroxidase and yeast cytochrome c for the pur pose of forming site-specific cross-linked intermolecular complexes, T his enables the formation of well-defined homogeneous covalently linke d complexes for the purpose of relating structure to intramolecular el ectron transfer. Two complexes have been prepared and analyzed, Comple x I has an engineered cysteine at position 290 near the C-terminus of the peroxidase linked to the naturally occurring Cys102 near the C-ter minus of yeast cytochrome c. This complex exhibits undetectable rates of intramolecular electron transfer. Complex II has Cys290 of the pero xidase linked to the engineered Cys73 of cyt c. This complex was desig ned to mimic the crystal structure of the peroxidase-cytochrome c nonc ovalent complex [Pelletier and Kraut (1992) Science 258, 1748-1755]. S topped-flow studies show that complex II carries out intramolecular el ectron transfer from ferrocytochrome c to peroxidase compound I at a r ate of approximate to 500-800 s(-1). This indicates that the binding o rientation observed in the crystal structure is competent in rapid int ramolecular electron transfer.