Structure-function studies on the redox-active tryptophan in cytochrome c peroxidase from Saccharomyces cerevisiae.

摘要

Cytochrome c peroxidase (CCP) is used as a model protein-engineering system to study structure-function relationships in heme peroxidases. In CCP compound I, a stable pi cation radical on tryptophan 191 (Trp191) forms during the catalytic cycle and is essential for activity. The redox active Trp191 functions in the electron transfer process. By installing a metal binding site into the proximal pocket of CCP approximately 8 angstroms from Trp191, it is possible to regulate the reactivity of Trp191 by varying the concentration of potassium. By increasing the concentration of potassium there is a decrease in the activity of the cation binding CCP mutant. The activity profile correlates with a decrease in the electron transfer rate as determined from stopped-flow analysis, and a reduction in the intensity of the compound I Trp191 radical signal as measured by electron paramagnetic resonance spectroscopy. The potassium dependent behavior is due to an electrostatic influence that the cation exerts on the electrostatic potential at Trp191, thus affecting the radical stability and consequently the activity.; The identical active site architecture of CCP exists in ascorbate peroxidase (APX), including a proximal Trp179. However, the reaction mechanism and substrate specificity of APX is very different than CCP, and does not involve a radical on Trp179. We reasoned that a nearby potassium ion found in the proximal pocket of APX was partly responsible for the different redox activity of the proximal Trp in APX. Electrostatic calculations and structural observations led us to design a functional metal binding site into CCP. The mutant presented here, CCPK2, has the same metal binding site as does the potassium binding site in APX. The metal cation occupying the binding site in CCP dramatically influences the activity of this mutant protein acting as an electrostatic "molecular switch". X-ray crystallographic analysis of CCPK2 confirms that potassium occupies the engineered site. Other metal binding sites have also been built onto the CCPK2 template to change the metal binding specificity. The structure based design strategy to control the activity of CCP by a concentration dependent metal ion response is described.