IDENTIFYING THE PHYSIOLOGICAL ELECTRON-TRANSFER SITE OF CYTOCHROME-C PEROXIDASE BY STRUCTURE-BASED ENGINEERING

A technique was developed to evaluate whether electron transfer (ET) c omplexes formed in solution by the cloned cytochrome c peroxidase [CcP (MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structura lly similar to those seen in the respective crystal structures, Site-d irected mutagenesis was used to convert the sole Cys of the parent enz yme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in t he crystal structure. Cys 193 was then modified with a bulky sulfhydry l reagent, 3-(N-maleimidylpropionyl)biocytin (MPB), to prevent yCc fro m binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20-100-fold decreases i n transient and steady-state ET reaction rates with yCc. The MPB modif ication causes only 2-3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies bas ed on the crystal structures and indicates that solution phase ET comp lexes closely resemble the crystalline complexes. The low rate of cata lysis of the MPB-enzyme was constant for yCc in buffers of 20-160 mM i onic strength. This indicates that the low affinity complex formed bet ween CcP(MI) and yCc at low ionic strength is not reactive in ET.